Markers for identifying and quantifying mutations, expression, splice variants, translocations, copy number, or methylation changes in nucleic acid sequences

Abstract

To provide techniques for virtually eliminating false positives in diagnostic tests aimed at finding very rare or low-abundance variant sequences.SOLUTION: The present invention relates to methods for identifying and / or quantifying low abundance, nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicing, exon insertion, exon deletion, intron insertion, or other rearrangements at the genomic level, and / or methylated nucleotide bases. A first aspect of the present application is directed to a method for identifying, in a sample, one or more parental nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequence of other parental nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 843,032, filed May 3, 2019, which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under Grant No. P41 EB020594 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Field This application relates to methods and markers for identifying and quantifying nucleic acid sequence, mutations, expression, splice variants, translocations, copy number, and / or methylation changes using bisulfite treatment, nucleases, ligation, and polymerase reactions in combination with carryover prevention. [Background technology]

[0004] background Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries. It accounts for 580,000 deaths annually in the United States, 1.3 million in Europe, and 2.8 million in China (Siegel et al., "Cancer Statistics, 2016," CA Cancer J. Clin. 66(1):7-30(2016)). Cancer is currently the leading cause of mortality worldwide, with an estimated 8.2 million cancer deaths in 2012 (Torre et al., "Global Cancer Statistics, 2012," CA Cancer J. Clin. 65(2):87-108(2015)). Global cancer cases are expected to increase by 75% over the next 20 years, reaching nearly 25 million cases. Women have a 19% lifetime risk of dying from invasive cancer, compared with a 23% lifetime risk for men. With the total annual cost of cancer treatment in the United States exceeding $400 billion, no other medical problem more urgently requires intelligent solutions.

[0005] In the United States, new cancer cases in men are primarily prostate cancer (21%), lung cancer (14%), colorectal cancer (8%), bladder cancer (7%), melanoma (6%), non-Hodgkin's lymphoma (5%), kidney cancer (5%), head and neck cancer (4%), leukemia (4%), and liver and bile duct cancer (3%). For women, most newly diagnosed cancers are breast cancer (29%), lung cancer (13%), colorectal cancer (8%), endometrial cancer (7%), thyroid cancer (6%), non-Hodgkin's lymphoma (4%), melanoma (3%), leukemia (3%), pancreatic cancer (3%), and kidney cancer (3%). The leading causes of cancer death in men and women, respectively, are lung cancer (27%), prostate cancer (8%), colorectal cancer (8%), and lung cancer (26%), breast cancer (14%), and colorectal cancer (8%). These cancers are caused by different biological processes, and although there have been great advances in treating some cancers, such as the advent of targeted drugs and immunotherapy, most cancers are found at a later stage when survival rates are low. The lack of reliable and affordable early detection tests means many types of cancer are diagnosed at a later stage, dropping survival rates for some cancers to less than 10%. Current screening technologies fail due to low patient compliance, high costs, and low sensitivity and specificity (Das et al., “Predictive and Prognostic Biomarkers in Colorectal Cancer: A Systematic Review of Recent Advances and Challenges,” Biomedicine & Pharmacotherapy 87:8-19 (2016)). For example, the high cost, discomfort, and invasiveness of colonoscopy are significant barriers to patient compliance with CRC screening (Beydoun et al., “Predictors of Colorectal Cancer Screening Behaviors Among Average-Risk Older Adults in the United States,” Cancer Causes & Control: CCC 19(4):339-359 (2008)).Similarly, patient aversion to handling feces has limited the success of FOBT / FIT and precluded stool-based testing as a remedy for low compliance. In contrast, the current proposal addresses these issues by developing a blood test with the potential to become widely adopted. Improving patient compliance with CRC testing will lead to earlier detection and ultimately increased patient survival.

[0006] Ultimately, there is an urgent need to develop noninvasive, highly sensitive, highly specific, and cost-effective tests for detecting early-stage cancer. Two relatively recent advances in cancer research provide guidance for these tasks. First, the use of modern genomic tools (e.g., genome-wide sequencing, transcription, and methylation profiling). Public access to the vast databases generated from these studies has accelerated the discovery of a broader list of molecular markers (e.g., promoter methylation, mutations, copy number, or expression levels of mRNA, microRNA, noncoding RNA (ncRNA), and long noncoding RNA (lncRNA) associated with cancer progression). Second, the discovery that cancer cells can release nucleic acids into a patient's bloodstream. Cancer cells can undergo apoptosis (induced cell death), releasing cell-free DNA (cfDNA) into a patient's bloodstream (Salvi et al., "Cell-free DNA as a Diagnostic Marker for Cancer: Current Insights," OncoTargets and Therapy 9:6549-6559 (2016)). Levels of cfDNA in the serum of cancer patients vary from vanishingly low to high but do not correlate with the stage of the cancer (Perlin et al., "Serum DNA Levels in Patients With Malignant Disease," American Journal of Clinical Pathology 58(5):601-602 (1972); Leon et al., "Free DNA in the Serum of Cancer Patients and the Effect of Therapy," Cancer Res. 37(3):646-650 (1977)). Furthermore, exosomes (lipid vesicles ranging from 30 to 100 nm) released into the blood by cancer cells may contain the same RNA molecules and serve as a transcriptional signature of the tumor. Exosomes or tumor-associated vesicles shield mRNA, lncRNA, ncRNA, and even mutant tumor DNA from exogenous nucleases, thus protecting the markers.Other protected states include, but are not limited to, DNA, RNA, and proteins within circulating tumor cells (CTCs), other non-cellular membranes, including within vesicles or particles, within nucleosomes, or within Argonaute or other protein complexes. Notably, cfDNA contains the same molecular abnormalities as solid tumors, such as hyper- or hypomethylated mutations, copy number alterations, or chromosomal rearrangements (Ignatiadis et al., "Circulating Tumor Cells and Circulating Tumor DNA for Precision Medicine: Dream or Reality?" Ann. Oncol. 25(12):2304-2313 (2014)).

[0007] Tumor-specific CpG methylation has been detected in plasma from patients with various solid tumors via various techniques involving bisulfite conversion of unmethylated cytosine, methylation-sensitive enzymes, or immunoprecipitation of 5-methylcytosine (Jorda et al., “Methods for DNA methylation analysis and applications in colon cancer,” Mutat. Res. 693(1-2):84-93(2010)). (Pratt VM, “Are We Ready for a Blood-Based Test to Detect Colon Cancer?” Clinical Chemistry 60(9):1141-1142(2014); Warton et al., “Methylation of Cell-Free Circulating DNA in the Diagnosis of Cancer,” Frontiers in Molecular Biosciences 2:13(2015)). Methylation signatures have better specificity for certain cancer types because methylation patterns are highly tissue-specific (Issa JP, "DNA Methylation as a Therapeutic Target in Cancer," Clin. Cancer Res. 13(6):1634-1637(2007)). The most well-studied blood-based methylation marker for CRC detection is located in the promoter region of the SEPT9 gene (Church et al., "Prospective Evaluation of Methylated SEPT9 in Plasma for Detection of Asymptomatic Colorectal Cancer," Gut 63(2):317-325(2014); Lofton-Day et al., "DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening," Clinical Chemistry 54(2):414-423(2008); Potter et al.,"Validation of a Real-time PCR-based Qualitative Assay for the Detection of Methylated SEPT9 DNA in Human Plasma," Clinical Chemistry 60(9):1183-1191(2014), Ravegnini et al., "Simultaneous Analysis of SEPT9 Promoter Methylation Status, Micronuclei Frequency, and Folate-Related Gene Polymorphisms: The Potential for a Novel Blood-Based Colorectal Cancer Biomarker," International Journal of Molecular Sciences 16(12):28486-28497(2015), Toth et al., "Detection of Methylated SEPT9 in Plasma is a Reliable Screening Method for Both Left- and Right-sided Colon Cancers," PloS One 7(9):e46000(2002), Toth et al., "Detection of Methylated Septin 9 in Tissue and Plasma of Colorectal Patients with Neoplasia and the Relationship to the Amount of Circulating Cell-Free DNA," PloS One 9(12):e115415(2014), Warren et al., "Septin 9 Methylated DNA is a Sensitive and Specific Blood Test for Colorectal Cancer," BMC Medicine 9:133(2011)). Other potential markers for CRC diagnosis include THBD (Lange et al., “Genome-scale Discovery of DNA-methylation Biomarkers for Blood-Based Detection of Colorectal Cancer,” PloS One 7(11):e50266(2012)), C9orf50(Lange et al., “Genome-scale Discovery of DNA-methylation Biomarkers for Blood-Based Detection of Colorectal Cancer,” PloS One 7(11):e50266(2012)), ZNF154(Margolin et al., “Robust Detection of DNA Hypermethylation of ZNF154 as a Pan-Cancer Locus with in Silico Modeling for Blood-Based Diagnostic Development,” The Journal of Molecular Diagnostics 18(2):283-298(2016)), and AGBL4, FLI1, and TWIST1 (Lin et al., “Clinical Relevance of Plasma DNA Methylation in Colorectal Cancer Patients Identified by Using a Examples include CpG sites in the promoter regions of tumor suppressor genes (including ATM, BRCA1, RASSF1, APC, and RARβ) detected in patient cfDNA (Tang et al., "Blood-Based DNA Methylation as a Biomarker for Breast Cancer: A Systematic Review," Clinical Epigenetics 8:115 (2016)). A caveat to using methylation markers is that bisulfite conversion tends to break down DNA, thus reducing the overall signal that can be detected. Methylation detection techniques can also result in false-positive signals due to incomplete conversion of unmethylated cytosines. As described herein, extensive bioinformatics analysis of public databases has been conducted to identify CRC- and tissue-specific methylation markers suitable for detecting cancer in plasma. It is predicted that methylation marker detection assays will enable higher levels of multiplexing with single-molecule detection capabilities, enabling greater sensitivity and specificity across a broad range of cancers.

[0008] The challenge in developing reliable diagnostic and screening tests is to distinguish markers expressed by tumors that are indicative of disease (e.g., early-stage cancer) from the presence of the same markers in normal tissue, which may result in false-positive signals. Also, the specificity and sensitivity of the assay must balance the number of markers tested against the cost of the test. Comprehensive molecular profiling (mRNA, methylation, copy number, miRNA, mutations) of thousands of tumors by The Cancer Genome Atlas Consortium (TCGA) has revealed that colorectal tumors are as distinct from each other as they are from breast, prostate, or other epithelial cancers (TCGA “Comprehensive Molecular Characterization of Human Colon and Rectal Cancer Nature 487:330-337(2014)). Furthermore, some markers they share are present in multiple cancer types, hindering the ability to identify the tissue of origin. BRAF mutations occur frequently in melanoma (42%) and thyroid cancer (41%), while KRAS is also highly mutated in pancreatic (55%) and lung (16%) cancers (Forbes et al., “COSMIC: Exploring the World's Knowledge of Somatic Mutations in Human Cancer,” Nucleic Acids Res. 43(Database issue):D805-811(2015)).Generally, CRC mutation markers such as KRAS and BRAF are found in late-stage primary cancers and metastases (Spindler et al., "Circulating free DNA as a Biomarker and Source for Mutation Detection in Metastatic Colorectal Cancer," PloS One 10(4):e0108247(2015); Gonzalez-Cao et al., "BRAF Mutation Analysis in Circulating Free Tumor DNA of Melanoma Patients Treated with BRAF Inhibitors," Melanoma Res. 25(6):486-495(2015); Sakai et al., "Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing," PloS One 10(5):e0121891(2015)). For early cancer detection, nucleic acid assays should primarily serve as screening tools and require the availability of secondary diagnostic follow-up (e.g., colonoscopy for colorectal cancer).

[0009] Complicating the biological problem is the need to reliably quantify mutations, CpG methylation, or DNA or RNA copy number from only a small number of early cells (i.e., from CTCs), or the cancer signal may be derived from cell-free DNA (cfDNA) in the blood and diluted by excess nucleic acid originating from normal cells or inadvertently released from normal blood cells during sample processing (Mateo et al., “The Promise of Circulating Tumor Cell Analysis in Cancer Management,” Genome Biol. 15:448 (2014); Haque et al., “Challenges in Using ctDNA to Achieve Early Detection of Cancer,” BioRxiv. 237578 (2017)).

[0010] Some IVD cancer companies are developing commercially available methylation detection tests. The aforementioned SEPT9 methylation is the basis of the Epi proColon test, a CRC detection assay by Epigenomics (Lofton-Day et al., “DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening,” Clinical Chemistry 54(2):414-423(2008)). Although early results on smaller sample sets have shown promise, a larger study using 1,544 plasma samples found a sensitivity of 64% and a specificity of 78%-82% for stages I-III CRC, effectively sending 180-220 out of 1,000 individuals to unnecessary colonoscopy (Potter et al., "Validation of a Real-time PCR-based Qualitative Assay for the Detection of Methylated SEPT9 DNA in Human Plasma," Clinical Chemistry 60(9):1183-1191 (2014)). Clinical genomics is currently developing blood-based CRC detection tests based on methylation of the BCAT1 and IKZF1 genes (Pedersen et al.(Young et al., "A Cross-sectional Study Comparing a Blood Test for Methylated BCAT1 and IKZF1 Tumor-derived DNA with CEA for Detection of Recurrent Colorectal Cancer," Cancer Medicine 5(10):2763-2772(2016)). A large-scale study of this two-marker test using 2,105 plasma samples demonstrated an overall sensitivity of 66%, a sensitivity of 38% for stage I CRC, and an impressive specificity of 94%. Exact Sciences and collaborators have slightly improved the sensitivity of CRC stool testing by adding K-ras mutations and methylation markers for BMP3 and NDRG4 (Lidgard et al., "Clinical Performance of an Automated Stool DNA Assay for Detection of Colorectal Neoplasia," Clin. Gastroenterol. Hepatol. 11(10):1313-1318(2013)) (Bosch et al., "Analytical Sensitivity and Stability of DNA Methylation Testing in Stool Samples for Colorectal Cancer Detection," Cell Oncol. (Dordr) 35(4):309-315(2012); Hong et al., "DNA Methylation Biomarkers of Stool and Blood for Early Detection of Colon Cancer," Genet. Test. Mol. Biomarkers 17(5):401-406(2013); Imperiale et al., “Multitarget Stool DNA Testing for Colorectal-Cancer Screening,” N. Engl. J.Med.370(14):1287-1297(2014), Xiao et al., “Validation of Methylation-Sensitive High-Resolution Melting (MS-HRM) for the Detection of Stool DNA Methylation in Colorectal Neoplasms,” Clin.Chim.Acta 431:154-163(2014), Yang et al., “Diagnostic Value of Stool DNA Testing for Multiple Markers of Colorectal Cancer and Advanced Adenoma: a Meta-Analysis,” Can.J.Gastroenterol.27(8):467-475(2013)). A large-scale study of 12,500 stool samples claimed a sensitivity of 93%, but specificity was still only 85%, essentially sending 150 out of 1,000 people to unnecessary colonoscopies. Despite the logistical challenges of handling feces, Exact Sciences recently sold its one millionth test. The Cologuard website warns that test results can be both false-positive and false-negative, and that the test should not be used if patients have hemorrhoids, menstrual periods, or bloody stools. The website also warns that the test should not be used by patients with ulcerative colitis (UC), Crohn's disease (CD), inflammatory bowel disease (IBD), or a family history of cancer. In other words, Exact Sciences is excluding patients who would most benefit from an accurate CRC detection test. Recently, the Laboratory for Advanded Medicine (based in Irvine, California, and associated with various Chinese academic institutions) demonstrated the feasibility of examining the methylation status of a single CpG site (cg10673833) for blood-based detection of colorectal cancer (Luo et al., “Circulating Tumor DNA Methylation Profiles Enable Early Diagnosis, Prognosis Prediction, and Screening for Colorectal Cancer,” Science Translational Medicine 12:(524)(2020)). . A range of diagnostic needs necessitates a range of diagnostic tests.

[0011] Most current molecular diagnostic efforts in cancer are centered on: (i) prognostic and predictive genomics, e.g., identifying genetic mutations in cancer predisposition genes such as BrCA1, BrCA2, (Ford et al., Am. J. Hum. Genet. 62:676-689 (1998)); (ii) personalized treatment, e.g., mutations in the EGFR gene leading to personalized medicine (Sequist and Lynch, Ann. Rev. Med. 59:429-442 (2008)); and (iii) recurrence monitoring, e.g., detecting KRAS mutations that appear in patients who develop resistance to drug treatment (Hiley et al., Genome Biol. 15:453 (2014); Amado et al., J. Clin. Oncol. 26:1626-1634 (2008)). However, this overlooks key opportunities in the cancer molecular diagnostic continuum: (i) more frequent screening of individuals with a family history, (ii) screening for early disease detection, and (iii) monitoring treatment effectiveness. To address these three unmet needs, a new metric for blood-based detection, termed "cancer marker load," analogous to viral load, is proposed herein.

[0012] DNA sequencing offers the ultimate ability to identify all nucleic acid changes associated with disease. However, this process still requires multiple prior sample and template preparations, and therefore DNA sequencing is not always cost-effective. DNA microarrays can provide substantial information about multiple sequence variants, such as SNPs or different RNA expression levels, and are therefore less cost-effective than sequencing, but they are not very suitable for obtaining highly quantitative results or detecting low-abundance mutations. At the other end of this spectrum is the TaqMan™ reaction, which provides real-time quantification of known genes, but is not very suitable for identifying multiple sequence variants or low-abundance mutations.

[0013] NGS requires substantial upfront sample preparation to polish ends and add linkers, and its current error rate of 0.7% is too high to identify 2–3 mutant sequence molecules among a 10,000-fold excess of wild-type molecules. "Deep sequencing" protocols have been developed to overcome this deficiency by adding unique molecular identifiers to both strands of individual fragments. These approaches are known as: Tam-Seq & CAPP-Seq (Roche), Circle-Seq (Guardant Health), Safe-SeqS (Personal Genome Diagnostics), ThruPlex (Rubicon Genomics), NEBNext (New England Biolabs), QIAseq (Qiagen), Oncomine (ThermoFisher), Duplex Barcoding (Schmitt), SMRT (Pacific Biosciences), SiMSen-Seq (Stahlberg), and smMIP (Shendure). However, these methods require 30–100x depth per mutation strand to validate each mutation and distinguish it from other types of sequencing errors. A recent study from MSKCC demonstrated that 60,000x coverage is required to accurately identify mutations in plasma from metastatic cancer patients (91% sensitivity, 508-gene panel, 60,000x coverage). Further complicating the challenge, a recent paper from NEB questions the quality of the most widely used databases for rare variants and somatic mutations (Chen et al., “DNA Damage is a Pervasive Cause of Sequencing Errors, Directly Confounding Variant Identification,” Science 355(6326):752–756 (2017)).

[0014] It is important to match each unmet diagnostic need with the appropriate diagnostic test. This combines the distinct goals of achieving both high sensitivity (i.e., low false negatives) and high specificity (i.e., low false positives) at low cost. For example, direct sequencing of EGFR exons from tumor biopsies to determine treatment for non-small cell lung cancer (NSCLC) is significantly more accurate and cost-effective than designing TaqMan™ probes for over 180 known mutations for which drug responses have already been cataloged (Jia et al., Genome Res. 23:1434-1445 (2013)). The most sensitive techniques for detecting point mutations, such as "BEAMing" (Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003)), rely on prior knowledge of which mutations to look for and are therefore best suited for monitoring disease recurrence rather than early detection. Similarly, for monitoring blood levels of Bcr-Abl translocations in CML patients treated with Gleevec (Jabbour et al., Cancer 112:2112-2118 (2008)), a simple quantitative reverse transcription PCR assay is far preferable to sequencing the whole genome DNA in 1 ml of blood (9 million cells x 3 GB = 27 million Gb of raw data).

[0015] Sequencing of 2.1 Gb of cell-free DNA (cfDNA) isolated from NSCLC patients was used to provide 10,000x coverage for 125 kb of targeted DNA (Kandoth et al., Nature 502:333-339 (2013)). This approach correctly identified mutations present in matched tumors, but only covered 50% of stage 1 tumors. While this approach is promising for NSCLC, where samples average 5-20 mutations / Mb, targeted NGS is not cost-effective for other cancers, such as breast and ovarian cancer, where samples average fewer than 1-2 mutations per Mb. The current pre-ligation, amplification, and / or capture steps required for highly accurate targeted deep sequencing remain more complex than multiplexed PCR-TaqMan™ or PCR-LDR assays.

[0016] Deep sequencing of cfDNA of 58 cancer-related genes at 30,000x coverage can detect stage 1 or 2 cancers with moderate sensitivity, but missed 29% of CRC, 41% of breast cancer, 41% of lung cancer, and 32% of ovarian cancer, respectively (Phallen et al., “Direct Detection of Early-Stage Cancers Using Circulating Tumor DNA,” Science Translational Medicine 9(403)(2017)).An alternative strategy relied on targeted sequencing of an average of 30 bases in 61 segments to examine "hotspot" mutations in 16 genes, including TP53, KRAS, APC, PIK3CA, and PTEN, missing earlier-stage cancers (Cohen et al., "Detection and Localization of Surgically Resectable Cancers with a Multi-analyte Blood Test," Science (2018)). To extend the sensitivity of mutation sequencing, the Hopkins team recently combined NGS with quantification of serum protein markers (e.g., CA-125, CA19-9, CEA, HGF, myeloperoxidase, OPN, prolactin, and TIMP-1) to improve detection of five cancer types (ovarian, liver, stomach, pancreatic, and esophageal) with sensitivities ranging from 69% to 98% (Cohen et al., "Detection and Localization of Surgically Resectable Cancers with a Multi-analyte Blood Test," Science (2018)). One caveat to using these protein markers is that a previous large-scale study using age-matched controls (n = 22,000) failed to demonstrate clinical utility (Jacobs et al., “Prevalence Screening for Ovarian Cancer in Postmenopausal Women by CA-125 Measurement and Ultrasonography,” BMJ 306(6884):1030-1034(1993)). Therefore, a 2018 JAMA report stated, “The USPSTF does not recommend [CA-125] screening for ovarian cancer in asymptomatic women. This recommendation applies to asymptomatic women who are not known to have a high-risk hereditary cancer syndrome” (USPSTF et al., “Screening for Ovarian Cancer: US Preventive Services Task Force Recommendation Statement,” JAMA 319(6):588-594(2018)).Another caveat to using these protein markers is that they reflect tissue damage and are likely to appear in patients with inflammatory diseases such as arthritis (Kaiser, "Liquid Biopsy for Cancer Promises Early Detection," Science 359(6373):259(2018)). With the obesity epidemic and aging population in the United States, the risk of false positives from protein markers increases, along with inflammation associated with obesity and aging.

[0017] More recently, NGS sequencing companies (Grail, Guardant Health, Natera, Freenome) have aggressively expanded their targeted sequencing panels to now include whole-genome sequencing (WGS) and whole-genome bisulfite sequencing (Bis-WGS). Recent results from Grail, published in an abstract at ASCO in 2018 (Klein et al., “Development of a Comprehensive Cell-Free DNA (cfDNA) Assay for Early Detection of Multiple Tumor Types: The Circulating Cell-Free Genome Atlas (CCGA) Study,” ASCO Annual Meeting 2018, Chicago, IL; Abstract 12021 #134), revealed a 63% sensitivity for detecting “early-stage” CRC, but that claim was based on only 27 samples, most of which were stage III. Mutation-rich lung cancer also provided a 50% sensitivity, and most of the samples were stage III. When most samples are stage I and II, such as prostate cancer, sensitivity for detecting "early cancer" drops to less than 5%. When attempting to detect the most common form of breast cancer (HR+ / HER2), sensitivity drops to less than 13%. Even worse, screening-diagnosed breast cancer yielded a sensitivity of less than 11%. In short, NGS approaches fail by consistently missing 30%-80% of early-stage cancers (i.e., stage I & II).In a study first reported at the 2019 ASCO meeting (Liu et al., “Simultaneous Multi-cancer Detection and Tissue of Origin (TOO) Localization Using Targeted Bisulfite Sequencing Plasma Cell-free DNA (cfDNA),” ASCO Breakthrough Presentation 2019), followed by a study published in 2020 (Liu et al., “Sensitive and Specific Multi-cancer Detection and Localization Using Methylation Signatures in Cell-free DNA,” Annals of Oncology; In Press (2020)), GRAIL demonstrated that their Multi-Cancer Early Detection Test demonstrated an overall detection rate of 76% (12 types of deadly cancer) with a specificity of 99.3%. A combined analysis of this group of cancers demonstrated robust detection across all stages, with detection rates of 39 percent (27-52%), 69 percent (56-80%), 83 percent (75-90%), and 92 percent (86-96%) for stage I (n=62), stage II (n=62), stage III (n=102), and stage IV (n=130), respectively. At another meeting, GRAIL and collaborators reported the results of an analysis of cell-free DNA (DNA that was once confined to cells but entered the bloodstream upon cell death) in blood samples from 3,583 individuals, including 1,530 patients diagnosed with cancer and 2,053 individuals without cancer (Oxnard et al., “Simultaneous Multi-cancer Detection and Tissue of Origin (TOO) Localization Using Targeted Bisulfite Sequencing of Plasma Cell-free DNA (cfDNA),” ESMO Congress (2019)).Patient samples included more than 20 cancer types, including hormone receptor-negative breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, head and neck cancer, lung cancer, lymphocytic leukemia, multiple myeloma, ovarian cancer, and pancreatic cancer. Overall specificity was 99.4%, meaning that only 0.6% of results falsely indicated the presence of cancer. The assay's sensitivity (the percentage of blood samples from these patients that tested positive for cancer) for detecting pre-specified high-mortality cancers was 76%. Within this group, sensitivity for patients with stage I cancer was 32%, stage II 76%, stage III 85%, and stage IV 93%. Sensitivity across all cancer types was 55%, with a similar increase in detection by stage. For 97% of samples that returned a tissue of origin result, the test correctly identified the organ or tissue of origin in 89% of cases. However, another 2019 study (reported by GRAIL and collaborators) questioned the validity of the aforementioned report (Razavi et al., “High-intensity Sequencing Reveals the Sources of Plasma Circulating Cell-free DNA Variants,” Nat Med 25(12):1928-1937 (2019)). Through sequencing (60,000x depth) of a 2Mb, 508-gene panel, the authors demonstrated that the majority of cell-free DNA mutations in both non-cancer controls and cancer patients had characteristics consistent with clonal hematopoiesis, a process by which white blood cells gradually accumulate somatic alterations without necessarily resulting in a hematologic condition or malignancy. Indeed, mutations were present in 93.6 percent of white blood cells from individuals without cancer and in 99.1 percent of individuals with cancer.At a recent conference, GRAIL and its collaborators reported that their blood-based test can detect multiple GI cancers with a sensitivity of just under 50% for stage I and 73% for stages I-III (Wolpin et al., “Performance of a Blood-Based Test for the Detection of Multiple Cancer Types,” in: Gastrointestinal Cancers Symposium 2020 (2020)). In the case of Freenome, a recent ASCO presentation showed that their platform (whole-genome sequencing, bisulfite sequencing, and plasma analysis with protein quantification) was able to achieve a mean sensitivity of 92% for early-stage (n=17) and 84% for late-stage (n=11) colorectal adenocarcinoma detection, with a specificity of 90%. Across all CRC pathological subtypes, the Freenome test achieved a specificity of 90% and a sensitivity of 80% and 83% for early-stage (n=19) and late-stage (n=12) CRC, respectively. In a private discussion with Imran Haque, who recently resigned as CSO of Freenome, which had a $70 million budget and 30 scientists sequencing the plasma of 817 CRC patients and matched controls, he admitted that Freenome (and GRAIL) were overcalling the data and that none of them had a robust approach to achieving cost-effective, true early cancer detection (Wan et al., “Machine Learning Enables Detection of Early-Stage Colorectal Cancer by Whole-genome Sequencing of Plasma Cell-free DNA,” BioRxiv 478065 (2018)).

[0018] A comprehensive data analysis of over 600 colorectal cancer samples, taking into account tumor heterogeneity, tumor clusters, and biological / technical false positives ranging from 3% to 10% per individual marker, showed that optimal early detection screening for colorectal cancer requires at least 5–6 positive markers out of 24 tested markers (Bacolod et al., Cancer Res. 69:723–727 (2009); Tsafrir et al., Cancer Res. 66:2129–2137 (2006); Weinstein et al., Nat. Genet. 45:1113–1120 (2013); Navin NE Genome Biol. 15:452 (2014); Hiley et al., Genome Biol. 15:453 (2014); Esserman et al. Lancet Oncol 15:e234–242 (2014)). Furthermore, marker distribution is biased across different tumor clades; for example, some tumors are heavily methylated, while others are barely methylated, indistinguishable from age-associated methylation in adjacent tissues. Therefore, to obtain sufficient coverage of all different tumor clades, a multidimensional approach is required, using a combination of 3–5 sets of mutation, methylation, miRNA, ncRNA, lncRNA, mRNA, copy variation, alternative splicing, or translocation markers. Similar to noninvasive prenatal screening for trisomy, based on performing sequencing or ligation detection on random fragments of fDNA (Benn et al., Ultrasound Obstet. Gynecol. 42(1):15-33(2013); Chiu et al., Proc. Natl. Acad. Sci. USA 105:20458-20463(2008); Juneau et al., Fetal Diagn. Ther. 36(4)(2014)), the actual markers scored in cancer screening are secondary to accurate quantification of those positive markers in plasma.

[0019] As noted above, cancer-specific RNA markers (including microRNAs, lncRNAs, and mRNAs) may be present in the blood, either compartmentalized (Souza et al., “Circulating mRNAs and miRNAs as Candidate Markers for the Diagnosis and Prognosis of Prostate Cancer,” PloS One 12(9):e0184094(2017)), or exosomes (Nedaeinia et al., “Circulating Exosomes and Exosomal microRNAs as Biomarkers in Gastrointestinal Cancer,” Cancer Gene Ther 24(2):48-56(2017); Lai et al., “A microRNA Signature in Circulating Exosomes is Superior to Exosomal Glypican-1 Levels for Diagnosing Pancreatic Cancer,” Cancer Lett 39:86-93(2017)) or contained in circulating tumor cells ("CTCs"), which have been tagged as potential indicators of early-stage cancer. Compared with markers derived from surrounding cells, there are numerous challenges regarding the use of plasma-derived nucleic acid markers (which contain minute amounts of these markers in the blood) for early cancer detection. Indeed, due to these limitations, these "early" detection assays appear more likely to detect late-stage primary and metastatic cancers (Pantel "Blood-Based Analysis of Circulating Cell-Free DNA and Tumor Cells for Early Cancer Detection," PLoS Med 13(12):e1002205(2016)). Technical challenges in developing cancer diagnostic tests.

[0020] Diagnostic tests aimed at finding very rare or low-abundance variant sequences face potential false positives arising from: (i) polymerase errors during replication of the wild-type target, (ii) DNA sequencing errors, (iii) misligation at the wild-type target, (iii) target-independent PCR products, and (iv) carryover contamination of PCR products arising from previous positive samples. The serious clinical implications of a positive test result when screening for cancer require such tests to use every possible means to virtually eliminate false positives.

[0021] The core concept of nucleic acid detection is the selective amplification or purification of a desired cancer-specific marker from the same or closely similar markers from normal cells. These approaches include (i) multiple primer binding regions for orthogonal amplification and detection, (ii) affinity selection of CTCs or exosomes, and (iii) spatial dilution of the sample.

[0022] The success of PCR-LDR, which uses four primer-binding regions to ensure sensitivity and specificity, has been demonstrated previously. The desired region is amplified using a pair of PCR primers, or even a tandem pair of PCR primers, followed by an orthogonal nested LDR primer pair for detection. One advantage of using PCR-LDR is that proportional PCR amplification of multiple fragments can be performed to enrich for low-copy targets, and then quantitative LDR can be used to directly identify cancer-specific mutations. Biofire / bioMerieux has developed a similar technology called "film array," in which the products of an initial multiplexed PCR reaction are redistributed into individual wells, followed by nested real-time PCR with SYBR Green dye detection.

[0023] Affinity purification of CTCs using antibody or aptamer capture has been demonstrated (Adams et al., J. Am. Chem. Soc. 130:8633-8641 (2008); Dharmasiri et al., Electrophoresis 30:3289-3300 (2009); Soper et al. Biosens. Bioelectron. 21:1932-1942 (2006)). Peptide affinity capture of exosomes has been reported in the literature. Enriching these tumor-specific fractions from blood allows for copy number quantification and simplifies screening and validation assays.

[0024] A final approach, spatial dilution of samples, is used in digital PCR and a related method known as BEAMing (Vogelstein and Kinzler, Proc. Natl. Acad. Sci. USA 96(16):9236-41 (1999); Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003)). The rationale for digital PCR is to overcome the limitations of enzyme discrimination when samples contain very few target molecules, including known mutations, in 1,000- to 10,000-fold excess over wild-type DNA. By diluting input DNA into 20,000 or more droplets or beads, distributing less than one target molecule per droplet, the DNA can be amplified by PCR and then detected by probe hybridization or TaqMan™ reactions, essentially yielding a digital score of 0 / 1. This approach is currently the most sensitive for finding point mutations in plasma, but it requires prior knowledge of the mutations to be scored and separate digital dilutions for each mutation, which can result in the depletion of the entire sample to score a small number of mutations (Alcaide et al., “A Novel Multiplex Droplet Digital PCR Assay to Identify and Quantify KRAS Mutations in Clinical Specimens,” J. Mol. Diagn. 21:28-33 (2019); Guibert et al., “Liquid Biopsy of Fine-Needle Aspiration Supernatant for Lung Cancer Genotyping,” Lung Cancer 1768:193-207 (2018); Yoshida et al., “Highly Sensitive Detection of ALK Resistance Mutations in Plasma Using Droplet Digital PCR,” BMC Cancer 18:1136 (2018)).

[0025] When developing a multiplexed assay, there is a difficult balance between performing enough preliminary cycles of PCR or other amplification techniques to generate enough copies of each variant or methylated region (so that when diluted into uniplex qPCR, multiplex qPCR, uniplex droplet PCR, or multiplex droplet PCR, there are enough copies to get a signal for a true positive) and performing too many PCR cycles (so that some markers are over-amplified while others are suppressed or relative quantitation is lost).

[0026] The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0027] A first aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules are then provided. One or more primary oligonucleotide primer sets are also provided. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer that contains a nucleotide sequence complementary to a sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence, and (b) a second primary oligonucleotide primer that contains a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. A sample, one or more first oligonucleotide primers of a primary oligonucleotide primer set, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures, as well as conditions suitable for performing one or more polymerase extension reaction cycles including denaturation, hybridization, and extension treatments, thereby forming primary extension products comprising a nucleotide sequence complementary to the target nucleotide sequence.The primary extension product, one or more second primary oligonucleotide primers of the primary oligonucleotide primer set, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix containing dUTP, and one or more polymerase extension reaction mixtures containing a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures. The method further includes subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more first polymerase chain reaction products containing the target nucleotide sequence or its complement. One or more oligonucleotide probe sets are then provided. Each probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion, and (b) a second oligonucleotide probe having a 5' target sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to complementary target nucleotide sequences of the secondary extension products. One or more first polymerase chain reaction products are blended with a ligase and one or more oligonucleotide probe sets to form one or more ligation reaction mixtures. The one or more ligation reaction mixtures are subjected to one or more ligation reaction cycles, thereby ligating the first and second oligonucleotide probes of the one or more oligonucleotide probe sets together when hybridized to their complementary sequences to form ligation product sequences in the ligation reaction mixture, each ligation product sequence comprising a 5' primer-specific portion, a target-specific portion, and a 3' primer-specific portion. The method further includes providing one or more secondary oligonucleotide primer sets.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer containing the same nucleotide sequence as the 5' primer-specific portion of the ligation product sequence, and (b) a second secondary oligonucleotide primer containing a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequence. The ligation product sequence, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. One or more second polymerase chain reaction products are formed by subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension. The method further includes detecting and identifying one or more second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0028] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, one or more nucleases capable of digesting nucleic acid molecules that do not contain modified nucleotides, and one or more first primary oligonucleotide primer(s) are provided. The one or more first primary oligonucleotide primer(s) contain a nucleotide sequence complementary to the sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence. The method further comprises providing one or more secondary oligonucleotide primer sets, a sample, one or more first primary oligonucleotide primers, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing one or more modified nucleotides that protect the extension products but not the target DNA from nuclease digestion, and a DNA polymerase to form one or more polymerase extension reaction mixtures, and subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures, and conditions suitable for performing one or more polymerase extension reaction cycles including denaturation, hybridization, and extension, thereby forming primary extension products containing the complement of the target nucleotide sequence.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a first 5' primer-specific portion and a 3' portion that is complementary to a portion of the primary extension product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a second 5' primer-specific portion and a 3' portion that includes a nucleotide sequence that is complementary to a portion of the extension product formed from the first secondary oligonucleotide primer. One or more polymerase extension reaction mixtures comprising the primary extension products, one or more secondary oligonucleotide primer sets, one or more nucleases, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures, and the one or more first polymerase chain reaction mixtures are subjected to suitable conditions for digesting nucleic acid molecules present in the first polymerase chain reaction mixture but not digesting the primary extension products comprising modified nucleotides, and suitable conditions for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension treatments to form one or more first polymerase chain reaction products comprising a first 5' primer-specific portion, a target-specific nucleotide sequence or its complement, and a complement of the second 5' primer-specific portion. One or more tertiary oligonucleotide primer sets are provided. Each tertiary oligonucleotide primer set includes (a) a first tertiary oligonucleotide primer that includes a nucleotide sequence identical to a first 5' primer-specific portion of one or more first polymerase chain reaction products, and (b) a second tertiary oligonucleotide primer that includes a nucleotide sequence complementary to a 3' primer-specific portion of one or more first polymerase chain reaction products.One or more second polymerase chain reaction mixtures are formed by blending one or more first polymerase chain reaction products, one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase, and subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixtures and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension treatments, thereby forming one or more second polymerase chain reaction products. The method further involves detecting and identifying one or more second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0029] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules and one or more nucleases capable of digesting existing nucleic acid molecules that do not contain modified nucleotides are provided. The method also involves providing one or more primary oligonucleotide primer sets. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer that contains a nucleotide sequence complementary to the sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence, and (b) a second primary oligonucleotide primer that contains a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. A sample, one or more first primary oligonucleotide primers of a primary oligonucleotide primer set, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including one or more modified nucleotides that protect the extension products from nuclease digestion but do not protect the target DNA, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixture, and conditions suitable for performing one or more polymerase extension reaction cycles including denaturation, hybridization, and extension treatments, thereby forming primary extension products that contain the complement of the target nucleotide sequence.The method further includes blending one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of one or more primary oligonucleotide primer sets, one or more nucleases, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures. The one or more first polymerase chain reaction mixtures are subjected to suitable conditions that digest nucleic acid molecules present in the polymerase chain reaction mixture but not the primary extension products containing modified nucleotides, and to suitable conditions for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products containing the target nucleotide sequence or its complement. One or more secondary oligonucleotide primer sets are then provided. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a 3' portion complementary to a portion of the extension product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 3' portion comprising a nucleotide sequence complementary to a portion of the extension product formed from the first secondary oligonucleotide primer. The first polymerase chain reaction product, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. The one or more second polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and conditions suitable for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming the second polymerase chain reaction product.The method further includes detecting and identifying second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0030] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more methylated residues, and subjecting the nucleic acid molecules in the sample to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules are then provided. The method further involves providing one or more primary oligonucleotide primer sets. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer containing a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence containing one or more methylated residues, and (b) a second primary oligonucleotide primer containing a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. The bisulfite-treated sample, one or more first primary oligonucleotide primers of one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for one or more polymerase extension reaction cycles including denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence. The one or more polymerase extension reaction mixtures including the primary extension products, one or more second primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures.One or more first polymerase chain reaction mixtures are subjected to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming a first polymerase chain reaction product containing a bisulfite-treated target nucleotide sequence or its complement. The method further includes providing one or more oligonucleotide probe sets. Each probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion, and (b) a second oligonucleotide probe having a 5' bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to the complementary nucleotide sequence of the first polymerase chain reaction product. The first polymerase chain reaction product is blended with a ligase and one or more oligonucleotide probe sets to form one or more ligation reaction mixtures. The one or more ligation reaction mixtures are subjected to one or more ligation reaction cycles, thereby ligating the first and second oligonucleotide probes of the one or more oligonucleotide probe sets together when hybridized to complementary sequences to form ligation product sequences in the ligation reaction mixture, each ligation product sequence comprising a 5' primer-specific portion, a bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion, and a 3' primer-specific portion. The method further comprises providing one or more secondary oligonucleotide primer sets.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer comprising the same nucleotide sequence as the 5' primer-specific portion of the ligation product sequence, and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequence. The second polymerase chain reaction products are formed by blending the ligation product sequence, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures, and subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension. The method further involves detecting and identifying second polymerase chain reaction products in one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues.

[0031] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more methylated residues. The nucleic acid molecules in the sample are subjected to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules are provided, and one or more first primary oligonucleotide primer(s) are provided. Each first primary oligonucleotide primer contains a nucleotide sequence complementary to the sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence containing one or more methylated residues. The bisulfite-treated sample, one or more first primary oligonucleotide primers, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for one or more polymerase extension reaction cycles, including denaturation, hybridization, and extension, to form primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence. The method further includes providing one or more secondary oligonucleotide primer sets, each of which includes (a) a first secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion complementary to a portion of the polymerase extension reaction product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion comprising a nucleotide sequence complementary to a portion of the extension product formed from the first secondary oligonucleotide primer.The method further comprises providing one or more tertiary oligonucleotide primer sets, one or more polymerase extension reaction mixtures comprising the primary extension products, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures, and subjecting the one or more first polymerase chain reaction mixtures to suitable conditions for digesting the nucleic acid molecules present in the first polymerase chain reaction mixture but not the primary extension products containing modified nucleotides, and suitable conditions for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products comprising the 5' primer-specific portion, the bisulfite-treated target nucleotide sequence-specific portion or complementary sequence-specific portion of the first secondary oligonucleotide primer, and the complement of the 5' primer-specific portion of the second secondary oligonucleotide primer. Each tertiary oligonucleotide primer set includes (a) a first tertiary oligonucleotide primer containing the same nucleotide sequence as the 5' primer-specific portion of the first polymerase chain reaction product, and (b) a second tertiary oligonucleotide primer containing a nucleotide sequence complementary to the 3' primer-specific portion of the first polymerase chain reaction product sequence. The first polymerase chain reaction product, one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. Secondary polymerase chain reaction products are formed by subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension.The method further involves detecting and identifying secondary polymerase chain reaction products in one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues.

[0032] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more methylated residues. The nucleic acid molecules in the sample are subjected to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample and one or more primary oligonucleotide primer sets are provided. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer containing a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence containing one or more methylated residues, and (b) a second primary oligonucleotide primer containing a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. The bisulfite-treated sample, one or more first primary oligonucleotide primers of one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures. The one or more polymerase extension reaction mixtures are subjected to conditions suitable for one or more polymerase extension reaction cycles including denaturation, hybridization, and extension, thereby forming primary extension products containing the complements of the bisulfite-treated target nucleotide sequences. The one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures.One or more first polymerase chain reaction mixtures are subjected to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming a first polymerase chain reaction product containing a bisulfite-treated target nucleotide sequence or its complement. The method further includes providing one or more secondary oligonucleotide primer sets. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a 3' portion complementary to a portion of the first polymerase chain reaction product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 3' portion comprising a nucleotide sequence complementary to a portion of the first polymerase chain reaction product formed from the first secondary oligonucleotide primer. The first polymerase chain reaction product, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. The one or more second polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and conditions suitable for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products. The method further involves detecting and identifying the second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues.

[0033] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more methylated residues, and subjecting the nucleic acid molecules in the sample to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample are provided. One or more primary oligonucleotide primer sets are also provided. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer having a 5' primer-specific portion and a 3' portion comprising a nucleotide sequence complementary to the sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence containing one or more methylated residues, and (b) a second primary oligonucleotide primer having a 5' primer-specific portion and a 3' portion comprising a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. The bisulfite-treated sample, one or more first primary oligonucleotide primers of one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures. The one or more polymerase extension reaction mixtures are subjected to conditions suitable for one or more polymerase extension reaction cycles including denaturation, hybridization, and extension, thereby forming primary extension products containing the complements of the bisulfite-treated target nucleotide sequences. The one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures.The method further includes forming a first polymerase chain reaction product containing a bisulfite-treated target nucleotide sequence or its complement by subjecting one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension. One or more secondary oligonucleotide primer sets are provided. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer containing the same nucleotide sequence as the 5' primer-specific portion of the first polymerase chain reaction product or its complement, and (b) a second secondary oligonucleotide primer containing a nucleotide sequence complementary to the 3' primer-specific portion of the first polymerase chain reaction product or its complement. The primary polymerase chain reaction product sequence, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. The one or more second polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, as well as conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products. The method further involves detecting and identifying the second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues.

[0034] Another aspect of the present application is directed to a method for identifying one or more parent ribonucleic acid molecules in a sample that contain a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in the sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertions, exon deletions, intron insertions, translocations, mutations, or other rearrangements at the genomic level. The method involves providing a sample containing one or more parent ribonucleic acid molecules that contain a target ribonucleic acid molecule potentially different in sequence from the other parent ribonucleic acid molecules, and providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample. The sample is contacted with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. One or more primary oligonucleotide primer sets are then provided. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer containing a nucleotide sequence complementary to the RNA sequence of a parent ribonucleic acid molecule adjacent to the target ribonucleotide sequence, and (b) a second primary oligonucleotide primer containing a nucleotide sequence complementary to a portion of a cDNA extension product formed from the first primary oligonucleotide primer. A contacted sample, one or more primary oligonucleotide primer sets, a deoxynucleotide mix containing dUTP, a reverse transcriptase, and a DNA polymerase or a DNA polymerase having reverse transcriptase activity are blended to form one or more reverse transcription / polymerase chain reaction mixtures. One or more different reverse transcription / polymerase chain reaction mixtures are subjected to conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target ribonucleic acid, as well as conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension. The method further includes providing one or more oligonucleotide probe sets.Each probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion, and (b) a second oligonucleotide probe having a 5' target sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to complementary portions of a reverse transcriptase / polymerase product corresponding to a target ribonucleic acid molecule sequence. The reverse transcriptase / polymerase product is contacted with a ligase and one or more oligonucleotide probe sets to form one or more ligation reaction mixtures, and the one or more ligation reaction mixtures are subjected to one or more ligation reaction cycles, thereby ligating the first and second probes of the one or more oligonucleotide probe sets together when hybridized to their complements to form ligation product sequences in the ligase reaction mixture, each ligation product sequence comprising a 5' primer-specific portion, a target-specific portion, and a 3' primer-specific portion. The method further includes providing one or more secondary oligonucleotide primer sets. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer comprising the same nucleotide sequence as the 5' primer-specific portion of the ligation product sequence, and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequence. The ligation product sequence, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules along with the one or more secondary oligonucleotide primer sets, a deoxynucleotide mix including dUTP, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures, and the one or more first polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products.The method further includes identifying the presence of one or more parent ribonucleic acid molecules that comprise a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in the sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertions, exon deletions, intron insertions, translocations, mutations, or other rearrangements at the genomic level, by detecting and identifying the first polymerase chain reaction product.

[0035] Another aspect of the present application is directed to a method for identifying one or more parent ribonucleic acid molecules in a sample that contain a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in the sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertion, exon deletion, intron insertion, translocation, mutation, or other rearrangements at the genomic level. The method involves providing a sample containing one or more parent ribonucleic acid molecules that contain a target ribonucleic acid molecule potentially different in sequence from the other parent ribonucleic acid molecules, and providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample. The sample is contacted with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The method further involves providing one or more primary oligonucleotide primer sets, each of which includes (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to an RNA sequence of a parent ribonucleic acid molecule adjacent to the target nucleotide sequence, and (b) a second primary oligonucleotide primer comprising a nucleotide sequence complementary to a portion of a cDNA extension product formed from the first primary oligonucleotide primer. The method further includes blending a contacted sample, one or more primary oligonucleotide primer sets, a deoxynucleotide mix, a reverse transcriptase, and a DNA polymerase or a DNA polymerase having reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures, and subjecting the one or more reverse transcription / polymerase chain reaction mixtures to conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target RNA, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct reverse transcription / primary polymerase chain reaction products. The method further includes providing one or more secondary oligonucleotide primer sets.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a 3' portion complementary to a portion of the reverse transcription / primary polymerase chain reaction product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 3' portion comprising a nucleotide sequence complementary to a portion of the reverse transcription / primary polymerase chain reaction product formed from the first secondary oligonucleotide primer. The first polymerase chain reaction product is formed by blending the reverse transcription / primary polymerase chain reaction product, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures, and subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and conditions suitable for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension. The method further includes identifying the presence of one or more parent ribonucleic acid molecules that comprise a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in the sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertions, exon deletions, intron insertions, translocations, mutations, or other rearrangements at the genomic level, by detecting and identifying the first polymerase chain reaction product.

[0036] Another aspect of the present application is directed to a method for identifying one or more target miRNAs in a sample whose sequences differ by one or more bases from other miRNA molecules in the sample. The method includes providing a sample containing one or more target miRNA molecules whose sequences potentially differ by one or more bases from other miRNA molecules in the sample, and providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample. The sample is contacted with the one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample, and the contacted sample is blended with a ligase and one or more first oligonucleotide pre-probes comprising a 5' phosphate, a 5' stem-loop portion, an internal primer-specific portion within the loop region, a blocking group, and a 3' nucleotide sequence complementary to the 3' portion of the target miRNA molecule sequence to form one or more first ligation reaction mixtures. The method further includes ligating one or more target miRNA molecules at their 3' ends to the 5' phosphate of one or more first oligonucleotide preparatory probes in one or more first ligation reaction mixtures to generate chimeric nucleic acid molecules containing the target miRNA molecule sequence, which, if present in the sample, is attached to one or more first oligonucleotide preparatory probes. One or more primary oligonucleotide primer sets are then provided. Each primer set includes (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to an internal primer-specific portion of the first oligonucleotide preparatory probe, and (b) a second primary oligonucleotide primer comprising a 5' primer-specific portion and a 3' portion, where the second primary oligonucleotide primer can be the same as or different from other second primary oligonucleotide primers in other sets.One or more first ligation reaction mixtures containing the chimeric nucleic acid molecule, one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in a sample, a deoxynucleotide mix containing dUTP, and a reverse transcriptase and a DNA polymerase or a DNA polymerase with reverse transcriptase activity are blended to form one or more reverse transcription / polymerase chain reaction mixtures. The one or more reverse transcription / polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the reverse transcription / polymerase chain reaction mixture, conditions suitable for generating a deoxyribonucleic acid (cDNA) molecule complementary to the chimeric nucleic acid molecule, and conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct primary reverse transcription / polymerase chain reaction products containing a 5' primer-specific portion, a nucleotide sequence corresponding to the target miRNA molecule sequence, and a complement of the internal primer-specific portion and its complement. The method further includes providing one or more oligonucleotide probe sets. Each probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion, and (b) a second oligonucleotide probe having a 5' target sequence-specific portion, a portion complementary to the primary extension product, and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner on complementary portions of the primary reverse transcription / polymerase chain reaction product corresponding to the target miRNA molecule sequence or its complement.The primary reverse transcription / polymerase chain reaction product is contacted with a ligase and one or more oligonucleotide probe sets to form one or more second ligation reaction mixtures, and the one or more second ligation reaction mixtures are subjected to one or more ligation reaction cycles, thereby ligating the first and second oligonucleotide probes of the one or more oligonucleotide probe sets together when hybridized to their complements to form ligation product sequences in the ligation reaction mixture, each ligation product sequence comprising a 5' primer-specific portion, a target-specific portion, and a 3' primer-specific portion. The method further comprises providing one or more secondary oligonucleotide primer sets. Each secondary oligonucleotide primer set comprises (a) a first secondary oligonucleotide primer comprising the same nucleotide sequence as the 5' primer-specific portion of the ligation product sequence, and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequence. The ligation product sequence and one or more secondary oligonucleotide primer sets are blended with one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures. Secondary polymerase chain reaction products are formed by subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension. The method further includes detecting and identifying the secondary polymerase chain reaction products in the one or more reactions to identify one or more target miRNA molecules whose sequence differs by one or more bases from other miRNA molecules in the sample.

[0037] Another aspect of the present application is directed to a method for identifying one or more target miRNAs in a sample whose sequences differ by one or more bases from other miRNA molecules in the sample. The method includes providing a sample containing one or more target miRNA molecules whose sequences potentially differ by one or more bases from other miRNA molecules in the sample, and providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample. The sample is contacted with the one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample, and the contacted sample is blended with a ligase and one or more first oligonucleotide probes comprising a 5' phosphate, a 5' stem-loop portion, an internal primer-specific portion within the loop region, a blocking group, and a 3' nucleotide sequence complementary to the 3' portion of the target miRNA molecule sequence to form one or more first ligation reaction mixtures. The method further involves ligating one or more target miRNA molecules at their 3' ends to the 5' phosphates of one or more first oligonucleotide probes in one or more ligation reaction mixtures to generate chimeric nucleic acid molecules containing the target miRNA molecule sequence, which, if present in the sample, are attached to one or more first oligonucleotide probes. Then, one or more primary oligonucleotide primer sets are provided. Each primer set includes (a) a first primary oligonucleotide primer containing a nucleotide sequence complementary to an internal primer-specific portion of the first oligonucleotide probe, and (b) a second primary oligonucleotide primer containing a 5' primer-specific portion and a 3' portion, where the second primary oligonucleotide primer may be the same as or different from other second primary oligonucleotide primers in other sets. The one or more ligation reaction mixtures containing the chimeric nucleic acid molecule, one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a reverse transcriptase and a DNA polymerase or a DNA polymerase having reverse transcriptase activity are blended to form one or more reverse transcription / polymerase chain reaction mixtures.One or more reverse transcription / polymerase chain reaction mixtures are subjected to conditions suitable for generating a deoxyribonucleic acid (cDNA) molecule complementary to the chimeric nucleic acid molecule, and to conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct primary reverse transcription / polymerase chain reaction products comprising a 5' primer-specific portion, a nucleotide sequence corresponding to the target miRNA molecule sequence, and a complement of the internal primer-specific portion and its complement. The method further includes providing one or more secondary oligonucleotide primer sets. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion complementary to a portion of the extension product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion comprising a nucleotide sequence complementary to a portion of the extension product formed from the first secondary oligonucleotide primer. The primary reverse transcription / polymerase chain reaction product, one or more secondary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures, and the one or more first polymerase chain reaction mixtures are subjected to conditions suitable for two or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, to form a first polymerase chain reaction product comprising a 5' primer-specific portion of a first secondary oligonucleotide primer, a nucleotide sequence corresponding to the target miRNA molecule sequence or its complement, and the complement of another 5' primer-specific portion of a second secondary oligonucleotide primer. The method further includes providing one or more tertiary oligonucleotide primer sets.Each tertiary oligonucleotide primer set includes (a) a first tertiary oligonucleotide primer comprising the same nucleotide sequence as the 5' primer-specific portion of a first polymerase chain reaction product or its complement, and (b) a second tertiary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer-specific portion of the first polymerase chain reaction product or its complement. The second polymerase chain reaction products are formed by blending the first polymerase chain reaction product, one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures, and subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension. The method further includes identifying one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases by detecting and identifying the second polymerase chain reaction product.

[0038] Another aspect of the present application is directed to a method for identifying one or more target miRNAs in a sample whose sequences differ by one or more bases from other miRNA molecules in the sample. The method includes providing a sample containing one or more target miRNA molecules whose sequences potentially differ by one or more bases from other miRNA molecules in the sample, and providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample. The sample is contacted with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The contacted sample is blended with ATP and Poly(A) polymerase to form a Poly(A) polymerase reaction mixture, and the Poly(A) polymerase reaction mixture is subjected to conditions suitable for adding a homopolymer A to the 3' end of one or more target miRNA molecules potentially present in the sample. The method further involves providing one or more primary oligonucleotide primer sets. Each primer set includes (a) a first primary oligonucleotide primer, the first primary oligonucleotide primer including a 5' primer-specific portion, an internal poly(dT) portion, and a 3' portion including 1 to 10 bases complementary to the 3' end of the target miRNA, and may be the same as or different from other first primary oligonucleotide primers in the other set, and (b) a second primary oligonucleotide primer, the first primary oligonucleotide primer including a 5' primer-specific portion and a 3' portion, and may be the same as or different from other second primary oligonucleotide primers in the other set. Poly(A) polymerase reaction mixtures, one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in a sample, a deoxynucleotide mix including dUTP, and a reverse transcriptase and a DNA polymerase or a DNA polymerase having reverse transcriptase activity, are blended to form one or more reverse transcription / polymerase chain reaction mixtures.One or more reverse transcription / polymerase chain reaction mixtures are subjected to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the reverse transcription / polymerase chain reaction mixture, followed by conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target miRNA sequence having a 3' polyA tail, and conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct reverse transcription / polymerase chain reaction products comprising the 5' primer-specific portion of the second primary oligonucleotide primer, a nucleotide sequence corresponding to the target miRNA molecule sequence, a poly dA region, and the complement of the 5' primer-specific portion of the first primary oligonucleotide primer, and its complement. The method further includes providing one or more oligonucleotide probe sets. Each probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion, and (b) a second oligonucleotide probe having a 5' target sequence-specific portion, a portion complementary to one or more reverse transcription / polymerase chain reaction products, and a 3' primer-specific portion, where the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to complementary portions of one or more reverse transcription / polymerase chain reaction products corresponding to a target miRNA molecule sequence or its complement. The one or more reverse transcription / polymerase chain reaction products are contacted with a ligase and one or more oligonucleotide probe sets to form one or more ligation reaction mixtures, and the one or more ligation reaction mixtures are subjected to one or more ligation reaction cycles, whereby the first and second oligonucleotide probes of the one or more oligonucleotide probe sets are ligated together when hybridized to their complements to form ligation product sequences in the ligation reaction mixture, where each ligation product sequence includes a 5' primer-specific portion, a target-specific portion, and a 3' primer-specific portion. The method further includes providing one or more secondary oligonucleotide primer sets.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer containing the same nucleotide sequence as the 5' primer-specific portion of the ligation product sequence, and (b) a second secondary oligonucleotide primer containing a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequence. The ligation product sequence and one or more secondary oligonucleotide primer sets are blended with one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures, and secondary polymerase chain reaction products are formed by subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension. The method further includes detecting and identifying the secondary polymerase chain reaction products to identify one or more target miRNA molecules whose sequence differs by one or more bases from other miRNA molecules in the sample.

[0039] Another aspect of the present application is directed to a method for identifying one or more target miRNAs in a sample whose sequences differ by one or more bases from other miRNA molecules in the sample. The method includes providing a sample containing one or more target miRNA molecules whose sequences potentially differ by one or more bases from other miRNA molecules in the sample, and providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample. The sample is contacted with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The contacted sample is blended with ATP and Poly(A) polymerase to form a Poly(A) polymerase reaction mixture, and the Poly(A) polymerase reaction mixture is subjected to conditions suitable for adding a homopolymer A to the 3' end of one or more target miRNA molecules potentially present in the sample. The method further involves providing one or more primary oligonucleotide primer sets. Each primer set includes (a) a first primary oligonucleotide primer, the first primary oligonucleotide primer comprising a 5' primer-specific portion, an internal poly(dT) portion, and a 3' portion comprising 1 to 10 bases complementary to the 3' end of the target miRNA, which may be the same as or different from other first primary oligonucleotide primers in the other set, and (b) a second primary oligonucleotide primer, the second primary oligonucleotide primer comprising a 5' primer-specific portion and a 3' portion, which may be the same as or different from other second primary oligonucleotide primers in the other set. A poly(A) polymerase reaction mixture potentially containing the target miRNA sequence is blended with a 3' poly(A) tail, one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a reverse transcriptase and a DNA polymerase or a DNA polymerase with reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures.One or more reverse transcription / polymerase chain reaction mixtures are subjected to conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target miRNA sequence with a 3' poly(A) tail, and conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct reverse transcription / polymerase chain reaction products comprising a 5' primer-specific portion of a second primary oligonucleotide primer, a nucleotide sequence corresponding to the target miRNA molecule sequence, a poly(dA) region, and the complement of the 5' primer-specific portion of the first primary oligonucleotide primer and its complement. The method further includes providing one or more secondary oligonucleotide primer sets. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion complementary to a portion of the reverse transcription / polymerase chain reaction product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion comprising a nucleotide sequence complementary to a portion of the reverse transcription / polymerase chain reaction product formed from the first secondary oligonucleotide primer. The reverse transcription / polymerase chain reaction product, one or more secondary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures, and the one or more first polymerase chain reaction mixtures are subjected to conditions suitable for two or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, to form first polymerase chain reaction products comprising a 5' primer-specific portion, a nucleotide sequence corresponding to the target miRNA molecule sequence or its complement, and a complement of another 5' primer-specific portion. The method further includes providing one or more tertiary oligonucleotide primer sets.Each tertiary oligonucleotide primer set includes (a) a first tertiary oligonucleotide primer containing the same nucleotide sequence as the 5' primer-specific portion of the first polymerase chain reaction product sequence, and (b) a second tertiary oligonucleotide primer containing a nucleotide sequence complementary to the 3' primer-specific portion of the first polymerase chain reaction product sequence. The first polymerase chain reaction product, one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. The one or more second polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, as well as conditions suitable for one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, to form the second polymerase chain reaction products. The method further includes identifying one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases by detecting and identifying a second polymerase chain reaction product in one or more reactions.

[0040] Another aspect of the present application is directed to a method of diagnosing or predicting a disease state of a cell or tissue based on identifying the presence or level of a plurality of disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in a biological sample from an individual. The plurality of markers is in a set of 6-12 markers, 12-24 markers, 24-36 markers, 36-48 markers, 48-72 markers, 72-96 markers, or more than 96 markers. Each marker in a given set is selected by having any one or more of the following criteria: being present in more than 50% of diseased cell or tissue biological samples from individuals diagnosed with the disease state, or above a cutoff level; being absent in more than 95% of normal cell or tissue biological samples from individuals not having the disease state, or being present in cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or samples from individuals diagnosed with the disease state. being present in more than 50% of biological samples containing those fractions or above the cutoff level; being absent in more than 95% of biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from individuals without the disease state; being present at a z-score of greater than 1.65 in biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from individuals diagnosed with the disease state. In biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from at least 50% of individuals diagnosed with the disease state, the markers of the set each contain one or more methylated residues, and / or at least 50% of the markers of the set that are present at or above the cutoff level or at a z-score of greater than 1.65 contain one or more methylated residues. The method involves obtaining a biological sample, which includes cells or tissues and cell-free DNA, RNA, and / or proteins derived from one or more other tissues or cells, and the biological sample is selected from the group consisting of cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodily fluids, bodily secretions, and bodily excretions, or fractions thereof.The sample is fractionated into one or more fractions, with at least one fraction containing exosomes, tumor-associated vesicles, other protected forms, or cell-free DNA, RNA, and / or protein. The nucleic acid molecules in one or more fractions are subjected to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. At least two enrichment steps are performed on the fractions and / or by performing nucleic acid amplification steps on 50% or more of the disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers. The method further involves performing one or more assays to detect and identify multiple disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers, thereby identifying their presence or levels in the sample. Here, a marker set consisting of 6 to 12 markers has at least 2 or 3 markers present or above the cutoff level, or a marker set consisting of 12 to 24 markers has at least 3, 4, or 5 markers present or above the cutoff level, or a marker set consisting of 24 to 36 markers has at least 3, 4, 5, or 6 markers present or above the cutoff level, or a marker set consisting of 36 to 48 markers has at least 4, 5, 6, 7, or 8 markers present or above the cutoff level. or if at least 6, 7, 8, 9, 10, 11, or 12 markers are present or above the cutoff level in a marker set of 48 to 72 markers, or if at least 7, 8, 9, 10, 11, 12, or 13 markers are present or above the cutoff level in a marker set of 72 to 96 markers, or if at least 8, 9, 10, 11, 12, 13, or "n" / 12 markers are present or above the cutoff level in a marker set of 96 to "n" markers (where "n" > 168).

[0041] Another aspect of the present application is directed to a method for diagnosing or predicting the disease status of solid tissue cancers, including colorectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, breast lobular and ductal carcinoma, uterine endometrial cancer, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine carcinosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, based on identifying the presence or level of multiple disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in an individual's biological sample. The multiple markers are in a set consisting of 48 to 72 total cancer markers, 72 to 96 total cancer markers, or 96 or more total cancer markers, with an average of more than one-quarter of such markers in a given set covering each of the aforementioned major cancers tested. Each marker in a given set for a given solid tissue cancer is selected by having any one or more of the following criteria for that solid tissue cancer: being present in more than 50% of biological samples of the given cancer tissue from individuals diagnosed with the given solid tissue cancer, or above a cutoff level; being absent in more than 95% of biological samples of normal tissue from individuals without the given solid tissue cancer, or below a cutoff level in cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, or the like from individuals diagnosed with the given solid tissue cancer. being present in more than 50% of biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, or being absent in more than 95% of biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from individuals without a given solid tissue cancer; being present at a z-score of greater than 1.65 in biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from individuals diagnosed with a given solid tissue cancer.In a biological sample containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from at least 50% of individuals diagnosed with a given solid tissue cancer, at least 50% of the markers in the set each contain one or more methylated residues, and / or at least 50% of the markers in the set, or present at a z-score above the cutoff level or greater than 1.65, contain one or more methylated residues. The method involves obtaining a biological sample containing cells or tissues and cell-free DNA, RNA, and / or protein derived from one or more other tissues or cells. The biological sample is selected from the group consisting of cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, and body excretions, or fractions thereof. The sample is fractionated into one or more fractions, and at least one fraction contains exosomes, tumor-associated vesicles, other protected forms, or cell-free DNA, RNA, and / or protein. The nucleic acid molecules in one or more fractions are subjected to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. At least two enrichment steps are performed on the fractions and / or by performing nucleic acid amplification steps for 50% or more of a given solid tissue cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein marker. The method further involves performing one or more assays to detect and identify multiple cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers, thereby identifying their presence or levels in the sample. Here, an individual is diagnosed or predicted to have solid tissue cancer if at least 4 markers are present or above a cutoff level in a marker set consisting of 48 to 72 total cancer markers, or if at least 5 markers are present or above a cutoff level in a marker set consisting of 72 to 96 total cancer markers, or if at least 6 or "n" / 18 markers are present or above a cutoff level in a marker set consisting of 96 to "n" total cancer markers (where "n" > 96 total cancer markers).

[0042] Another aspect of the present application is directed to a method of diagnosing or predicting solid tissue cancer disease status and identifying the particular tissue(s) most likely to be a solid tissue cancer in the following groups: Group 1 (colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma), Group 2 (lobular and ductal breast carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, squamous cell carcinoma and adenocarcinoma of the cervix, uterine carcinosarcoma), Group 3 (lung adenocarcinoma, squamous cell lung carcinoma, squamous cell carcinoma of the head and neck), Group 4 (prostate adenocarcinoma, invasive bladder urothelial carcinoma), and / or Group 5 (hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma). This is based on identifying the presence or level of multiple disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in an individual's biological sample, where the multiple markers are in a set consisting of 36-48 group-specific cancer markers, 48-64 group-specific cancer markers, or 64 or more group-specific cancer markers, with an average of more than one-third of such markers in a given set covering each of the aforementioned cancers tested within that group. Each marker in a given set for a given solid tissue cancer is selected by having any one or more of the following criteria for that solid tissue cancer: being present in more than 50% of the given cancer tissue biological samples from individuals diagnosed with the given solid tissue cancer, or above a cutoff level; being absent in more than 95% of normal tissue biological samples from individuals without the given solid tissue cancer, or being present in cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, or other biological samples from individuals diagnosed with the given solid tissue cancer. being present in more than 50% of biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, or being absent in more than 95% of biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from individuals without a given solid tissue cancer; being present at a z-score of greater than 1.65 in biological samples containing cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from individuals diagnosed with a given solid tissue cancer.In a biological sample comprising cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from at least 50% of individuals diagnosed with a given solid tissue cancer, at least 50% of the markers in the set each comprise one or more methylated residues, and / or are present, or are present at a z-score above the cutoff level or greater than 1.65. The method involves obtaining a biological sample. The biological sample comprises cells or tissues, and cell-free DNA, RNA, and / or proteins derived from one or more other tissues or cells. The biological sample is selected from the group consisting of cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, and body excretions, or fractions thereof. The sample is fractionated into one or more fractions, and at least one fraction comprises exosomes, tumor-associated vesicles, other protected forms, or cell-free DNA, RNA, and / or proteins. The nucleic acid molecules in one or more fractions are subjected to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. At least two enrichment steps are performed on the fractions and / or by performing nucleic acid amplification steps for 50% or more of a given solid tissue cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein marker. The method further involves performing one or more assays to detect and identify multiple cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers, thereby identifying their presence or levels in the sample. wherein an individual is diagnosed or predicted to have solid tissue cancer if at least four markers are present or above a cutoff level in a marker set of 36 to 48 group-specific cancer markers, or if at least five markers are present or above a cutoff level in a marker set of 48 to 64 group-specific cancer markers, or if 6 or "n" / 12 markers are present or above a cutoff level in a marker set of 64 to "n" total cancer markers (where "n" > 64 group-specific cancer markers).

[0043] This application describes several approaches for detecting mutations, expression, splice variants, translocations, copy number, and / or methylation changes in target nucleic acid molecules using nuclease, ligase, and polymerase reactions. This application solves the problem of carryover prevention and enables spatial multiplexing to provide relative quantification, similar to digital PCR. Such technologies can be used for noninvasive early detection of cancer, noninvasive prognosis of cancer, and monitoring of cancer recurrence from plasma or serum samples.

[0044] This application provides a comprehensive roadmap of nucleic acid methylation, miRNA, lncRNA, ncRNA, mRNA, exon, and cancer-associated protein markers specific to solid tissue cancers and matched normal tissues. This application teaches techniques for selecting the desired number and type of markers for both pan-tumor and specific cancers (i.e., colorectal cancer) to guide physicians in improving patient care. Details of primer design and optimized primer sequences are provided to enable rapid validation of these tests for both pan-tumor and specific cancers. A two-step procedure is designed to broadly search and initially identify the majority of individuals with early-stage cancer, followed by a more stringent second step to improve specificity and narrow the patient population to those most likely to have occult cancer (patients are then referred for imaging and follow-up). The advantage of this two-step approach is that it is designed not only to identify potential tissues of origin but also to provide the highest positive predictive value (PPV). Therefore, if a rare cancer result comes back as a presumptive positive (i.e., early-stage ovarian cancer), physicians can focus their attention on providing imaging and follow-up to patients who need it most, and the test minimizes false positives that create unnecessary anxiety and unwanted invasive procedures.

[0045] This application provides a robust approach for detecting cancer markers (mutations, expression, splice variants, translocations, copy number, and / or methylation changes) using either qPCR or dPCR readouts, using protocols that are amenable to automation and work with readily available commercial equipment. This approach offers the advantages of being integrated and convenient for laboratory setup, allowing for cost savings, scalability, and compatibility with medical and laboratory workflows in a CLIA-compliant automated setting. The benefits in lives saved worldwide would be immeasurable. [Brief explanation of the drawings]

[0046] [Figure 1A] Figures A-B show a conditional logic tree for an early detection colon cancer test based on the analysis of a patient's blood sample. A one-step colon cancer assay is shown, using 24 markers with an average sensitivity of 50%. [Figure 1B] AB show a conditional logic tree for early detection colon cancer testing based on analysis of patient blood samples. [Figure 1C] (C-D) Conditional logic tree for a two-step assay for early detection pan-tumor cancer testing based on analysis of patient blood samples. A two-step pan-tumor assay is shown, where the first step uses 96 group-specific markers with an average sensitivity of 50%, followed by the second step using 64 type-specific markers for each of group 1 or group 2 with an average sensitivity of 50%. [Figure 1D] (C-D) Conditional logic tree for a two-step assay for early detection pan-tumor cancer testing based on analysis of patient blood samples. A two-step pan-tumor assay is shown, where the first step uses 96 group-specific markers with an average sensitivity of 66%, followed by the second step using 64 type-specific markers for each of group 1 or group 2 with an average sensitivity of 66%. [Figure 2] 1 shows an exPCR-LDR-qPCR carryover prevention reaction with Taqman™ detection to identify or relatively quantitate target(s) and / or low-level mutations. [Figure 3]Figure 1 shows an exPCR-LDR-qPCR carryover prevention reaction with UniTaq detection for specific or relative quantification of target(s) and / or low-level mutations. [Figure 4] 1 shows an exPCR-qPCR carryover prevention reaction with Taqman™ detection to identify or relatively quantitate target(s) and / or low-level mutations. [Figure 5] Figure 1 shows an exPCR-qPCR carryover prevention reaction with UniTaq detection for specific or relative quantification of target(s) and / or low-level mutations. [Figure 6] 1 shows a variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection to identify or relatively quantitate target(s) and / or low-level mutations. [Figure 7] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection to identify or relatively quantitate target(s) and / or low-level mutations. [Figure 8] 1 shows a variation of the exPCR-qPCR carryover prevention reaction with UniTaq detection to identify or relatively quantitate target(s) and / or low-level mutations. [Figure 9] Figure 1 shows an exPCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 10] 1 shows a variation of the exPCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 11] Figure 1 shows an exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 12]1 shows a variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 13] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 14] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 15] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 16] 1 shows another variation of the exPCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 17] 1 shows another variation of the exPCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 18] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 19] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 20] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 21] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 22] 1 shows another variation of the exPCR-qPCR carryover prevention reaction with Taqman™ detection for specific or relative quantification of low levels of methylation. [Figure 23] Figure 1 shows an RT-PCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating translocation events at the mRNA level. [Figure 24] Figure 1 shows an RT-PCR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating translocation events at the mRNA level. [Figure 25] Figure 1 shows an RT-PCR-PCR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating translocation events at the mRNA level. [Figure 26] Figure 1 shows an RT-PCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating translocation events at the mRNA level. [Figure 27] 1 shows an RT-PCR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating RNA copy number. [Figure 28] 1 shows an RT-PCR-PCR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating RNA copy number. [Figure 29] Figure 1 shows a ligation-RT-PCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for the detection and enumeration of miRNAs. [Figure 30] Figure 1 shows a ligation-RT-PCR-qPCR carryover prevention reaction with Taqman™ detection for the detection and enumeration of miRNAs. [Figure 31] RT-PCR-LDR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating miRNAs. [Figure 32]1 shows an RT-PCR-qPCR carryover prevention reaction with Taqman™ detection for detecting and enumerating miRNAs. [Figure 33] The overall sensitivity and specificity results calculated for the 24-marker assay are shown. The average sensitivity of individual markers is 50% (A), and the average false positive rate of individual markers is 2%-5% (B). [Figure 34] The overall sensitivity and specificity results calculated for the 36-marker assay are shown. The average sensitivity of individual markers is 50% (A), and the average false positive rate for individual markers is 2%-5% (B). [Figure 35] The overall sensitivity and specificity results calculated for the 48-marker assay are shown. The average sensitivity of individual markers is 50% (A), and the average false positive rate for individual markers is 2%-5% (B). [Figure 36] The overall sensitivity and specificity results calculated for the 96-marker assay are shown. The average sensitivity of individual markers is 50% (A), and the average false positive rate for individual markers is 2%-5% (B). [Figure 37] ROC curves for the 48-marker assay (where the average sensitivity of each individual marker is 50%) are shown, as well as AUC calculations (where the average number of molecules per marker in the blood ranges from 150 to 600 molecules). For A and B, the calculations are based on average false positive rates for each individual marker of 2% and 3%, respectively. [Figure 38] ROC curves for the 48-marker assay (with an individual mean marker sensitivity of 50%), as well as AUC calculations (when the mean number of molecules per marker in blood ranges from 150 to 600 molecules) are shown. For A and B, the calculations are based on an individual marker mean false positive rate of 4% and 5%, respectively. [Figure 39A] We provide a list of blood-based colon cancer-specific microRNA markers derived through analysis of the TCGA microRNA dataset, which may be present in exosomes or other protected states in the blood. [Figure 39B]We provide a list of blood-based colon cancer-specific microRNA markers derived through analysis of the TCGA microRNA dataset, which may be present in exosomes or other protected states in the blood. [Figure 40A] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40B] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40C] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40D] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40E] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40F] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40G] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40H] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40I] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40J]We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40K] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40L] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40M] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40N] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40O] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40P] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40Q] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40R] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40S] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40T]We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40U] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40V] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40W] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 40X] We provide a list of blood-based colon cancer-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 41A] We provide a candidate list of blood-based colon cancer-specific exon transcripts that can be enriched in exosomes or other protected conditions in the blood. [Figure 41B] We provide a candidate list of blood-based colon cancer-specific exon transcripts that can be enriched in exosomes or other protected conditions in the blood. [Figure 41C] We provide a candidate list of blood-based colon cancer-specific exon transcripts that can be enriched in exosomes or other protected conditions in the blood. [Figure 42A] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42B]We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42C] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42D] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42E] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42F] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42G]We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42H] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42I] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 42J] We provide a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colorectal tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 43] A list of protein markers that can be secreted into the blood by colorectal tumors is provided. [Figure 44A] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44B]We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44C] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44D] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44E] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44F] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44G] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44H] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44I]We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44J] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44K] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44L] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44M] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44N] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44O] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44P]We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44Q] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44R] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44S] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44T] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44U] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44V] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44W]We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44X] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 44Y] We provide a list of key CpG sites that are markers for colorectal cancer and colon tissue-specific markers that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 45A] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45B] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45C] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45D]We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45E] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45F] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45G] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45H] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45I] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45J]We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45K] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45L] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45M] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45N] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45O] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 45P]We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for colorectal cancer and colon tissue-specific markers, that can be used to identify the presence of colorectal cancer from cfDNA, or DNA in exosomes, or DNA in other protected states in the blood (e.g., in CTCs). [Figure 46] Overall sensitivity and specificity results calculated for the 24-marker assay are shown. The average sensitivity of individual markers was 50%, and the average false positive rate for individual markers ranged from 2% to 5%, including one marker with a 90% sensitivity (A) and a 10% false positive rate (B). [Figure 47] Overall sensitivity and specificity results calculated for the 24-marker assay are shown. The average sensitivity of each individual marker was 50%, and the average false positive rate for each individual marker ranged from 2% to 5%, including two markers with a sensitivity of 90% (A) and a false positive rate of 10% (B). [Figure 48] Overall sensitivity and specificity results calculated for the 48-marker assay are shown. The average sensitivity of individual markers was 50%, and the average false positive rate for individual markers ranged from 2% to 5%, including one marker with a 90% sensitivity (A) and a 10% false positive rate (B). [Figure 49] Overall sensitivity and specificity results calculated for the 48-marker assay are shown. The average sensitivity of each individual marker was 50%, and the average false positive rate for each individual marker ranged from 2% to 5%, including two markers with a sensitivity of 90% (A) and a false positive rate of 10% (B). [Figure 50] The overall sensitivity and specificity results calculated for the 24-marker assay are shown. The average sensitivity of individual markers is 66% (A), and the average false positive rate for individual markers is 2%-5% (B). [Figure 51] The overall sensitivity and specificity results calculated for the 36-marker assay are shown. The average sensitivity of individual markers was 66% (A), and the average false positive rate for individual markers was 2%-5% (B). [Figure 52]The overall sensitivity and specificity results calculated for the 48-marker assay are shown. The average sensitivity of individual markers was 66% (A), and the average false positive rate for individual markers was 2%-5% (B). [Figure 53] We provide a list of blood-based solid tumor-specific ncRNA and lncRNA markers that may be present in exosomes or other protected states in the blood. [Figure 54A] We provide a candidate list of blood-based solid tumor-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 54B] We provide a candidate list of blood-based solid tumor-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 54C] We provide a candidate list of blood-based solid tumor-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 54D] We provide a candidate list of blood-based solid tumor-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 54E] We provide a candidate list of blood-based solid tumor-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 54F] We provide a candidate list of blood-based solid tumor-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 55A] Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 55B]Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 55C] Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 55D] Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 55E] Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 55F] Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 55G]Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 55H] Provides a list of protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products, in cancers arising from solid tumors, which can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 56A] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56B] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56C] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56D] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56E]We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56F] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56G] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56H] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56I] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56J] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56K] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56L]We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56M] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56N] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56O] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56P] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56Q] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56R] We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 56S]We provide a list of key CpG sites that are solid tumor markers and tissue-specific markers that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57A] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57B] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57C] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57D] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57E] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57F]We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57G] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57H] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57I] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 57J] We provide a list of chromosomal regions or subregions, among which are key CpG sites that are markers for solid tumors and tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 58] Provided is a list of cancer protein markers, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, that can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 59A] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59B] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59C] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59D] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59E] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59F] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59G]We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59H] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59I] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59J] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59K] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59L] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59M]We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59N] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59O] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59P] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59Q] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59R] We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 59S]We provide a list of key CpG sites that are markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60A] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60B] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60C] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60D] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60E]Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60F] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60G] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60H] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60I] Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 60J]Provides a list of chromosomal regions or subregions, including markers for colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 61A] We provide a list of key CpG sites that are markers and tissue-specific markers for breast lobular and ductal carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, which can be used to identify the presence of solid tumor cancer from cfDNA, DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 61B] We provide a list of key CpG sites that are markers and tissue-specific markers for breast lobular and ductal carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, which can be used to identify the presence of solid tumor cancer from cfDNA, DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 61C] We provide a list of key CpG sites that are markers and tissue-specific markers for breast lobular and ductal carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, which can be used to identify the presence of solid tumor cancer from cfDNA, DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 62A] Provides a list of chromosomal regions or subregions, including key CpG sites that are markers and tissue-specific markers for breast lobular and ductal carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 62B]Provides a list of chromosomal regions or subregions, including key CpG sites that are markers and tissue-specific markers for breast lobular and ductal carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 63] We provide a list of key CpG sites that are markers and tissue-specific markers for lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, which can be used to identify the presence of solid tumor cancer from cfDNA, DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 64] We provide a list of chromosomal regions or subregions, including key CpG sites that are markers for lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 65] We provide a list of key CpG sites that are markers for prostate adenocarcinoma or invasive bladder urothelial carcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 66] We provide a list of chromosomal regions or subregions, including key CpG sites that are markers for prostate adenocarcinoma or invasive bladder urothelial carcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 67] We provide a list of blood-based hepatocellular carcinoma-, pancreatic ductal adenocarcinoma-, or gallbladder adenocarcinoma-specific ncRNA and lncRNA markers that may exist in exosomes or other protected states in the blood. [Figure 68A] We provide a candidate list of blood-based hepatocellular carcinoma-, pancreatic ductal adenocarcinoma-, or gallbladder adenocarcinoma-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 68B] We provide a candidate list of blood-based hepatocellular carcinoma-, pancreatic ductal adenocarcinoma-, or gallbladder adenocarcinoma-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 68C] We provide a candidate list of blood-based hepatocellular carcinoma-, pancreatic ductal adenocarcinoma-, or gallbladder adenocarcinoma-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 68D] We provide a candidate list of blood-based hepatocellular carcinoma-, pancreatic ductal adenocarcinoma-, or gallbladder adenocarcinoma-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 68E] We provide a candidate list of blood-based hepatocellular carcinoma-, pancreatic ductal adenocarcinoma-, or gallbladder adenocarcinoma-specific exon transcripts that can be enriched in exosomes or other protected states in the blood. [Figure 69A] A list of cancer protein markers is provided, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 69B] A list of cancer protein markers is provided, identified via mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products arising from hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and can be identified in the blood either in exosomes, other protected states, tumor-associated vesicles, or free in plasma. [Figure 70A]We provide a list of key CpG sites that are markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., within CTCs). [Figure 70B] We provide a list of key CpG sites that are markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., within CTCs). [Figure 70C] We provide a list of key CpG sites that are markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., within CTCs). [Figure 70D] We provide a list of key CpG sites that are markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., within CTCs). [Figure 70E] We provide a list of key CpG sites that are markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., within CTCs). [Figure 71A] We provide a list of chromosomal regions or subregions, including markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 71B]We provide a list of chromosomal regions or subregions, including markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 71C] We provide a list of chromosomal regions or subregions, including markers for hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, as well as key CpG sites that are tissue-specific markers, that can be used to identify the presence of solid tumor cancer from cfDNA, or DNA in exosomes, or DNA in another protected state in the blood (e.g., in CTCs). [Figure 72] Figure 1 shows real-time PCR amplification plots obtained in a pixelated bisulfite-PCR-LDR-qPCR experiment for enumeration of single molecules of methylated DNA in the presence of excess unmethylated DNA (Roche DNA). [Figure 73] Figure 1 shows real-time PCR amplification plots obtained for multiplex detection of 10 CRC methylation markers by bisulfite-PCR-LDR-qPCR using HT29 cell line DNA, with an average of 20 molecules of each marker per 10,000 molecules of normal (e.g., unmethylated) DNA (Roche DNA). [Figure 74] Figure 1 shows real-time PCR amplification plots obtained for multiplex detection of seven CRC methylation markers by bisulfite-exPCR-PCR-LDR-qPCR using HT29 cell line DNA, with an average of 30 molecules of each marker among 3,000 molecules of normal (e.g., unmethylated) DNA (Roche DNA). [Figure 75] Figure 1 shows real-time PCR amplification plots obtained for multiplex detection of seven CRC methylation markers by bisulfite-exPCR-LDR-qPCR using cfDNA isolated from CRC plasma (A) and normal plasma (B). [Figure 76]Figure 1 shows real-time PCR amplification plots obtained for multiplex detection of seven CRC methylation markers by bisulfite-exPCR-LDR-qPCR using cfDNA isolated from CRC plasma (A) and normal plasma (B). [Figure 77] Real-time PCR amplification plots obtained for multiplex detection of seven CRC methylation markers by bisulfite-exPCR-LDR-qPCR using cfDNA isolated from CRC plasma (A) and normal plasma (B). [Figure 78] Figure 1 shows real-time PCR amplification plots obtained for multiplex detection of 20 CRC methylation markers by bisulfite-exPCR-LDR-qPCR using HT29 cell line DNA, with 1,500 genome equivalents of HT29 cell line DNA in 7,500 genome equivalents of normal (e.g., unmethylated) DNA (Roche DNA, A) compared to 7,500 genome equivalents of normal (e.g., unmethylated) DNA (B). [Figure 79] Real-time PCR amplification plots for multiplex detection of 20 CRC methylation markers by bisulfite-exPCR-LDR-qPCR using tailed reverse primers were shown, with HT29 cell line DNA containing 200 genome equivalents of HT29 cell line DNA in 7,500 genome equivalents of normal (e.g., unmethylated) DNA (Roche DNA, A) compared to 7,500 genome equivalents of normal (e.g., unmethylated) DNA (B). [Figure 80] Figure 1 shows real-time PCR amplification plots obtained for multiplex detection of 20 CRC methylation markers by bisulfite-exPCR-LDR-qPCR using untailed reverse primers, using HT29 cell line DNA with 200 genome equivalents of HT29 cell line DNA in 7,500 genome equivalents of normal (e.g., unmethylated) DNA (Roche DNA, A) compared with 7,500 genome equivalents of normal (e.g., unmethylated) DNA (B). DETAILED DESCRIPTION OF THE INVENTION

[0047] Universal Design for Early Cancer Detection Using "Cancer Marker Load" The most cost-effective cancer early detection test could combine an initial multiplex amplification and ligation assay to determine the "cancer burden," which would achieve a sensitivity of >95% for all cancers (pan-tumor), with a specificity of >97% for detecting cancer.

[0048] Figure 1 shows several flowcharts of cancer tumor burden assays. In its simplest form, this assay is a one-step assay for identifying individuals with early-stage colorectal cancer (CRC). Blood samples are fractionated into plasma and other components as needed, assayed for a set of 24 markers with an average sensitivity of 50%, and the results are recorded (Figure 1A). For example, initial multiplex PCR / LDR screening assay scoring for mutations, methylation, miRNA, mRNA, alternative splicing, and / or translocations identifies those samples with positive results. Physicians provide simple instructions without considering which specific markers are positive. Patients with 0–2 positive markers are told, "Don't worry. You don't have cancer. Go home." Patients with 5 or more positive markers out of 24 are instructed to "have a colonoscopy." Patients with an intermediate number of positive markers (3–4) are instructed to "return for a re-examination in 3–6 months." Thus, this test is based on overall cancer marker burden and does not depend on the specific markers that test positive.

[0049] In advanced versions of the test, a two-step assay is used to identify whether a patient has colorectal cancer. The rationale for the two-step test is to first maximize sensitivity by broadly screening and identify most individuals with potential cancer. The second step maximizes specificity for only positive samples (including both true and false positives), effectively eliminating all false positives and focusing on individuals most likely to have cancer. In the first step, the blood sample is fractionated into plasma and other components as needed, followed by an assay that tests an initial set of 24 markers with an average sensitivity of 50% (Figure 1B). The first-step assay can be scored for mutations, methylation, miRNA, mRNA, alternative splicing, and / or translocation events using multiplex PCR / LDR or digital PCR screening. As with the one-step assay, patients with 0–2 positive markers are presumed to be cancer-free. On the other hand, patients with three or more positive markers undergo a second step in which 48 (new) markers are assayed and scored as follows: 0-3 positive markers are considered cancer-free, 4-5 positive markers are recommended to return in 3-6 months for retesting, and 6 or more positive markers are instructed to undergo a colonoscopy.

[0050] In the pan-tumor version of the test, in the first step, the assay screens 96 markers; on average, 36 or more such markers would demonstrate a mean sensitivity of 50% for most major cancers (see Figure 1C). These cancers are clustered into specific groups, including group 1 (colorectal cancer, gastric cancer, esophageal cancer), group 2 (breast cancer, endometrial cancer, ovarian cancer, cervical cancer, uterine cancer), group 3 (lung cancer, head and neck cancer), group 4 (prostate cancer, bladder cancer), and group 5 (liver cancer, pancreatic cancer, gallbladder cancer). Patients who test positive for 0 to 4 markers are presumed cancer-free, while patients who test positive for 5 or more markers undergo the second step. Presumed positive samples are then assayed in the second step, testing one or two groups using 64 markers per group. On average, 36 or more such markers would demonstrate an average sensitivity of 50% for each specific cancer type within that group, including using tissue-specific markers to validate the initial results and identify the tissue of origin. Results are scored as follows: 0–3 positive markers are considered cancer-free, 4 positive markers are recommended to return in 3–6 months for retesting, and 5 or more positive markers are recommended to undergo imaging consistent with the cancer type(s) most likely to be the tissue of origin. At higher sensitivity, both the initial 96 markers in the first step and the group-specific markers in the second step would have an average sensitivity of 66% (Figure 1D). The physician can then prescribe targeted sequencing to further guide the patient's treatment decisions.

[0051] The present application is directed to a universal diagnostic approach that seeks to combine the best features of digital polymerase chain reaction (PCR) or quantitative polymerase chain reaction (qPCR) with bisulfite conversion, ligation detection reaction (LDR), and quantitative detection of multiple disease markers (e.g., cancer markers).

[0052] Multiplexing, avoidance of false positives, and carryover protection There is a technical challenge in distinguishing true signals generated from desired disease-specific nucleic acid differences from false signals generated from normal nucleic acids present in the sample, and from false signals generated in the absence of disease-specific nucleic acid differences (i.e., somatic mutations).

[0053] Below are some solutions to these challenges, which share some common themes.

[0054] The first theme is multiplexing. PCR is most effective when primer concentrations are relatively high, between 50 nM and 500 nM, limiting multiplexing. Furthermore, the more PCR primer pairs added, the greater the chance of amplifying erroneous products or generating primer dimers. In contrast, for LDR probes, low concentrations of between 4 nM and 20 nM are used, and probe dimers are limited by the requirement for adjacent hybridization on the target to allow for ligation events. Using gene-specific PCR primers or LDR probes containing a low concentration of universal primer sequence "tail" allows subsequent addition of higher concentrations of universal primer to achieve proportional amplification of the initial PCR or LDR product. Another way to avoid or minimize incorrect PCR amplicons or primer dimers is to use PCR primers that contain a few extra bases and a blocking group, which are released to form a free 3'OH by cleavage with a nuclease only when hybridized to the target (e.g., ribonucleotide bases as blocking groups and RNase H2 as the cleaving nuclease).

[0055] The second theme is signal variability due to low input target nucleic acid. Often, the target nucleic acid originates from a few cells captured as either CTCs or tumor cells that have undergone apoptosis and released their DNA into the serum as small fragments (140–160 bp). Under these conditions, when distributing a small number of starting molecules into individual wells (for real-time or droplet PCR quantification), some proportional amplification is preferable to avoid missing the entire signal due to variability or reporting inaccurate copy numbers. As long as these initial amplifications are maintained at reasonable levels (approximately 12–20 cycles), the risk of carryover contamination is minimized while the tubes are open and during subsequent distribution of amplicons for detection / quantification (using real-time or droplet PCR). Other schemes use even smaller, more limited amplifications (approximately 8–12 cycles).

[0056] The third theme is target-independent signals, also known as "no-template controls" (NTCs). These arise from either polymerase or ligase reactions occurring in the absence of the correct target. Some of this signal can be minimized by careful primer design. In ligation reactions, the 5'->3' nuclease activity of the polymerase can be used to liberate the 5' phosphate of the downstream ligation primer (only when hybridized to the target), making it suitable for ligation. Further specificity for identifying the presence of low-level mutations using LDRs can be achieved by: (i) using an upstream mutation-specific LDR probe containing a mismatch at the second or third position from the 3'OH base; (ii) using an LNA or PNA probe against the wild-type sequence to reduce hybridization of the mutation-specific LDR probe to the wild-type sequence; (iii) using an LDR probe against the wild-type sequence to ligate (optionally) without additional amplification; and (iv) using an upstream LDR probe containing several extra bases and a blocking group to form a free 3'OH by nuclease cleavage only when hybridized to a complementary target (e.g., RNase H2 and a ribonucleotide base). Similar approaches to improving the specificity for identifying the presence of low-level mutations using PCR can be achieved by: (i) using a mutation-specific PCR primer that contains a mismatch at the second or third position from the 3'OH base; (ii) using an LNA or PNA probe to the wild-type sequence that reduces hybridization of the mutation-specific PCR primer to the wild-type sequence; (iii) using a PCR primer to the wild-type sequence that blocks the wild-type sequence and prevents further amplification; and (iv) using an upstream PCR primer that contains several extra bases and a blocking group that is liberated to form a free 3'OH by cleavage with a nuclease (e.g., RNase H2 and a ribonucleotide base) only when hybridized to a complementary target.

[0057] The fourth theme is either suppressed (reduced) amplification or incorrect (incorrect) amplification due to the use of primers in the reaction. One approach to eliminate such unused primers is to capture genomic DNA or target DNA or amplified target DNA on a solid support, hybridize and ligate to ligation probes, and then remove unhybridized probes or products. Alternative solutions include pre-amplification followed by a subsequent nested LDR and / or PCR step (thus providing a second selection level in the process).

[0058] The fifth theme is carryover prevention. Carryover signals can be eliminated by incorporating standard uracil during the universal PCR amplification step and by using UDG (and optionally AP endonuclease) during the pre-amplification workup procedure. Incorporation of carryover prevention is at the heart of the method of the present application, as described in more detail below. The initial PCR amplification is performed using uracil incorporation. The LDR reaction is performed using an LDR probe lacking uracil. Thus, when the LDR product is subjected to real-time PCR quantification, the addition of UDG destroys the initial PCR product but not the LDR product. Furthermore, because LDR is a linear process and the tag primer uses a sequence not present in the human genome, accidental carryover of the LDR product back into the original PCR does not result in template-independent amplification. Additional schemes that provide carryover prevention from methylated targets include using restriction endonucleases to destroy unmethylated DNA prior to PCR amplification, or using methyl-specific DNA-binding proteins or antibodies to capture and enrich methylated DNA.

[0059] The sixth theme is achieving uniform amplification of many mutation-specific or methylation-specific targets in a multiplexed reaction. One approach, as already described above, is to perform limited initial PCR amplification (8–12 or 12–20 cycles). However, different products may amplify at different rates, particularly when using mutation-specific or methylation-specific primers, or when using blocking LNA or PNA probes or other means to suppress amplification of wild-type DNA. This is because a typical PCR reaction involves the simultaneous action of both forward and reverse primers. For example, there may be preferential amplification using the forward methylation-specific primer (i.e., after bisulfite treatment), while the reverse primer amplifies both methylated and unmethylated DNA (again, after bisulfite treatment), thus magnifying the difference in the initial rate of forward primer amplification. Furthermore, this also means that when using mutation-specific forward primers, the use of a nonselective reverse primer means that the initial amplification product still contains a significant amount of wild-type DNA sequence, which can lead to undesirable false positives in subsequent amplification steps. One approach is to perform an initial one-sided linear amplification using primers that amplify only one strand of the target DNA. This is particularly useful when amplifying bisulfite-treated DNA, where the resulting two strands are no longer complementary to each other. An important variation on this theme is to destroy the initial target DNA after the linear amplification step. This can be achieved by incorporating one or more modified nucleotides, such as α-thio-dNTPs, which protect the initial extension products (but not the original cfDNA or genomic DNA) from exonuclease I digestion. When using bisulfite-converted DNA, after an initial one-sided linear amplification (i.e., polymerase extension reaction) with normal dNTPs (i.e., no dUTP), the original bisulfite-converted DNA can be destroyed using UDG.

[0060] Methods for identifying cancer markers A first aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules are then provided. One or more primary oligonucleotide primer sets are also provided. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer that contains a nucleotide sequence complementary to a sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence, and (b) a second primary oligonucleotide primer that contains a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. A sample, one or more first oligonucleotide primers of a primary oligonucleotide primer set, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures, as well as conditions suitable for performing one or more polymerase extension reaction cycles including denaturation, hybridization, and extension treatments, thereby forming primary extension products comprising a nucleotide sequence complementary to the target nucleotide sequence.The primary extension product, one or more second primary oligonucleotide primers of the primary oligonucleotide primer set, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix containing dUTP, and one or more polymerase extension reaction mixtures containing a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures. The method further includes subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more first polymerase chain reaction products containing the target nucleotide sequence or its complement. One or more oligonucleotide probe sets are then provided. Each probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion, and (b) a second oligonucleotide probe having a 5' target sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to complementary target nucleotide sequences of the secondary extension products. One or more first polymerase chain reaction products are blended with a ligase and one or more oligonucleotide probe sets to form one or more ligation reaction mixtures. The one or more ligation reaction mixtures are subjected to one or more ligation reaction cycles, thereby ligating the first and second oligonucleotide probes of the one or more oligonucleotide probe sets together when hybridized to their complementary sequences to form ligation product sequences in the ligation reaction mixture, each ligation product sequence comprising a 5' primer-specific portion, a target-specific portion, and a 3' primer-specific portion. The method further includes providing one or more secondary oligonucleotide primer sets.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer containing the same nucleotide sequence as the 5' primer-specific portion of the ligation product sequence, and (b) a second secondary oligonucleotide primer containing a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequence. The ligation product sequence, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. One or more second polymerase chain reaction products are formed by subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension. The method further includes detecting and identifying one or more second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0061] Figures 2 and 3 illustrate various embodiments of this aspect of the present application, abbreviated as exPCR-LDR-qPCR carryover prevention reaction for detecting low-level mutations (exPCR is an abbreviation for single-sided extension using a primer for one strand of a locus, followed by PCR using either the same primer from the initial extension or an additional primer for the PCR step). Genomic or cfDNA is isolated (Step A in Figure 2), and the isolated DNA sample is treated with UDG to digest dU-containing nucleic acid molecules that may be present in the sample (Step B in Figure 2). Suitable enzymes include, but are not limited to, E. coli uracil DNA glycosylase (UDG), Antarctic thermolabile UDG, or human single-strand-selective monofunctional uracil DNA glycosylase (hSMUG1). The region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing the wild-type sequence, and a deoxynucleotide mix containing dUTP. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, located several bases upstream of the mutation, freeing a 3'-OH group suitable for polymerase extension (step B in Figure 2 or Figure 3; see, e.g., Dobosy et al., "RNase H-Dependent PCR (rhPCR): Improved Specificity and Single Nucleotide Polymorphism Detection Using Blocked Cleavable Primers," BMC Biotechnology 11(80):1011 (2011), the entire contents of which are incorporated herein by reference). A blocking LNA or PNA probe containing a wild-type sequence that partially overlaps with the upstream PCR primer will preferentially compete with the upstream primer for binding to the wild-type sequence, but to a lesser extent to the mutant DNA, thus suppressing the extension of the wild-type DNA during each round of primer extension.Samples are optionally aliquoted into 12, 24, 36, 48, or 96 wells before the first extension step. Locus-specific downstream primers are then added, followed by limited (8-20 cycles) or complete (20-40 cycles) PCR. Optionally, the downstream primers contain identical 8-11 base tails to prevent primer dimerization. Furthermore, such tails provide the option for asymmetric PCR at the end of the PCR cycles by raising the hybridization temperature above the hybridization temperature of the forward primer but below the hybridization temperature of the reverse primer (which will be longer than 8-11 bases and have a higher Tm). This generates more bottom-strand products, which are suitable substrates for the subsequent LDR step. In an alternative embodiment, the initial extension products incorporate one or more modified nucleotides, such as α-thio-dNTPs, which protect the initial extension products (but not the original cfDNA or genomic DNA) from Exonuclease I digestion. After exonuclease I digestion, a downstream locus-specific primer (optionally containing an identical 8-11 base tail) is added, followed by another limited (8-20 cycles) or complete (20-40 cycles) PCR. As shown in step D of Figure 2 or 3, the amplified product contains dU, allowing for subsequent treatment with UDG or a similar enzyme for carryover prevention.

[0062] As shown in step E of Figure 2, a target-specific oligonucleotide probe hybridizes to the amplification product, and a ligase (filled circle) covalently closes the two oligonucleotides when hybridized to their complementary sequences. In this embodiment, the upstream oligonucleotide probe, which has a sequence specific for detecting the mutation of interest, further contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product. Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence prevents ligation to the wild-type target sequence (if present after enrichment of the mutant sequence during the PCR amplification step). The downstream oligonucleotide probe, which has a sequence common to both the mutant and wild-type sequences, contains a 3' primer-specific portion (Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream probe, which has a sequence specific for detecting the mutation, allows subsequent amplification and detection of only the mutant ligation product. Further specificity can be incorporated into this method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe, as shown in step E of Figure 2. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base to generate a ligation-competent 3'-OH group (step D of Figure 2).

[0063] As shown in step F of Figure 2, target-specific oligonucleotide probes hybridize to the amplification product, and a ligase (filled circle) covalently closes the two oligonucleotides when hybridized to their complementary sequences. The upstream oligonucleotide probe contains a 5' primer-specific portion (Ai), and the downstream oligonucleotide probe contains a 3' primer-specific portion (Ci'), allowing for subsequent amplification of the ligation product. After ligation, the ligation product is aliquoted into separate wells, micropores, or droplets containing one or more tag-specific primer pairs, each pair containing matched primers Ai and Ci, and treated with UDG or a similar enzyme to remove dU-containing amplicons or contaminants, amplify, and detect the PCR product. As shown in steps G & H of Figure 2, detection of the ligation product can be performed using a conventional TaqMan™ detection assay (see U.S. Patent No. 6,270,967 to Whitcombe et al. and U.S. Patent No. 7,601,821 to Anderson et al., which are incorporated by reference in their entireties). For detection using TaqMan™, an oligonucleotide probe spanning the ligation junction is used in conjunction with a primer suitable for hybridization on a primer-specific portion of the ligation product for amplification and detection. The TaqMan™ probe contains a fluorescent reporter group (F1) at one end and a quencher molecule (Q) at the other end, which are sufficiently close to each other in the intact probe that the quencher molecule quenches the fluorescence of the reporter group. During amplification, the TaqMan™ probe and the upstream primer hybridize to complementary regions of the ligation product. The 5' to 3' nuclease activity of the polymerase extends the hybridized primer, liberating the fluorescent group of the TaqMan™ probe and generating a detectable signal (Step H in Figure 2). In a preferred embodiment, the TaqMan probe contains a second quencher group (ZEN) approximately 9 bases from the fluorescent reporter group, and the probe is designed so that the ZEN group is at or adjacent to the mutant base.The use of dUTP during the amplification reaction generates products containing dU, which can then be destroyed using UDG for carryover prevention.

[0064] As shown in step D of Figure 3, a target-specific oligonucleotide probe hybridizes to the amplification product, and a ligase (filled circle) covalently closes the two oligonucleotides when hybridized to complementary sequences. In this embodiment, the upstream oligonucleotide probe, which has a sequence specific for detecting the mutation of interest, further contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product. Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence suppresses ligation to the wild-type target sequence (if present after enrichment of the mutant sequence during the PCR amplification step). The downstream oligonucleotide probe, which has a sequence common to both the mutant and wild-type sequences, contains a 3' primer-specific portion (Bi-Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream s probe, which has a sequence specific for detecting the mutation, allows subsequent amplification and detection of only the mutant ligation product. Further specificity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe, as shown in step D of Figure 3. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base to generate a ligation-competent 3'-OH group (step D of Figure 3).

[0065] In this embodiment, the ligation probe is designed to include a UniTaq primer and tag sequence to facilitate detection. The UniTaq system is fully described in U.S. Patent Application Publication No. 2011 / 0212846 to Spier, incorporated herein by reference in its entirety. The UniTaq system involves the use of three unique "tag" sequences, with at least one unique tag sequence (Ai) present in a first oligonucleotide probe and a second and third unique tag moiety (Bi' and Ci') present in a second oligonucleotide probe sequence, as shown in steps D&E of Figure 3. Upon ligation of the oligonucleotide probes of a probe set, the resulting ligation product comprises the Ai sequence-target-specific sequence-Bi' sequence-Ci' sequence. The essence of the UniTaq approach is that both oligonucleotide probes of a ligation probe set must be correct to obtain a positive signal, thereby enabling highly multiplexed nucleic acid detection. For example, as described herein, this is achieved by requiring two moieties (i.e., two tags) to hybridize to each other.

[0066] Prior to detecting the ligation products, the sample is treated with UDG to destroy the original target amplicon, ensuring that only authentic ligation products are detected. After ligation, the ligation products are aliquoted into separate wells, micropores, or droplets containing one or more tag-specific primer pairs. For the detection step, the ligation products, which contain Ai (first primer-specific portion), Bi' (UniTaq detection portion), and Ci' (second primer-specific portion), are primed on both strands using a first oligonucleotide primer with the same nucleotide sequence as Ai and a second oligonucleotide primer (i.e., Ci) complementary to Ci'. The first oligonucleotide primer also contains a UniTaq detection probe (Bi) with a detectable label F1 at one end and a quencher molecule (Q) at the other end (F1-Bi-Q-Ai). Optionally, a polymerase blocking unit (e.g., HEG, THF, Sp-18, ZEN, or any other blocking agent known in the art sufficient to terminate polymerase extension) is placed proximal to the quencher. In another embodiment, the ZEN quencher group is also placed approximately 9 bases from the fluorescent reporter group to ensure more complete quenching. As shown in step G of Figure 3, PCR amplification results in the formation of a double-stranded product. In this example, the polymerase blocking unit prevents the polymerase from replicating the 5' portion (Bi) of the first universal primer, so that a hairpin cannot form when the bottom strand of the product becomes single-stranded. Formation of such a hairpin results in the annealing of the 3' end of the stem to the amplicon, and therefore polymerase extension of this 3' end terminates the PCR reaction.

[0067] When the double-stranded PCR product is denatured and then the temperature is reduced, the top strand of the product forms a hairpin with a stem between the 5' portion of the first oligonucleotide primer (Bi) and the portion Bi' at the opposite end of the strand (Step H in Figure 3). Also during this step, a second oligonucleotide primer anneals to the 5' primer-specific portion (Ci') of the hairpinned product. During extension of the second universal primer in Step H, the 5' nuclease activity of the polymerase cleaves the detectable label D1 or quencher molecule from the 5' end of the amplicon, thereby increasing the distance between the label and quencher and allowing detection of the label.

[0068] Ligation reactions used in the methods of the present application are well known in the art. Ligases suitable for ligating together the oligonucleotide probes of a probe set (optionally after cleavage of the 3' ribose and blocking group on the first oligonucleotide probe or the 5' flap on the second oligonucleotide probe) include, but are not limited to, Thermus aquaticus ligase, E. coli ligase, T4 DNA ligase, T4 RNA ligase, Taq ligase, 9N ligase, and Pyrococcus ligase, or any other thermostable ligase known in the art. According to the present application, the nuclease ligation process of the present application is performed using an oligonucleotide ligation assay (OLA) reaction (Landegren, et al., "A Ligase-Mediated Gene Detection Technique," Science 241:1077-80 (1988); Landegren, et al., "DNA Diagnostics -- Molecular Techniques and Automation," Science 241:1077-80 (1988)). 242:229-37 (1988), and U.S. Patent No. 4,988,617 to Landegren et al., which are incorporated herein by reference in their entireties), a ligation detection reaction (LDR) that utilizes one set of complementary oligonucleotide probes (see, e.g., WO 90 / 17239 to Barany et al., which are incorporated herein by reference in their entireties), or a ligation chain reaction (LCR) that utilizes two sets of complementary oligonucleotide probes (see, e.g., WO 90 / 17239 to Barany et al., which are incorporated herein by reference in their entireties).

[0069] The oligonucleotide probes of the probe set can be in the form of ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleotide analogs, modified peptide nucleotide analogs, modified phosphate-sugar backbone oligonucleotides, nucleotide analogs, and mixtures thereof.

[0070] The hybridization step in the ligase detection reaction is preferably a thermal hybridization process, which distinguishes between nucleotide sequences based on the nucleotides at the ligation junction.The difference between the target nucleotide sequences can be, for example, a single nucleic acid base difference, a nucleic acid deletion, a nucleic acid insertion, or a nucleic acid rearrangement.Such sequence differences involving two or more bases can also be detected.Preferably, the oligonucleotide probe sets have substantially the same length, so that they hybridize to the target nucleotide sequence under substantially similar hybridization conditions.

[0071] Ligase discrimination can be further enhanced by employing various probe design features. For example, intentional mismatches or nucleotide analogs (e.g., inosine, nitroindole, or nitropyrrole) can be incorporated into the first oligonucleotide probe at the second or third base from the 3' junction end. A perfectly matched 3' end slightly destabilizes hybridization at the 3' end, whereas a mismatched 3' end significantly destabilizes hybridization at the 3' end. This design reduces inappropriate misligation when a mutant probe hybridizes to a wild-type target. Alternatively, RNA bases that are cleaved by RNases can be incorporated into the oligonucleotide probe to ensure template-dependent product formation. For example, Dobosy et al. "RNase H-Dependent PCR (rhPCR): Improved Specificity and Single Nucleotide Polymorphism Detection Using Blocked Cleavable Primers," BMC Biotechnology 11(80):1011 (2011) (incorporated herein by reference in its entirety) describes using an RNA base near the 3' end of an oligonucleotide probe with a 3'-blocked end for cleavage with RNase H2 to generate a PCR-extendable and ligatable 3'-OH. This approach can be used to generate either a ligation-competent 3'-OH (as in the case of standard DNA ligases) or a 5'-P, or both; in the latter case, a ligase capable of ligating 5'-RNA bases is utilized.

[0072] Other potential modifications included abasic sites, such as internal abasic furan or oxo-G. These unusual "bases" are removed by specific enzymes to generate ligation-competent 3'-OH or 5'-P sites. Endonuclease IV, Tth EndoIV (NEB), removes abasic residues after the ligation oligonucleotide anneals to the target nucleic acid, but not from single-stranded DNA. Similarly, Fpg can be used for oxo-G, or EndoV for inosine / uracil, or EndoVIII for thymine glycol.

[0073] Ligation discrimination can also be enhanced by using a coupled nuclease-ligase reaction, as described in WO 2013 / 123220 to Barany et al. or U.S. Patent Application Publication No. 2006 / 0234252 to Anderson et al. (the entire contents of which are incorporated herein by reference). In this embodiment, the first oligonucleotide probe has a ligation-competent 3' OH group, and the second oligonucleotide probe has a ligation-incompetent 5' end (i.e., an oligonucleotide probe without a 5' phosphate). The oligonucleotide probes of the probe set are designed so that the 3'-terminal base of the first oligonucleotide probe overlaps with the immediately adjacent 5'-most base of the second oligonucleotide probe complementary to the target nucleic acid molecule. The overlapping nucleotide is referred to as a "flap." If the overlapping flap nucleotide of the second oligonucleotide probe is complementary to the target nucleic acid molecule sequence and the terminating 3' nucleotide of the first oligonucleotide probe, the phosphodiester bond immediately upstream of the flap nucleotide of the second oligonucleotide probe is recognized and cleaved by a flap endonuclease (FEN) or enzyme with 5' nuclease activity. The specific FEN activity generates a new, ligation-competent 5' phosphate end on the second oligonucleotide probe, which is precisely aligned with the adjacent 3' OH of the first oligonucleotide probe, thereby allowing ligation of the two probes to occur. According to this embodiment, flap endonucleases or 5' nucleases suitable for cleaving the 5' flap of the second oligonucleotide probe prior to ligation include, but are not limited to, E. coli DNA polymerase and polymerases derived from Taq and T. thermophilus, as well as polymerases with 5' nuclease activity, such as T4 RNase H and TaqExo. In another embodiment, the second probe of the probe set has a 3' primer-specific portion, a target-specific portion, and a 5' nucleotide sequence.wherein the 5' nucleotide sequence is complementary to at least a portion of the 3' primer, and the 5' nucleotide sequence hybridizes to its complementary portion of the 3' primer-specific portion to form a hairpin-shaped second oligonucleotide probe when the second probe is not hybridized to the target nucleotide sequence.

[0074] In the case of an insertion or deletion, incorporating a corresponding base or nucleotide analog (e.g., 5-amino-dA or 5-propynyl-dC) into the first oligonucleotide probe at the second or third position from the junction can improve stability and improve discrimination of such frameshift mutations from wild-type sequences. In the case of an insertion, using one or more phosphorothioate-modified nucleotides downstream from the desired dissociative phosphate bond in the second oligonucleotide probe prevents inappropriate cleavage by a 5' nuclease enzyme when the probe hybridizes to wild-type DNA, thereby reducing false-positive ligation to wild-type targets. Similarly, in the case of a deletion, using one or more phosphorothioate-modified nucleotides upstream from the desired dissociative phosphate bond in the second oligonucleotide probe prevents inappropriate cleavage by a 5' nuclease enzyme when the probe hybridizes to wild-type DNA, thereby reducing false-positive ligation to wild-type targets.

[0075] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, one or more nucleases capable of digesting nucleic acid molecules that do not contain modified nucleotides, and one or more first primary oligonucleotide primer(s) are provided. The one or more first primary oligonucleotide primer(s) contain a nucleotide sequence complementary to the sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence. The method further comprises providing one or more secondary oligonucleotide primer sets, a sample, one or more first primary oligonucleotide primers, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing one or more modified nucleotides that protect the extension products but not the target DNA from nuclease digestion, and a DNA polymerase to form one or more polymerase extension reaction mixtures, and subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures, and conditions suitable for performing one or more polymerase extension reaction cycles including denaturation, hybridization, and extension, thereby forming primary extension products containing the complement of the target nucleotide sequence.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a first 5' primer-specific portion and a 3' portion that is complementary to a portion of the primary extension product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a second 5' primer-specific portion and a 3' portion that includes a nucleotide sequence that is complementary to a portion of the extension product formed from the first secondary oligonucleotide primer. One or more polymerase extension reaction mixtures comprising the primary extension products, one or more secondary oligonucleotide primer sets, one or more nucleases, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures, and the one or more first polymerase chain reaction mixtures are subjected to suitable conditions for digesting nucleic acid molecules present in the first polymerase chain reaction mixture but not digesting the primary extension products comprising modified nucleotides, and suitable conditions for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension treatments to form one or more first polymerase chain reaction products comprising a first 5' primer-specific portion, a target-specific nucleotide sequence or its complement, and a complement of the second 5' primer-specific portion. One or more tertiary oligonucleotide primer sets are provided. Each tertiary oligonucleotide primer set includes (a) a first tertiary oligonucleotide primer that includes a nucleotide sequence identical to a first 5' primer-specific portion of one or more first polymerase chain reaction products, and (b) a second tertiary oligonucleotide primer that includes a nucleotide sequence complementary to a 3' primer-specific portion of one or more first polymerase chain reaction products.One or more second polymerase chain reaction mixtures are formed by blending one or more first polymerase chain reaction products, one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase, and subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixtures and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension treatments, thereby forming one or more second polymerase chain reaction products. The method further involves detecting and identifying one or more second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0076] Figures 4-8 show various embodiments of this aspect of the application.

[0077] Figure 4 shows an exemplary exPCR-qPCR carryover prevention reaction for detecting low-level mutations. Genomic or cfDNA is isolated (Step A of Figure 4), and the isolated DNA sample is treated with UDG to digest dU-containing nucleic acid molecules that may be present in the sample (Step A of Figure 4). The sample is then subjected to a linear amplification reaction, e.g., one or more polymerase extension reactions, to generate complementary copies of the mutation containing the region of interest. The region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing the wild-type sequence, and a deoxynucleotide mix containing one or more modified nucleotides. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, located several bases upstream of the mutation, freeing a 3'-OH group suitable for polymerase extension (Step B of Figure 4). A blocking LNA or PNA probe containing a wild-type sequence that partially overlaps with the upstream PCR primer preferentially competes with the upstream primer for binding to the wild-type sequence, but to a lesser extent to the mutant DNA, thus suppressing the extension of the wild-type DNA during each round of primer extension. Optionally, prior to the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells.

[0078] The initial extension products incorporate one or more modified nucleotides, such as α-thio-dNTPs, which protect the initial extension products (but not the original cfDNA or genomic DNA) from exonuclease I digestion (Figure 4, step C). By simply using an upstream locus-specific primer in the presence of a blocking LNA or PNA probe, mutation-containing extension products are enriched in each extension cycle. Exonuclease digestion destroys wild-type DNA present in the original genomic or cfDNA sample, and therefore, the enriched extension products are not diluted by subsequent extension or amplification from the original wild-type DNA (see step D below).

[0079] As shown in step D of Figure 4, mutation-specific and locus-specific oligonucleotide primers are added, followed by limited-cycle nested PCR to amplify mutation-containing sequences, if present in the sample. In this embodiment, the upstream mutation-specific primer, which has a sequence specific for detecting the mutation of interest, further contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the nested PCR product. Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits extension of the wild-type target sequence (if present after enrichment of the mutant sequence during the initial extension step). The reverse locus-specific primer, which has a sequence common to both the mutant and wild-type sequences, contains a 5' primer-specific portion (Ci), which, together with the 5' primer-specific portion (Ai) of the upstream primer, which has a sequence specific for detecting the mutation, allows subsequent amplification and detection of only the mutant PCR product. As shown in step D of Figure 4, further specificity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the mutation-specific and locus-specific primers. Upon target-specific hybridization, RNase H (star) removes the RNA base, generating a polymerase-extendable 3'-OH group (step D of Figure 4). In the initial primer extension (step B), the free 3'-OH base is located several bases upstream from the mutation position and, therefore, if cleaved, will extend both the wild-type and mutant sequences (although the blocking LNA or PNA should limit cleavage of the primer hybridized to the wild-type sequence). In contrast, in nested PCR (step D), the mutation-specific base of the primer is located at the 3'-OH base, thereby reducing the likelihood of extension on the wild-type sequence due to the base mismatch.The specificity of polymerase extension of the mutant sequence relative to the wild-type sequence is further improved by: (i) using a mutation-specific PCR primer containing a mismatch at the second or third position from the 3'OH base; (ii) using an LNA or PNA probe to the wild-type sequence, which reduces hybridization of the mutation-specific PCR primer to the wild-type sequence; (iii) using a PCR primer to the wild-type sequence, which blocks the wild-type sequence and prevents further amplification; and (iv) avoiding G:T or T:G mismatches between the primer and the wild-type sequence at the 3'OH base.

[0080] As shown in Figure 4, step E, the nested PCR product contains a 5' primer-specific portion (Ai), a target-specific sequence, and a 3' primer-specific portion (Ci'), allowing for subsequent amplification of the nested PCR product. After limited-cycle PCR, the PCR product is aliquoted into separate wells, micropores, or droplets containing one or more tag-specific primer pairs, each pair containing corresponding primers Ai and Ci, treated with UDG or a similar enzyme to remove dU-containing amplification products or contaminants, PCR amplified, and detected. As shown in steps F&G of Figure 4, detection of the ligation product can be performed using a conventional TaqMan™ detection assay (see U.S. Patent No. 6,270,967 to Whitcombe et al. and U.S. Patent No. 7,601,821 to Anderson et al., both of which are incorporated by reference in their entireties). For detection using TaqMan™, an oligonucleotide probe spanning the mutation-specific region is used in conjunction with a primer suitable for hybridization to the primer-specific portion of the nested PCR product for amplification and detection. The TaqMan™ probe contains a fluorescent reporter group (F1) at one end and a quencher molecule (Q) at the other end, which are sufficiently close to each other in the intact probe that the quencher molecule quenches the fluorescence of the reporter group. During amplification, the TaqMan™ probe and upstream primer hybridize to their complementary regions of the nested PCR product. The 5' to 3' nuclease activity of the polymerase extends the hybridized primer, liberating the fluorescent group of the TaqMan™ probe to generate a detectable signal (Step G in Figure 4). In a preferred embodiment, the TaqMan™ probe contains a second quencher group (ZEN) approximately 9 bases from the fluorescent reporter group, and the probe is designed so that the ZEN group is at or adjacent to the mutant base. The use of dUTP during the amplification reaction generates products containing dU, which can then be destroyed using UDG for carryover prevention.

[0081] Figure 5 shows another exPCR-qPCR carryover prevention reaction for detecting low-level mutations. Genomic or cfDNA is isolated (Step A of Figure 5), and the isolated DNA sample is treated with UDG to digest any dU-containing nucleic acid molecules that may be present in the sample (Step A of Figure 5). The sample is then subjected to a linear amplification reaction, e.g., one or more polymerase extension reactions, to generate complementary copies of the mutation containing the region of interest. The region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing the wild-type sequence, and a deoxynucleotide mix containing one or more modified nucleotides. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, located several bases upstream of the mutation, freeing a 3'-OH group suitable for polymerase extension (Step B of Figure 5). A blocking LNA or PNA probe containing a wild-type sequence that partially overlaps with the upstream PCR primer preferentially competes with the upstream primer for binding to the wild-type sequence, but to a lesser extent to the mutant DNA, thus suppressing the extension of the wild-type DNA during each round of primer extension. Optionally, prior to the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells.

[0082] The initial extension products incorporate one or more modified nucleotides, such as α-thio-dNTPs, which protect the initial extension products (but not the original cfDNA or genomic DNA) from exonuclease I digestion (Step C in Figure 5). By simply using an upstream locus-specific primer in the presence of a blocking LNA or PNA probe, mutation-containing extension products are enriched in each extension cycle. Exonuclease digestion destroys wild-type DNA present in the original genomic or cfDNA sample, and therefore, the enriched extension products are not diluted by subsequent extension or amplification from the original wild-type DNA (see Step D below).

[0083] As shown in step D of Figure 5, mutation-specific and locus-specific oligonucleotide primers are added, followed by limited-cycle nested PCR to amplify mutation-containing sequences, if present in the sample. In this embodiment, the upstream mutation-specific primer, which has a sequence specific for detecting the mutation of interest, further contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the nested PCR product. Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits extension of the wild-type target sequence (if present after enrichment of the mutant sequence during the initial extension step). The reverse locus-specific primer, which has a sequence common to both the mutant and wild-type sequences, contains a 3' primer-specific portion (Bi-Ci), which, together with the 5' primer-specific portion (Ai) of the upstream primer, which has a sequence specific for detecting the mutation, allows subsequent amplification and detection of only the mutant PCR product. As shown in step D of this figure, further specificity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the mutation-specific and locus-specific primers. Upon target-specific hybridization, RNase H (star) removes the RNA base, generating a polymerase-extendable 3'-OH group (step D of Figure 5). In the initial primer extension (step B), the free 3'-OH base is located several bases upstream from the mutation position and, therefore, if cleaved, will extend both the wild-type and mutant sequences (although the blocking LNA or PNA should limit cleavage of the primer hybridized to the wild-type sequence). In contrast, in nested PCR (step D), the mutation-specific base of the primer is located at the 3'-OH base, thereby reducing the likelihood of extension on the wild-type sequence due to the base mismatch.The specificity of polymerase extension of the mutant sequence relative to the wild-type sequence is further improved by: (i) using a mutation-specific PCR primer containing a mismatch at the second or third position from the 3'OH base; (ii) using an LNA or PNA probe to the wild-type sequence, which reduces hybridization of the mutation-specific PCR primer to the wild-type sequence; (iii) using a PCR primer to the wild-type sequence, which blocks the wild-type sequence and prevents further amplification; and (iv) avoiding G:T or T:G mismatches between the primer and the wild-type sequence at the 3'OH base.

[0084] As shown in step E of Figure 5, the nested PCR product contains a 5' primer-specific portion (Ai), a target-specific sequence, and a 3' primer-specific portion (Bi'-Ci'), allowing for subsequent amplification of the nested PCR product. After limited-cycle PCR, the PCR product is aliquoted into separate wells, micropores, or droplets containing one or more tag-specific primer pairs, each pair containing corresponding primers F1-Bi-Q-Ai and Ci, treated with UDG or a similar enzyme to remove dU-containing amplification products or contaminants, PCR amplified (step F of Figure 5), and detected. As shown in step G of Figure 5, PCR amplification results in the formation of a double-stranded product. In this example, the polymerase blocking unit prevents the polymerase from replicating the 5' portion (Bi) of the first universal primer, so that the bottom strand of the product cannot form a hairpin when it becomes single-stranded. Formation of such a hairpin results in the annealing of the 3' end of the stem to the amplicon, and therefore polymerase extension of this 3' end will terminate the PCR reaction.

[0085] When the double-stranded PCR product is denatured and then the temperature is reduced, the top strand of the product forms a hairpin with a stem between the 5' portion of the first oligonucleotide primer (Bi) and the portion Bi' at the opposite end of the strand (Step H in Figure 5). Also during this step, a second oligonucleotide primer anneals to the 5' primer-specific portion (Ci') of the hairpinned product. During extension of the second universal primer in Step H, the 5' nuclease activity of the polymerase cleaves the detectable label D1 or quencher molecule from the 5' end of the amplicon, thereby increasing the distance between the label and quencher and allowing detection of the label.

[0086] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules and one or more nucleases capable of digesting existing nucleic acid molecules that do not contain modified nucleotides are provided. The method also involves providing one or more primary oligonucleotide primer sets. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer that contains a nucleotide sequence complementary to the sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence, and (b) a second primary oligonucleotide primer that contains a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. A sample, one or more first primary oligonucleotide primers of a primary oligonucleotide primer set, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including one or more modified nucleotides that protect the extension products from nuclease digestion but do not protect the target DNA, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixture, and conditions suitable for performing one or more polymerase extension reaction cycles including denaturation, hybridization, and extension treatments, thereby forming primary extension products that contain the complement of the target nucleotide sequence.The method further includes blending one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of one or more primary oligonucleotide primer sets, one or more nucleases, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures. The one or more first polymerase chain reaction mixtures are subjected to suitable conditions that digest nucleic acid molecules present in the polymerase chain reaction mixture but not the primary extension products containing modified nucleotides, and to suitable conditions for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products containing the target nucleotide sequence or its complement. One or more secondary oligonucleotide primer sets are then provided. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a 3' portion complementary to a portion of the extension product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 3' portion comprising a nucleotide sequence complementary to a portion of the extension product formed from the first secondary oligonucleotide primer. The first polymerase chain reaction product, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. The one or more second polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and conditions suitable for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming the second polymerase chain reaction product.The method further includes detecting and identifying second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues.

[0087] Figures 6, 7 and 8 show various embodiments of this aspect of the application.

[0088] Figure 6 shows another exemplary exPCR-qPCR carryover prevention reaction for detecting low-level mutations. Genomic or cfDNA is isolated (Step A of Figure 6), and the isolated DNA sample is treated with UDG to digest any dU-containing nucleic acid molecules that may be present in the sample (Step A of Figure 6). The sample is then subjected to a linear amplification reaction, e.g., one or more polymerase extension reactions, to generate complementary copies of the mutation containing the region of interest. The region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing the wild-type sequence, and a deoxynucleotide mix containing one or more modified nucleotides. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, located several bases upstream of the mutation, freeing a 3'-OH group suitable for polymerase extension (Step B of Figure 6). A blocking LNA or PNA probe containing a wild-type sequence that partially overlaps with the upstream PCR primer preferentially competes with the upstream primer for binding to the wild-type sequence, but to a lesser extent to the mutant DNA, thus suppressing the extension of the wild-type DNA during each round of primer extension. The initial extension product incorporates one or more modified nucleotides, such as α-thio-dNTPs, which protect the initial extension product (but not the original cfDNA or genomic DNA) from Exonuclease I digestion (Step B in Figure 6). Optionally, prior to the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells.

[0089] A locus-specific downstream primer is then added, followed by limited cycle PCR (8-12 cycles) (Step C in Figure 6). In a preferred embodiment, the locus-specific downstream primer is approximately 20-40 bases downstream from the locus-specific upstream primer. Optionally, the downstream primer contains an identical 8-11 base tail to prevent primer dimerization.

[0090] After limited-cycle PCR, the PCR products are aliquoted into separate wells, micropores, or droplets containing Taqman™ probes, mutation-specific primers, and locus-specific primers to amplify mutation-containing sequences (if present in the sample) (Step D in Figure 6). Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits extension of the wild-type target sequence (if present after enrichment of mutant sequences during the initial extension step) (Steps B and C in Figure 6). As shown in Step D in Figure 6, further specificity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the mutation-specific and locus-specific primers. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, generating a 3'-OH group competent for polymerase extension (Step D in Figure 6). In the initial primer extension (Step B), the free 3'OH is located several bases upstream from the mutation position, and therefore, if cleaved, will extend both the wild-type and mutant sequences (although the blocking LNA or PNA should limit cleavage of the primer hybridized to the wild-type sequence). In contrast, in nested PCR (Step D), the mutation-specific base of the primer is at the 3'OH, which reduces the likelihood of extension on the wild-type sequence due to the mismatched base. The specificity of the mutant sequence relative to the wild-type sequence for polymerase extension is further improved by: (i) using a mutation-specific PCR primer that contains a mismatch at the second or third position from the 3'OH base; (ii) using an LNA or PNA probe to the wild-type sequence, which reduces hybridization of the mutation-specific PCR primer to the wild-type sequence; (iii) using a PCR primer to the wild-type sequence, which blocks the wild-type sequence and prevents further amplification; and (iv) avoiding G:T or T:G mismatches between the primer and the wild-type sequence at the 3'OH base.The TaqMan™ probe spans the mutation region and contains a fluorescent reporter group (F1) at one end and a quencher molecule (Q) at the other end, which are sufficiently close to each other in the intact probe that the quencher molecule quenches the fluorescence of the reporter group. During amplification, the TaqMan™ probe and upstream primer hybridize to their complementary regions of the initial PCR product. The 5' to 3' nuclease activity of the polymerase extends the hybridized primer, liberating the fluorescent group of the TaqMan™ probe and generating a detectable signal (Step E in Figure 6). In a preferred embodiment, the TaqMan™ probe contains a second quencher group (ZEN) approximately 9 bases from the fluorescent reporter group, and the probe is designed so that the ZEN group is at or adjacent to the mutant base. The use of dUTP during the amplification reaction generates a product containing dU, which can then be destroyed using UDG to prevent carryover.

[0091] Figures 7 and 8 show additional exemplary exPCR-qPCR carryover prevention reactions for detecting low-level mutations. Genomic or cfDNA is isolated (Step A in Figures 7 and 8), and the isolated DNA sample is treated with UDG to digest dU-containing nucleic acid molecules that may be present in the sample (Step A in Figure 7). The region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing the wild-type sequence, and a deoxynucleotide mix containing one or more modified nucleotides. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, located several bases upstream of the mutation, freeing a 3'-OH group suitable for polymerase extension (Step B in Figures 7 and 8). A blocking LNA or PNA probe containing a wild-type sequence that partially overlaps the upstream PCR primer preferentially competes with the upstream primer for binding to the wild-type sequence but to a lesser extent to the mutant DNA, thus suppressing the extension of the wild-type DNA during each round of primer extension. The initial extension product incorporates one or more modified nucleotides, such as α-thio-dNTPs, which protect the initial extension product (but not the original cfDNA or genomic DNA) from Exonuclease I digestion (Step B in Figures 7 and 8). Optionally, before the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells. A locus-specific downstream primer is then added, followed by limited-cycle PCR (8–12 cycles) (Step B in Figures 7 and 8).

[0092] For the protocol shown in Figure 7, after limited-cycle PCR, the PCR product is aliquoted into separate wells, micropores, or droplets containing a Taqman™ probe, a mutation-specific primer containing a 5' primer-specific portion (Ai), a locus-specific primer containing a 5' primer-specific portion (Ci), and the corresponding primers Ai and Ci. These primers combine to amplify mutation-containing sequences (if present in the sample) (Step C of Figure 7). Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits extension of the wild-type target sequence (if present after enrichment of mutant sequences during the initial extension step) (Step B of Figure 7). Further specificity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the mutation-specific and locus-specific primers, as shown in Step C of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, generating a polymerase-extendable 3'OH group (Step C in Figure 7). In the initial primer extension (Step B), the free 3'OH is located several bases upstream from the mutation position and, therefore, if cleaved, will extend both the wild-type and mutant sequences (although a blocking LNA or PNA should limit cleavage of the primer hybridized to the wild-type sequence). In contrast, in the combinatorial Taqman™ Universal Tag PCR amplification (Steps C-F), the mutation-specific base of the upstream primer is located at the 3'OH, making extension on the wild-type sequence less likely due to the mismatched base. Following mutation- and locus-specific extension, a product comprising the Ai tag sequence, the target-specific sequence, and the Ci' tag sequence is generated (Step D of Figure 7), which can be detected by paired corresponding primers Ai and Ci and a TaqMan™ probe spanning the ligation junction (see Steps E and F of Figure 7), as described above for Steps F-G of Figure 4, or using other suitable means known in the art.The specificity of polymerase extension of mutant sequences relative to wild-type sequences is further improved by: (i) using mutation-specific PCR primers containing a mismatch at the second or third position from the 3'OH base, (ii) using an LNA or PNA probe to the wild-type sequence, which reduces hybridization of the mutation-specific PCR primer to the wild-type sequence, (iii) using a PCR primer to the wild-type sequence, which blocks the wild-type sequence and prevents further amplification, and (iv) avoiding G:T or T:G mismatches between the primer and the wild-type sequence at the 3'OH base. Furthermore, the longer target-specific primers are at significantly lower concentrations than the Taqman™ probe and tag-specific primers (Ai, Ci), thereby depleting the longer mutation-specific primers and allowing the Taqman™ probe and tag-specific primers to hybridize, enabling target-dependent detection.

[0093] For the protocol shown in Figure 8, after limited-cycle PCR, the PCR product is aliquoted into separate wells, micropores, or droplets containing a Taqman™ probe, a mutation-specific primer containing a 5' primer-specific portion (Ai), a locus-specific primer containing a 5' primer-specific portion (Bi-Ci), and the corresponding UniTaq primers F1-Bi-Q-Ai and Ci. These primers combine to amplify mutation-containing sequences (if present in the sample) (Step C of Figure 8). Again, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits extension of the wild-type target sequence (if present after enrichment of mutant sequences during the initial extension step) (Step B of Figure 8). Further specificity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the mutation-specific and locus-specific primers, as shown in Step C of this figure. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, generating a polymerase-extendable 3'OH group (Step C in Figure 8). In the initial primer extension (Step B), the free 3'OH is located several bases upstream from the mutation position and, therefore, if cleaved, will extend both the wild-type and mutant sequences (although a blocking LNA or PNA should limit cleavage of the primer hybridized to the wild-type sequence). In contrast, in the combined Taqman™-UniTaq PCR amplification (Steps C-G), the mutation-specific base of the upstream primer is located at the 3'OH, making extension on the wild-type sequence less likely due to the base mismatch. Following mutation- and locus-specific extension, a product comprising the Ai tag sequence, the target-specific sequence, and the Bi'-Ci' tag sequence is generated (Step D of Figure 8), which can be detected by paired corresponding UniTaq primers (i.e., F1-Bi-Q-Ai and Ci) (see Steps E-G of Figure 8), as described above for Steps F-H of Figure 5, or using other suitable means known in the art.The specificity of polymerase extension of mutant sequences relative to wild-type sequences is further improved by: (i) using mutation-specific PCR primers containing a mismatch at the second or third position from the 3'OH base; (ii) using an LNA or PNA probe to the wild-type sequence, which reduces hybridization of the mutation-specific PCR primer to the wild-type sequence; (iii) using a PCR primer to the wild-type sequence, which blocks the wild-type sequence and prevents further amplification; and (iv) avoiding G:T or T:G mismatches between the primer and the wild-type sequence at the 3'OH base. Furthermore, the longer target-specific primers are at significantly lower concentrations than the UniTaq primers (F1-Bi-Q-Ai, Ci), thereby depleting the longer mutation-specific primers and allowing the UniTaq primers to hybridize, enabling target-dependent detection.

[0094] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more methylated residues, and subjecting the nucleic acid molecules in the sample to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules are then provided. The method further involves providing one or more primary oligonucleotide primer sets. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer containing a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence containing one or more methylated residues, and (b) a second primary oligonucleotide primer containing a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. The bisulfite-treated sample, one or more first primary oligonucleotide primers of one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for one or more polymerase extension reaction cycles including denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence. The one or more polymerase extension reaction mixtures including the primary extension products, one or more second primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures.One or more first polymerase chain reaction mixtures are subjected to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming a first polymerase chain reaction product containing a bisulfite-treated target nucleotide sequence or its complement. The method further includes providing one or more oligonucleotide probe sets. Each probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion, and (b) a second oligonucleotide probe having a 5' bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to the complementary nucleotide sequence of the first polymerase chain reaction product. The first polymerase chain reaction product is blended with a ligase and one or more oligonucleotide probe sets to form one or more ligation reaction mixtures. The one or more ligation reaction mixtures are subjected to one or more ligation reaction cycles, thereby ligating the first and second oligonucleotide probes of the one or more oligonucleotide probe sets together when hybridized to complementary sequences to form ligation product sequences in the ligation reaction mixture, each ligation product sequence comprising a 5' primer-specific portion, a bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion, and a 3' primer-specific portion. The method further comprises providing one or more secondary oligonucleotide primer sets.Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer comprising the same nucleotide sequence as the 5' primer-specific portion of the ligation product sequence, and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequence. The second polymerase chain reaction products are formed by blending the ligation product sequence, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures, and subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension. The method further involves detecting and identifying second polymerase chain reaction products in one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues.

[0095] Figures 9 and 10 show an exPCR-LDR-qPCR carryover prevention reaction for detecting low-level methylation according to this embodiment of the present application. These steps are similar to those described in Figure 2, with two key differentiators. First, genomic DNA or cfDNA is isolated and then, optionally, treated with a DNA repair kit prior to bisulfite conversion (Step A in Figures 9 and 10). Bisulfite converts unmethylated cytosines, neither 5-methylcytosine (5meC) nor 5-hydroxymethylcytosine (5hmC), to uracil bases (which form base pairs with A). Thus, after a single cycle of PCR amplification, unmethylated Cm, neither 5meC nor 5hmC, is converted to a "T" base, thus enabling both modified forms of cytosine to be distinguished from unmodified cytosine. Second, the region of interest is selectively extended using a locus-specific downstream primer (optionally with an identical 8-11 base tail) and a dUTP-free deoxynucleotide mix. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the downstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, freeing a 3'OH group suitable for polymerase extension (Step B in Figure 9). UDG is added to destroy bisulfite-converted DNA (but not primer extension products). Optionally, prior to the first extension step, samples are aliquoted into 12, 24, 36, 48, or 96 wells. A locus-specific upstream primer is then added, followed by limited (8–20 cycles) or complete (20–40 cycles) PCR using a deoxynucleotide mix containing dUTP (Step C in Figure 9). Upon target-specific hybridization, RNase H removes the RNA base located a few bases upstream of the bisulfite-converted methylated target base, freeing a 3'OH group suitable for polymerase extension (Step C in Figure 9).A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) that partially overlaps the upstream PCR primer preferentially competes for binding to the bisulfite-converted unmethylated sequence over the upstream primer, thus suppressing the extension of the bisulfite-converted unmethylated sequence DNA during each round of PCR. Optionally, the downstream primer contains an identical 8- to 11-base tail to prevent primer dimerization. Furthermore, such a tail provides an option for asymmetric PCR at the end of the PCR cycle by raising the hybridization temperature above the hybridization temperature of the forward primer but below that of the reverse primer (which is longer than 8- to 11-base pairs and has a higher Tm). This generates more bottom-strand product, which is a suitable substrate for the subsequent LDR step. As shown in step D of Figure 9, the amplified product contains dU, allowing for subsequent treatment with UDG or a similar enzyme to prevent carryover.

[0096] Alternatively, as shown in Figure 10, the region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement), and a deoxynucleotide mix that does not contain dUTP. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, several bases upstream of the bisulfite-converted methylated target base, freeing a 3'-OH group suitable for polymerase extension (Step B in Figure 10). The blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) that partially overlaps the upstream PCR primer preferentially competes for binding to the bisulfite-converted unmethylated sequence over the upstream primer, thus suppressing the extension of the bisulfite-converted unmethylated sequence DNA during each round of PCR. UDG is added to destroy bisulfite-converted DNA (but not primer extension products). Optionally, before the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells. Locus-specific downstream primers are then added, followed by limited (8-20 cycles) or complete (20-40 cycles) PCR using a deoxynucleotide mix containing dUTP (Step C in Figure 14). Optionally, the downstream primers contain identical 8-11 base tails to prevent primer dimerization.

[0097] 9 and 10, the methylation-specific upstream probe and locus-specific downstream probe containing tails (Ai, Ci') allow for the formation of ligation products in the presence of bisulfite-converted methylated base-containing PCR products. After ligation, the ligation products can be detected using the pair of corresponding primers Ai and Ci and a TaqMan™ probe spanning the ligation junction (see steps E-H of FIG. 9), as described above with respect to FIG. 2, or using other suitable means known in the art.

[0098] Alternatively, a methylation-specific upstream probe and a locus-specific downstream probe containing tails (Ai, Bi'-Ci') allow for the formation of ligation products in the presence of PCR products containing bisulfite-converted methylated bases. After ligation, the ligation products are amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as described above with respect to Figure 3 or using other suitable means known in the art.

[0099] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more methylated residues. The nucleic acid molecules in the sample are subjected to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules are provided, and one or more first primary oligonucleotide primer(s) are provided. Each first primary oligonucleotide primer contains a nucleotide sequence complementary to the sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence containing one or more methylated residues. The bisulfite-treated sample, one or more first primary oligonucleotide primers, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures, and the one or more polymerase extension reaction mixtures are subjected to conditions suitable for one or more polymerase extension reaction cycles, including denaturation, hybridization, and extension, to form primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence. The method further includes providing one or more secondary oligonucleotide primer sets, each of which includes (a) a first secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion complementary to a portion of the polymerase extension reaction product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 5' primer-specific portion and a 3' portion comprising a nucleotide sequence complementary to a portion of the extension product formed from the first secondary oligonucleotide primer.The method further comprises providing one or more tertiary oligonucleotide primer sets, one or more polymerase extension reaction mixtures comprising the primary extension products, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures, and subjecting the one or more first polymerase chain reaction mixtures to suitable conditions for digesting the nucleic acid molecules present in the first polymerase chain reaction mixture but not the primary extension products containing modified nucleotides, and suitable conditions for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products comprising the 5' primer-specific portion, the bisulfite-treated target nucleotide sequence-specific portion or complementary sequence-specific portion of the first secondary oligonucleotide primer, and the complement of the 5' primer-specific portion of the second secondary oligonucleotide primer. Each tertiary oligonucleotide primer set includes (a) a first tertiary oligonucleotide primer containing the same nucleotide sequence as the 5' primer-specific portion of the first polymerase chain reaction product, and (b) a second tertiary oligonucleotide primer containing a nucleotide sequence complementary to the 3' primer-specific portion of the first polymerase chain reaction product sequence. The first polymerase chain reaction product, one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. Secondary polymerase chain reaction products are formed by subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture and conditions suitable for performing one or more polymerase chain reaction cycles, including denaturation, hybridization, and extension.The method further involves detecting and identifying secondary polymerase chain reaction products in one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues.

[0100] Figures 11, 12, 18, and 19 show various embodiments of this aspect of the present application.

[0101] Figure 11 shows an exemplary exPCR-qPCR carryover prevention reaction for detecting low levels of methylation. Genomic DNA or cfDNA is isolated and, optionally, treated with a DNA repair kit prior to bisulfite conversion (Step A in Figure 11). The region of interest is selectively extended using a locus-specific downstream primer (optionally with an identical 8- to 11-base tail) and a dUTP-free deoxynucleotide mix. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the downstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, freeing a 3'-OH group suitable for polymerase extension (Step B in Figure 11). If the locus-specific downstream primer covers one or more methylation sites, further specificity can be added by using a blocking primer whose sequence corresponds to the bisulfite-converted, unmethylated sequence. After the extension cycle, UDG is added to destroy bisulfite converted DNA (but not the primer extension products). Optionally, before the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells.

[0102] Alternatively, as shown in Figure 12, the region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement), and a deoxynucleotide mix that does not contain dUTP. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, several bases upstream of the bisulfite-converted methylated target base, freeing a 3'-OH group suitable for polymerase extension (Step B in Figure 12). The blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) that partially overlaps the upstream PCR primer preferentially competes for binding to the bisulfite-converted unmethylated sequence over the upstream primer, thus suppressing the extension of the bisulfite-converted unmethylated sequence DNA during each round of PCR. UDG is added to destroy bisulfite converted DNA (but not primer extension products). Optionally, before the first extension step, samples are aliquoted into 12, 24, 36, 48, or 96 wells.

[0103] As shown in step C of Figures 11 and 12, a bisulfite-converted methylated base-specific primer (containing a 5' primer-specific portion Ai) and a bisulfite-converted locus-specific primer (containing a 5' primer-specific portion Ci) are added, followed by limited-cycle nested PCR to amplify the bisulfite-converted methylation-containing sequence, if present in the sample. A blocking LNA or PNA probe containing the bisulfite-converted unmethylated sequence (or its complement) allows amplification of the originally methylated allele, but not the originally unmethylated allele. The primers are unblocked with RNase H2 only when they bind to the correct target. After PCR, the product can be detected using the paired corresponding primers Ai and Ci and a TaqMan™ probe spanning the bisulfite-converted methylated target region (see steps D-F of Figures 11 and 12), as described above with respect to Figure 4, or using other suitable means known in the art.

[0104] Alternatively, a bisulfite-converted methylated base-specific primer (containing a 5' primer-specific portion Ai) and a bisulfite-converted locus-specific primer (containing a 5' primer-specific portion Bi-Ci) are added, followed by limited-cycle nested PCR to amplify bisulfite-converted methylation-containing sequences, if present in the sample. A blocking LNA or PNA probe containing the bisulfite-converted unmethylated sequence (or its complement) allows amplification of the originally methylated allele, but not the originally unmethylated allele. The primers are unblocked with RNase H2 only when they bind to the correct target. After PCR, the product is amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as described above with reference to Figure 5 or using other suitable means known in the art.

[0105] Another aspect of the present application is directed to a method for identifying one or more parent nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues. The method involves providing a sample containing one or more parent nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules by one or more methylated residues. The nucleic acid molecules in the sample are subjected to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues. One or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample and one or more primary oligonucleotide primer sets are provided. Each primary oligonucleotide primer set includes (a) a first primary oligonucleotide primer containing a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence containing one or more methylated residues, and (b) a second primary oligonucleotide primer containing a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer. The bisulfite-treated sample, one or more first primary oligonucleotide primers of one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more polymerase extension reaction mixtures. The one or more polymerase extension reaction mixtures are subjected to conditions suitable for one or more polymerase extension reaction cycles including denaturation, hybridization, and extension, thereby forming primary extension products containing the complements of the bisulfite-treated target nucleotide sequences. The one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix, and a DNA polymerase are blended to form one or more first polymerase chain reaction mixtures.One or more first polymerase chain reaction mixtures are subjected to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and conditions suitable for performing one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming a first polymerase chain reaction product containing a bisulfite-treated target nucleotide sequence or its complement. The method further includes providing one or more secondary oligonucleotide primer sets. Each secondary oligonucleotide primer set includes (a) a first secondary oligonucleotide primer having a 3' portion complementary to a portion of the first polymerase chain reaction product formed from the first primary oligonucleotide primer, and (b) a second secondary oligonucleotide primer having a 3' portion comprising a nucleotide sequence complementary to a portion of the first polymerase chain reaction product formed from the first secondary oligonucleotide primer. The first polymerase chain reaction product, one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix containing dUTP, and a DNA polymerase are blended to form one or more second polymerase chain reaction mixtures. The one or more second polymerase chain reaction mixtures are subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and conditions suitable for performing two or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products. The method further involves detecting and identifying the second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues.

[0106] Figures 13-15, 20, and 21 show various embodiments of this aspect of the application.

[0107] Figure 13 shows another exemplary exPCR-qPCR carryover prevention reaction for detecting low levels of methylation. Genomic DNA or cfDNA is isolated and, optionally, treated with a DNA repair kit prior to bisulfite conversion (Step A in Figure 13). The region of interest is selectively extended using a locus-specific downstream primer (optionally with an identical 8- to 11-base tail) and a dUTP-free deoxynucleotide mix. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the downstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, freeing a 3'OH group suitable for polymerase extension (Step B in Figure 13). If the locus-specific downstream primer covers one or more methylation sites, further specificity can be added by using a blocking primer whose sequence corresponds to the bisulfite-converted, unmethylated sequence. After the extension cycle, UDG is added to destroy the bisulfite-converted DNA (but not the primer extension product). Optionally, before the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells. It is then selectively extended in limited-cycle PCR (8–20 cycles) using a locus-specific upstream primer, a blocking LNA or PNA probe containing the bisulfite-converted unmethylated sequence (or its complement), and a deoxynucleotide mix without dUTP. In this embodiment, further selectivity can be incorporated into the method by including a 3′-cleavable blocking group (Blk3′, e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, located several bases upstream of the bisulfite-converted methylated target base, freeing a 3′-OH group suitable for polymerase extension (step C in Figure 13).A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) that partially overlaps with the upstream PCR primer will preferentially compete for binding to the bisulfite-converted unmethylated sequence over the upstream primer, thus suppressing the extension of the bisulfite-converted unmethylated sequence DNA during each round of PCR.

[0108] After limited cycle PCR, the PCR products are aliquoted into separate wells, micropores, or droplets containing Taqman™ probes, mutation-specific primers, bisulfite-converted methylated base-specific primers, and bisulfite-converted locus-specific primers to amplify bisulfite-converted methylation-containing sequences (if present in the sample) (Step D of Figure 13). The bisulfite-converted methylation-containing products are amplified and detected as described above with respect to Figure 6 (see Steps D-E of Figure 13), or using other suitable means known in the art.

[0109] Figures 14 and 15 show additional exemplary exPCR-qPCR carryover prevention reactions for detecting low levels of methylation. Genomic or cfDNA is isolated and, optionally, treated with a DNA repair kit prior to bisulfite conversion (Step A in Figures 14 and 15). The region of interest is selectively extended using a locus-specific downstream primer (optionally with an identical 8- to 11-base tail) and a deoxynucleotide mix that does not contain dUTP. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the downstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, freeing a 3'OH group suitable for polymerase extension (Step B in Figure 14). If the locus-specific downstream primer covers one or more methylation sites, further specificity can be added by using a blocking primer whose sequence corresponds to the bisulfite-converted, unmethylated sequence. After the extension cycle, UDG is added to destroy the bisulfite-converted DNA (but not the primer extension product). Optionally, before the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells. It is then selectively extended in limited-cycle PCR (8–20 cycles) using a locus-specific upstream primer, a blocking LNA or PNA probe containing the bisulfite-converted unmethylated sequence (or its complement), and a deoxynucleotide mix without dUTP. In this embodiment, further selectivity can be incorporated into the method by including a 3′-cleavable blocking group (Blk3′, e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, located several bases upstream of the bisulfite-converted methylated target base, freeing a 3′-OH group suitable for polymerase extension (step C in Figure 14).A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) that partially overlaps with the upstream PCR primer will preferentially compete for binding to the bisulfite-converted unmethylated sequence over the upstream primer, thus suppressing the extension of the bisulfite-converted unmethylated sequence DNA during each round of PCR.

[0110] Alternatively, as shown in Figure 15, the region of interest is selectively extended using a locus-specific upstream primer, a blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement), and a deoxynucleotide mix that does not contain dUTP. In this embodiment, further selectivity can be incorporated into the method by including a 3'-cleavable blocking group (Blk3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base, several bases upstream of the bisulfite-converted methylated target base, freeing a 3'-OH group suitable for polymerase extension (Step B in Figure 15). The blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) that partially overlaps the upstream PCR primer preferentially competes for binding to the bisulfite-converted unmethylated sequence over the upstream primer, thus suppressing the extension of the bisulfite-converted unmethylated sequence DNA during each round of PCR. UDG is added to destroy bisulfite-converted DNA (but not primer extension products). A locus-specific downstream primer is then added, followed by limited-cycle PCR (8–12 cycles) (Step C in Figure 15). If the locus-specific downstream primer covers one or more methylation sites, further specificity can be added by using a blocking primer whose sequence corresponds to the bisulfite-converted, unmethylated sequence. Optionally, before the first extension step, the sample is aliquoted into 12, 24, 36, 48, or 96 wells.

[0111] For the protocol shown in Figures 14 and 15, after limited-cycle PCR, the PCR product is aliquoted into separate wells, micropores, or droplets containing a Taqman™ probe, a bisulfite-converted methylated base-specific primer containing a 5' primer-specific portion (Ai), a bisulfite-converted locus-specific primer containing a 5' primer-specific portion (Ci), and the corresponding primers Ai and Ci. These primers combine to amplify bisulfite-converted methylation-containing sequences (if present in the sample) (Step D in Figures 14 and 15). A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) allows amplification of the originally methylated allele, but not the originally unmethylated allele. The primers are unblocked with RNase H2 only when they bind to the correct target. After PCR, the products can be detected using a pair of corresponding primers Ai and Ci and a TaqMan™ probe spanning the bisulfite-converted methylated target region (see steps E-G of Figure 14), as described above with respect to Figure 4, or using other suitable means known in the art.

[0112] Alternatively, after limited-cycle PCR, the PCR products are aliquoted into separate wells, micropores, or droplets containing a Taqman™ probe, a bisulfite-converted methylated base-specific primer containing a 5' primer-specific portion (Ai), a bisulfite-converted locus-specific primer containing a 5' primer-specific portion (Bi-Ci), and the corresponding UniTaq primers F1-Bi-Q-Ai and Ci. A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complement) allows amplification of the originally methylated allele, but not the originally unmethylated allele. The primers are unblocked with RNase H2 only when they bind to the correct target. After PCR, the products are amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and detected as described above with respect to Figure 5 or using other suitable means known in the art.

[0113] Figures 16 and 17 show additional exemplary exPCR-LDR-qPCR carryover prevention reactions for detecting low-level methylation. Genomic or cfDNA is isolated and then treated with either: (i) a methyl-sensitive restriction endonuclease, e.g., Bsh1236I (CG^CG), to completely digest unmethylated DNA and prevent carryover, or (ii) methylated DNA is captured and concentrated, followed by (iii) a DNA repair kit (Step A in Figures 16 and 17). The DNA is treated with bisulfit...

Claims

1. 1. A method for identifying, in a sample, one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues, said method comprising: providing a sample comprising one or more parent nucleic acid molecules potentially comprising said target nucleotide sequence that differs from said nucleotide sequence of another parent nucleic acid molecule by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence; and (b) a second primary oligonucleotide primer comprising a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer; blending the sample, one or more first primary oligonucleotide primers of the primary oligonucleotide primer set, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures, and to conditions suitable for performing one or more polymerase extension reaction cycles comprising denaturation, hybridization, and extension, thereby forming primary extension products comprising nucleotide sequences complementary to the target nucleotide sequences; blending the one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of the primary oligonucleotide primer set, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming one or more first polymerase chain reaction products comprising the target nucleotide sequence or its complement; providing one or more oligonucleotide probe sets, each probe set comprising: (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion; and (b) a second oligonucleotide probe having a 5' target sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize in a base-specific manner on a complementary target nucleotide sequence of a secondary extension product; blending the one or more first polymerase chain reaction products with a ligase and the one or more oligonucleotide probe sets to form one or more ligation reaction mixtures; subjecting the one or more ligation reaction mixtures to one or more ligation reaction cycles, whereby the first and second oligonucleotide probes of the one or more oligonucleotide probe sets, when hybridized to their complementary sequences, are ligated together to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises the 5' primer-specific portion, the target-specific portion, and the 3' primer-specific portion; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the ligation product sequence; and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the ligation product sequence; blending the ligation product sequences, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming one or more second polymerase chain reaction products; detecting and identifying the one or more second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues; The method comprising:

2. 1. A method for identifying, in a sample, one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues, said method comprising: providing a sample comprising one or more parent nucleic acid molecules potentially comprising said target nucleotide sequence that differs from said nucleotide sequence of another parent nucleic acid molecule by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules; providing one or more nucleases capable of digesting nucleic acid molecules that do not contain modified nucleotides; providing one or more first primary oligonucleotide primer(s) comprising a nucleotide sequence complementary to a sequence of said parent nucleic acid molecule that flanks said target nucleotide sequence; blending the sample, the one or more first primary oligonucleotide primers, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix comprising one or more modified nucleotides that protect extension products but not target DNA from nuclease digestion, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures, and to conditions suitable for performing one or more polymerase extension reaction cycles comprising denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the target nucleotide sequence; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer having a first 5′ primer-specific portion and a 3′ portion that is complementary to a portion of a primary extension product formed from the first primary oligonucleotide primer; and (b) a second secondary oligonucleotide primer having a second 5′ primer-specific portion and a 3′ portion that comprises a nucleotide sequence that is complementary to a portion of an extension product formed from the first secondary oligonucleotide primer; blending the one or more polymerase extension reaction mixtures comprising the primary extension products, the one or more secondary oligonucleotide primer sets, the one or more nucleases, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to suitable conditions that digest nucleic acid molecules present in the first polymerase chain reaction mixture but not primary extension products comprising modified nucleotides, and to suitable conditions for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension treatments, thereby forming one or more first polymerase chain reaction products comprising the first 5' primer-specific portion, a target-specific nucleotide sequence or its complement, and the complement of the second 5' primer-specific portion; providing one or more tertiary oligonucleotide primer sets, each tertiary oligonucleotide primer set comprising: (a) a first tertiary oligonucleotide primer comprising the same nucleotide sequence as the first 5' primer-specific portion of the one or more first polymerase chain reaction products; and (b) a second tertiary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer-specific portion of the one or more first polymerase chain reaction products; blending the one or more first polymerase chain reaction products, the one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming one or more second polymerase chain reaction products; detecting and identifying the one or more second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues; The method comprising:

3. 1. A method for identifying, in a sample, one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues, said method comprising: providing a sample comprising one or more parent nucleic acid molecules potentially comprising said target nucleotide sequence that differs from said nucleotide sequence of another parent nucleic acid molecule by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules; providing one or more nucleases capable of digesting existing nucleic acid molecules that do not contain modified nucleotides; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a sequence of the parent nucleic acid molecule adjacent to the target nucleotide sequence; and (b) a second primary oligonucleotide primer comprising a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer; blending the sample, one or more first primary oligonucleotide primers of the primary oligonucleotide primer set, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix comprising one or more modified nucleotides that protect extension products but not target DNA from nuclease digestion, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures, and to conditions suitable for performing one or more polymerase extension reaction cycles comprising denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the target nucleotide sequence; blending the one or more polymerase extension reaction mixtures comprising the primary extension products, one or more second primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, the one or more nucleases, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to suitable conditions that digest nucleic acid molecules present in the polymerase chain reaction mixture but not primary extension products comprising modified nucleotides, and to suitable conditions for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension treatments, thereby forming first polymerase chain reaction products comprising the target nucleotide sequence or its complement; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer having a 3′ portion complementary to a portion of an extension product formed from the first primary oligonucleotide primer; and (b) a second secondary oligonucleotide primer having a 3′ portion comprising a nucleotide sequence complementary to a portion of an extension product formed from the first secondary oligonucleotide primer; blending the first polymerase chain reaction product, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecule, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products; detecting and identifying the second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylated residues; The method comprising:

4. 1. A method for identifying, in a sample, one or more parent nucleic acid molecules that comprise a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues, the method comprising: providing a sample containing one or more parent nucleic acid molecules potentially containing the target nucleotide sequence that differs from the nucleotide sequence of the other parent nucleic acid molecules by one or more methylated residues; subjecting the nucleic acid molecules in the sample to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence comprising the one or more methylated residues; and (b) a second primary oligonucleotide primer comprising a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer; blending the bisulfite-treated sample, one or more first primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for one or more polymerase extension reaction cycles comprising denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence; blending the one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products comprising the bisulfite-treated target nucleotide sequence or its complement; providing one or more oligonucleotide probe sets, each probe set comprising: (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion; and (b) a second oligonucleotide probe having a 5' bisulfite-treated target nucleotide sequence-specific portion or a complementary sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize in a base-specific manner on a complementary nucleotide sequence of a first polymerase chain reaction product; blending the first polymerase chain reaction product with a ligase and the one or more oligonucleotide probe sets to form one or more ligation reaction mixtures; subjecting the one or more ligation reaction mixtures to one or more ligation reaction cycles, whereby the first and second oligonucleotide probes of the one or more oligonucleotide probe sets, when hybridized to complementary sequences, are ligated together to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises the 5' primer-specific portion, the bisulfite-treated target nucleotide sequence-specific portion or complementary sequence-specific portion, and the 3' primer-specific portion; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the ligation product sequence; and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the ligation product sequence; blending the ligation product sequences, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products; detecting and identifying said second polymerase chain reaction products in said one or more second polymerase chain reaction mixtures to identify the presence of one or more nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in said sample by one or more methylated residues; The method comprising:

5. 1. A method for identifying, in a sample, one or more parent nucleic acid molecules that comprise a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues, the method comprising: providing a sample containing one or more parent nucleic acid molecules potentially containing the target nucleotide sequence that differs from the nucleotide sequence of the other parent nucleic acid molecules by one or more methylated residues; subjecting the nucleic acid molecules in the sample to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules; providing one or more first primary oligonucleotide primer(s) comprising a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence comprising the one or more methylated residues; blending the bisulfite-treated sample, the one or more first primary oligonucleotide primers, a deoxynucleotide mix, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for one or more polymerase extension reaction cycles comprising denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion that is complementary to a portion of the polymerase extension reaction product formed from the first primary oligonucleotide primer; and (b) a second secondary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion that comprises a nucleotide sequence that is complementary to a portion of the extension product formed from the first secondary oligonucleotide primer; blending the primary extension products, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and one or more polymerase extension reaction mixtures comprising a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to suitable conditions that digest nucleic acid molecules present in the first polymerase chain reaction mixture but not primary extension products comprising modified nucleotides, and to suitable conditions for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension treatments, thereby forming first polymerase chain reaction products comprising the 5' primer-specific portion of the first secondary oligonucleotide primer, the bisulfite-treated target nucleotide sequence-specific portion or complementary sequence-specific portion, and the complement of the 5' primer-specific portion of the second secondary oligonucleotide primer; providing one or more tertiary oligonucleotide primer sets, each tertiary oligonucleotide primer set comprising: (a) a first tertiary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the first polymerase chain reaction product; and (b) a second tertiary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the first polymerase chain reaction product sequence; blending the first polymerase chain reaction product, the one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming secondary polymerase chain reaction products; detecting and identifying said secondary polymerase chain reaction products in said one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in said sample by one or more methylated residues; The method comprising:

6. 1. A method for identifying, in a sample, one or more parent nucleic acid molecules that comprise a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues, the method comprising: providing a sample containing one or more parent nucleic acid molecules potentially containing the target nucleotide sequence that differs from the nucleotide sequence of the other parent nucleic acid molecules by one or more methylated residues; subjecting the nucleic acid molecules in the sample to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence comprising the one or more methylated residues; and (b) a second primary oligonucleotide primer comprising a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer; blending the bisulfite-treated sample, one or more first primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for one or more polymerase extension reaction cycles comprising denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence; blending the one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products comprising the bisulfite-treated target nucleotide sequence or its complement; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer having a 3′ portion complementary to a portion of a first polymerase chain reaction product formed from the first primary oligonucleotide primer; and (b) a second secondary oligonucleotide primer having a 3′ portion comprising a nucleotide sequence complementary to a portion of a first polymerase chain reaction product formed from the first secondary oligonucleotide primer; blending the first polymerase chain reaction product, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecule, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products; detecting and identifying said second polymerase chain reaction products in said one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in said sample by one or more methylated residues; The method comprising:

7. 1. A method for identifying, in a sample, one or more parent nucleic acid molecules that comprise a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in the sample by one or more methylated residues, the method comprising: providing a sample containing one or more parent nucleic acid molecules potentially containing the target nucleotide sequence that differs from the nucleotide sequence of the other parent nucleic acid molecules by one or more methylated residues; subjecting the nucleic acid molecules in the sample to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising: (a) a first primary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion comprising a nucleotide sequence complementary to a sequence of the bisulfite-treated parent nucleic acid molecule adjacent to the bisulfite-treated target nucleotide sequence comprising the one or more methylated residues; and (b) a second primary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion comprising a nucleotide sequence complementary to a portion of an extension product formed from the first primary oligonucleotide primer; blending the bisulfite-treated sample, one or more first primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for one or more polymerase extension reaction cycles comprising denaturation, hybridization, and extension, thereby forming primary extension products comprising the complement of the bisulfite-treated target nucleotide sequence; blending the one or more polymerase extension reaction mixtures containing the primary extension products, one or more second primary oligonucleotide primers of the one or more primary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixture, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products comprising the bisulfite-treated target nucleotide sequence or its complement; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the first polymerase chain reaction product or its complement; and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the first polymerase chain reaction product or its complement; blending the primary polymerase chain reaction product sequences, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products; detecting and identifying said second polymerase chain reaction products in said one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequences of other parent nucleic acid molecules in said sample by one or more methylated residues; The method comprising:

8. 8. The method of any one of claims 1 to 7, further comprising contacting the sample with a DNA repair enzyme to repair damaged DNA, abasic sites, oxidized bases, or nicks in the DNA.

9. prior to or concurrently with the blending step to form one or more polymerase extension reaction mixtures, contacting the sample with at least a first methylation-sensitive enzyme to form a restriction enzyme reaction mixture, wherein the first methylation-sensitive enzyme cleaves nucleic acid molecules in the sample that contain one or more unmethylated residues within at least one methylation-sensitive enzyme recognition sequence, whereby the detecting step involves detecting one or more parent nucleic acid molecules that contain the target nucleotide sequence, wherein the parent nucleic acid molecules originally contain one or more methylated residues. The method of any one of claims 4 to 7, further comprising:

10. 8. The method of any one of claims 4 to 7, further comprising contacting the sample with an immobilized methylated nucleic acid binding protein or antibody to selectively bind to and enrich for methylated nucleic acids in the sample.

11. 8. The method of any one of claims 1 to 7, wherein primers from the one or more primary or secondary oligonucleotide primer sets have no or one nucleotide sequence mismatch when hybridized in a base-specific manner to the target nucleic acid sequence or bisulfite converted methylated nucleic acid sequence or its complementary sequence, but comprise a portion with one or more additional nucleotide sequence mismatches that interfere with polymerase extension when hybridized in a base-specific manner to a corresponding nucleotide sequence portion of a wild-type nucleic acid sequence or bisulfite converted unmethylated nucleic acid sequence or its complementary sequence.

12. one or both primary oligonucleotide primers of said primary oligonucleotide primer set and / or one or both secondary oligonucleotide primers of said secondary oligonucleotide primer set have a 3' portion comprising a cleavable nucleotide or nucleotide analogue and a blocking group, such that the 3' end of the primer(s) is / are not suitable for polymerase extension; 8. The method of any one of claims 1 to 7, wherein the method further comprises cleaving the cleavable nucleotide or nucleotide analog of one or both of the oligonucleotide primers during the hybridization step, thereby liberating a free 3' OH terminus on one or both of the oligonucleotide primers prior to the extension step.

13. 13. The method of claim 12, wherein a primer from the one or more primary or secondary oligonucleotide primer sets comprises a sequence that differs from the target nucleic acid sequence or bisulfite-converted methylated nucleic acid sequence or its complementary sequence, and the difference is located at the second or third nucleotide base from the released free 3' OH terminus.

14. 13. The method of claim 12, wherein the cleavable nucleotide comprises one or more RNA bases.

15. providing one or more blocking oligonucleotide primers, the one or more blocking oligonucleotide primers comprising one or more mismatched bases at the 3' end or one or more nucleotide analogs and a blocking group at the 3' end, such that the 3' end of the blocking oligonucleotide primer is not suitable for polymerase extension when hybridized in a base-specific manner to a wild-type nucleic acid sequence or a bisulfite converted unmethylated nucleic acid sequence or its complementary sequence, wherein the blocking oligonucleotide primer comprises a portion having the same nucleotide sequence as the nucleotide sequence portion of the wild-type nucleic acid sequence or the bisulfite converted unmethylated nucleic acid sequence or its complementary sequence to which the blocking oligonucleotide primer hybridizes, but has one or more nucleotide sequence mismatches with the corresponding nucleotide sequence portion of the target nucleic acid sequence or the bisulfite converted methylated nucleic acid sequence or its complementary sequence; blending the one or more blocking oligonucleotide primers with the sample or its subsequent products prior to a polymerase extension reaction, polymerase chain reaction, or ligation reaction, whereby during hybridization, the one or more blocking oligonucleotide primers preferentially hybridize in a base-specific manner to a wild-type nucleic acid sequence or a bisulfite-converted unmethylated nucleic acid sequence or its complementary sequence, thereby preventing polymerase extension or ligation during a reaction of a primer or probe hybridized in a base-specific manner to the wild-type sequence or the bisulfite-converted unmethylated sequence or its complementary sequence; The method of any one of claims 1 to 7, further comprising:

16. 7. The method of claim 3, wherein the first secondary oligonucleotide primer has a 5' primer-specific portion, the second secondary oligonucleotide primer has a 5' primer-specific portion, and the one or more secondary oligonucleotide primer sets further comprise: (d) a third secondary oligonucleotide primer comprising the same nucleotide sequence as the 5' primer-specific portion of the first secondary oligonucleotide primer; and (d) a fourth secondary oligonucleotide primer comprising the same nucleotide sequence as the 5' primer-specific portion of the second secondary oligonucleotide primer.

17. 1. A method for identifying, in a sample, one or more parent ribonucleic acid molecules comprising a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in said sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertions, exon deletions, intron insertions, translocations, mutations, or other rearrangements at the genomic level, said method comprising: providing a sample containing one or more parent ribonucleic acid molecules, including a target ribonucleic acid molecule that potentially differs in sequence from other parent ribonucleic acid molecules; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to an RNA sequence of the parent ribonucleic acid molecule adjacent to the target ribonucleotide sequence; and (b) a second primary oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the cDNA extension product formed from the first primary oligonucleotide primer; blending the contacted sample, the one or more primary oligonucleotide primer sets, a deoxynucleotide mix including dUTP, a reverse transcriptase, and a DNA polymerase or a DNA polymerase having reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures; subjecting the one or more reverse transcription / polymerase chain reaction mixtures to conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target ribonucleic acid, and for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming one or more distinct reverse transcription / polymerase chain reaction products; providing one or more oligonucleotide probe sets, each probe set comprising: (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion; and (b) a second oligonucleotide probe having a 5' target sequence-specific portion and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize in a base-specific manner on a complementary portion of a reverse transcriptase / polymerase product corresponding to the target ribonucleic acid molecule sequence; contacting the reverse transcriptase / polymerase product with a ligase and the one or more oligonucleotide probe sets to form one or more ligation reaction mixtures; subjecting the one or more ligation reaction mixtures to one or more ligation reaction cycles, whereby the first and second probes of the one or more oligonucleotide probe sets, when hybridized to their complements, ligate together to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises the 5' primer-specific portion, the target-specific portion, and the 3' primer-specific portion; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the ligation product sequence; and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the ligation product sequence; blending the ligation product sequences, the one or more secondary oligonucleotide primer sets with one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products; detecting and identifying said first polymerase chain reaction product, thereby identifying the presence of one or more parent ribonucleic acid molecules comprising a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in said sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertions, exon deletions, intron insertions, translocations, mutations, or other rearrangements at the genomic level; The method comprising:

18. 1. A method for identifying, in a sample, one or more parent ribonucleic acid molecules comprising a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in said sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertions, exon deletions, intron insertions, translocations, mutations, or other rearrangements at the genomic level, said method comprising: providing a sample containing one or more parent ribonucleic acid molecules, including a target ribonucleic acid molecule that potentially differs in sequence from other parent ribonucleic acid molecules; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to an RNA sequence of the parent ribonucleic acid molecule adjacent to the target nucleotide sequence; and (b) a second primary oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the cDNA extension product formed from the first primary oligonucleotide primer; blending the contacted sample, the one or more primary oligonucleotide primer sets, a deoxynucleotide mix, a reverse transcriptase, and a DNA polymerase or a DNA polymerase having reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures; subjecting the one or more reverse transcription / polymerase chain reaction mixtures to conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target RNA, and for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming one or more distinct reverse transcription / primary polymerase chain reaction products; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer having a 3′ portion that is complementary to a portion of a reverse transcription / primary polymerase chain reaction product formed from the first primary oligonucleotide primer; and (b) a second secondary oligonucleotide primer having a 3′ portion that comprises a nucleotide sequence that is complementary to a portion of a reverse transcription / primary polymerase chain reaction product formed from the first secondary oligonucleotide primer; blending the reverse transcription / primary polymerase chain reaction product, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecule, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and to conditions suitable for performing two or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming first polymerase chain reaction products; detecting and identifying said first polymerase chain reaction product, thereby identifying the presence of one or more parent ribonucleic acid molecules comprising a target ribonucleic acid sequence that differs from the ribonucleic acid sequences of other parent ribonucleic acid molecules in said sample due to alternative splicing, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, exon insertions, exon deletions, intron insertions, translocations, mutations, or other rearrangements at the genomic level; The method comprising:

19. 1. A method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence by one or more bases from other miRNA molecules in the sample, the method comprising: providing a sample containing one or more target miRNA molecules that potentially differ in sequence by one or more bases from other miRNA molecules in the sample; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample; blending the contacted sample with a ligase and one or more first oligonucleotide preparatory probes comprising a 5' phosphate, a 5' stem-loop portion, an internal primer-specific portion within the loop region, a blocking group, and a 3' nucleotide sequence complementary to a 3' portion of the target miRNA molecule sequence to form one or more first ligation reaction mixtures; ligating the one or more target miRNA molecules at their 3' ends to the 5' phosphates of the one or more first oligonucleotide preparatory probes in the one or more first ligation reaction mixtures to generate chimeric nucleic acid molecules comprising the target miRNA molecule sequence, if present in the sample, attached to the one or more first oligonucleotide preparatory probes; providing one or more primary oligonucleotide primer sets, each primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to the internal primer-specific portion of the first oligonucleotide preparatory probe; and (b) a second primary oligonucleotide primer comprising a 5′ primer-specific portion and a 3′ portion, wherein the second primary oligonucleotide primer may be the same as or different from other second primary oligonucleotide primers of other sets; blending the one or more first ligation reaction mixtures containing chimeric nucleic acid molecules, the one or more primary oligonucleotide primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the sample, a deoxynucleotide mix containing dUTP, and a reverse transcriptase and a DNA polymerase or a DNA polymerase having reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures; subjecting the one or more reverse transcription / polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the reverse transcription / polymerase chain reaction mixture, conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the chimeric nucleic acid molecules, and conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension treatments, thereby forming one or more distinct primary reverse transcription / polymerase chain reaction products comprising the 5' primer-specific portion, a nucleotide sequence corresponding to the target miRNA molecule sequence, and the complement of the internal primer-specific portion, and its complement; providing one or more oligonucleotide probe sets, each probe set comprising: (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion; and (b) a second oligonucleotide probe having a 5' target sequence-specific portion, a portion complementary to a primary extension product, and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize in a base-specific manner on a complementary portion of a primary reverse transcription / polymerase chain reaction product corresponding to the target miRNA molecule sequence or its complement; contacting the primary reverse transcription / polymerase chain reaction product with a ligase and the one or more oligonucleotide probe sets to form one or more second ligation reaction mixtures; subjecting the one or more second ligation reaction mixtures to one or more ligation reaction cycles, whereby the first and second oligonucleotide probes of the one or more oligonucleotide probe sets, when hybridized to their complements, ligate together to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises the 5' primer-specific portion, the target-specific portion, and the 3' primer-specific portion; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the ligation product sequence; and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the ligation product sequence; blending the ligation product sequences and the one or more secondary oligonucleotide primer sets with one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming secondary polymerase chain reaction products; detecting and identifying the secondary polymerase chain reaction products in the one or more reactions, thereby identifying one or more target miRNA molecules that differ in sequence by one or more bases from other miRNA molecules in the sample; The method comprising:

20. 1. A method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence by one or more bases from other miRNA molecules in the sample, the method comprising: providing a sample containing one or more target miRNA molecules that potentially differ in sequence by one or more bases from other miRNA molecules in the sample; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample; blending the contacted sample with a ligase and one or more first oligonucleotide probes comprising a 5' phosphate, a 5' stem-loop portion, an internal primer-specific portion within the loop region, a blocking group, and a 3' nucleotide sequence complementary to a 3' portion of the target miRNA molecule sequence to form one or more ligation reaction mixtures; ligating the one or more target miRNA molecules at their 3' ends to the 5' phosphates of the one or more first oligonucleotide probes in the one or more ligation reaction mixtures to generate chimeric nucleic acid molecules comprising the target miRNA molecule sequence, if present in the sample, attached to the one or more first oligonucleotide probes; providing one or more primary oligonucleotide primer sets, each primer set comprising: (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to the internal primer-specific portion of the first oligonucleotide probe; and (b) a second primary oligonucleotide primer comprising a 5′ primer-specific portion and a 3′ portion, wherein the second primary oligonucleotide primer may be the same as or different from other second primary oligonucleotide primers of other sets; blending the one or more ligation reaction mixtures containing the chimeric nucleic acid molecule, the one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a reverse transcriptase and a DNA polymerase or a DNA polymerase having reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures; subjecting the one or more reverse transcription / polymerase chain reaction mixtures to conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the chimeric nucleic acid molecules, and to conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct primary reverse transcription / polymerase chain reaction products comprising the 5' primer-specific portion, a nucleotide sequence corresponding to the target miRNA molecule sequence, and a complement of the internal primer-specific portion, and their complements; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion that is complementary to a portion of an extension product formed from the first primary oligonucleotide primer; and (b) a second secondary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion that comprises a nucleotide sequence that is complementary to a portion of an extension product formed from the first secondary oligonucleotide primer; blending the primary reverse transcription / polymerase chain reaction product, the one or more secondary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for two or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, thereby forming a first polymerase chain reaction product comprising the 5' primer-specific portion of the first secondary oligonucleotide primer, a nucleotide sequence corresponding to the target miRNA molecule sequence or its complement, and the complement of another 5' primer-specific portion, a second secondary oligonucleotide primer; providing one or more tertiary oligonucleotide primer sets, each tertiary oligonucleotide primer set comprising: (a) a first tertiary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the first polymerase chain reaction product or its complement; and (b) a second tertiary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the first polymerase chain reaction product or its complement; blending the first polymerase chain reaction process product, the one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products; detecting and identifying the second polymerase chain reaction product, thereby identifying one or more target miRNA molecules that differ in sequence by one or more bases from other miRNA molecules in the sample; The method comprising:

21. 1. A method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence by one or more bases from other miRNA molecules in the sample, the method comprising: providing a sample containing one or more target miRNA molecules that potentially differ in sequence by one or more bases from other miRNA molecules in the sample; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample; blending the contacted sample with ATP and poly(A) polymerase to form a poly(A) polymerase reaction mixture; subjecting the poly(A) polymerase reaction mixture to conditions suitable for adding a homopolymer A to the 3' end of the one or more target miRNA molecules potentially present in the sample; providing one or more primary oligonucleotide primer sets, each primer set comprising: (a) a first primary oligonucleotide primer comprising a 5' primer-specific portion, an internal poly-dT portion, and a 3' portion comprising 1-10 bases complementary to the 3' end of the target miRNA, said first primary oligonucleotide primer being the same as or different from other first primary oligonucleotide primers of another set; and (b) a second primary oligonucleotide primer comprising a 5' primer-specific portion and a 3' portion, said second primary oligonucleotide primer being the same as or different from other second primary oligonucleotide primers of another set; blending the poly(A) polymerase reaction mixture, the one or more primary oligonucleotide primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the sample, a deoxynucleotide mix including dUTP, and a reverse transcriptase and a DNA polymerase or a DNA polymerase having reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures; subjecting the one or more reverse transcription / polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the reverse transcription / polymerase chain reaction mixture, followed by conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target miRNA sequence having a 3' poly-A tail, and conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct reverse transcription / polymerase chain reaction products comprising the 5' primer-specific portion of the second primary oligonucleotide primer, a nucleotide sequence corresponding to the target miRNA molecule sequence, a poly-dA region, and the complement of the 5' primer-specific portion of the first primary oligonucleotide primer, and its complement; providing one or more oligonucleotide probe sets, each probe set comprising: (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target sequence-specific portion; and (b) a second oligonucleotide probe having a 5' target sequence-specific portion, a portion complementary to the one or more reverse transcription / polymerase chain reaction products, and a 3' primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize in a base-specific manner to complementary portions of the one or more reverse transcription / polymerase chain reaction products that correspond to the target miRNA molecule sequence or its complement; contacting the one or more reverse transcription / polymerase chain reaction products with a ligase and the one or more oligonucleotide probe sets to form one or more ligation reaction mixtures; subjecting the one or more ligation reaction mixtures to one or more ligation reaction cycles, whereby the first and second oligonucleotide probes of the one or more oligonucleotide probe sets, when hybridized to their complements, ligate together to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises the 5' primer-specific portion, the target-specific portion, and the 3' primer-specific portion; and subjecting the one or more second ligation reaction mixtures to one or more ligation reaction cycles. providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the ligation product sequence; and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the ligation product sequence; blending the ligation product sequences and the one or more secondary oligonucleotide primer sets with one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and to conditions suitable for performing one or more polymerase chain reaction cycles comprising denaturation, hybridization, and extension, thereby forming secondary polymerase chain reaction products; detecting and identifying the secondary polymerase chain reaction products, thereby identifying one or more target miRNA molecules that differ in sequence by one or more bases from other miRNA molecules in the sample; The method comprising:

22. 1. A method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence by one or more bases from other miRNA molecules in the sample, the method comprising: providing a sample containing one or more target miRNA molecules that potentially differ in sequence by one or more bases from other miRNA molecules in the sample; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in said sample; contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample; blending the contacted sample with ATP and poly(A) polymerase to form a poly(A) polymerase reaction mixture; subjecting the poly(A) polymerase reaction mixture to conditions suitable for adding homopolymer A to the 3' ends of the one or more target miRNA molecules potentially present in the sample; providing one or more primary oligonucleotide primer sets, each primer set comprising: (a) a first primary oligonucleotide primer comprising a 5' primer-specific portion, an internal poly-dT portion, and a 3' portion comprising 1-10 bases complementary to the 3' end of the target miRNA, said first primary oligonucleotide primer being the same as or different from other first primary oligonucleotide primers of another set; and (b) a second primary oligonucleotide primer comprising a 5' primer-specific portion and a 3' portion, said second primary oligonucleotide primer being the same as or different from other second primary oligonucleotide primers of another set; blending the poly(A) polymerase reaction mixture potentially containing a target miRNA sequence with a 3' poly-A tail, the one or more primary oligonucleotide primer sets, a deoxynucleotide mix, and a reverse transcriptase and a DNA polymerase or a DNA polymerase having reverse transcriptase activity to form one or more reverse transcription / polymerase chain reaction mixtures; subjecting the one or more reverse transcription / polymerase chain reaction mixtures to conditions suitable for generating deoxyribonucleic acid (cDNA) molecules complementary to the target miRNA sequence having a 3' poly-A tail, and to conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming one or more distinct reverse transcription / polymerase chain reaction products comprising the 5' primer-specific portion of the second primary oligonucleotide primer, a nucleotide sequence corresponding to the target miRNA molecule sequence, a poly-dA region, and the complement of the 5' primer-specific portion of the first primary oligonucleotide primer, and its complement; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising: (a) a first secondary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion that is complementary to a portion of a reverse transcription / polymerase chain reaction product formed from the first primary oligonucleotide primer; and (b) a second secondary oligonucleotide primer having a 5′ primer-specific portion and a 3′ portion that comprises a nucleotide sequence that is complementary to a portion of a reverse transcription / polymerase chain reaction product formed from the first secondary oligonucleotide primer; blending the reverse transcription / polymerase chain reaction product, the one or more secondary oligonucleotide primer sets, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for two or more polymerase chain reaction cycles, including denaturation, hybridization, and extension, thereby forming a first polymerase chain reaction product comprising a 5' primer-specific portion, a nucleotide sequence corresponding to the target miRNA molecule sequence or its complement, and a complement of another 5' primer-specific portion; providing one or more tertiary oligonucleotide primer sets, each tertiary oligonucleotide primer set comprising: (a) a first tertiary oligonucleotide primer comprising a nucleotide sequence identical to the 5′ primer-specific portion of the first polymerase chain reaction product sequence; and (b) a second tertiary oligonucleotide primer comprising a nucleotide sequence complementary to the 3′ primer-specific portion of the first polymerase chain reaction product sequence; blending the first polymerase chain reaction product, the one or more tertiary oligonucleotide primer sets, one or more enzymes capable of digesting the deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the first polymerase chain reaction mixture, and to conditions suitable for one or more polymerase chain reaction cycles including denaturation, hybridization, and extension, thereby forming second polymerase chain reaction products; detecting and identifying the second polymerase chain reaction products in the one or more reactions, thereby identifying one or more target miRNA molecules that differ in sequence by one or more bases from other miRNA molecules in the sample; The method comprising:

23. 23. The method of any one of claims 19 to 22, wherein the 3' portion of the second primary oligonucleotide primer comprises ribo-G and / or G nucleotide analogs, and the reverse transcriptase adds two or three cytosine nucleotides to the 3' end of the complementary deoxyribonucleic acid product of the target miRNA, allows transient hybridization of the second primary oligonucleotide primer to the 3' end, and allows the reverse transcriptase to cause strand switching and extend the complementary deoxyribonucleic acid product to include the complementary sequence of the 5' primer-specific portion of the second primary oligonucleotide primer, to form the one or more different first polymerase chain reaction products comprising a 5' primer-specific portion, a nucleotide sequence portion corresponding to the target miRNA molecule sequence or its complement, a further portion, and the complement of another 5' primer-specific portion.

24. 23. The method of any one of claims 19 to 22, wherein the 3' portion of the second primary oligonucleotide primer comprises, from 5' to 3', 6 to 14 bases, including three ribo-G or G bases followed by the same additional base as the 5' end of the target miRNA sequence; the reverse transcriptase adds two or three cytosine residues to the 3' end of an initial complementary deoxyribonucleic acid extension product of the target miRNA; and once the denaturation step of the polymerase chain reaction is initiated, the conditions are then adjusted to allow transient hybridization of the 3' end of the second primary oligonucleotide primer to the 3' end of the complementary deoxyribonucleic acid extension product, extending either or both of the second primary oligonucleotide primer and the complementary deoxyribonucleic acid extension product to form the one or more different primary reverse transcription / polymerase chain reaction products comprising a 5' primer-specific portion, a nucleotide sequence portion corresponding to the target miRNA molecule sequence or its complementary portion, a further portion, and a complement of another 5' primer-specific portion.

25. the second oligonucleotide probe of the oligonucleotide probe set further comprises a unitaq detection portion, thereby forming a ligation product sequence comprising the 5' primer-specific portion, the target-specific portion, the unitaq detection portion, and the 3' primer-specific portion; The method comprises: providing one or more unitaq detection probes, each unitaq detection probe hybridizing to a complementary unitaq detection moiety, said detection probe comprising a quencher molecule and a detectable label separated from said quencher molecule; adding the one or more unitaq detection probes to the second polymerase chain reaction mixture; hybridizing the one or more unitaq detection probes to complementary unitaq detection moieties on the ligation product sequence or its complement during the step of subjecting the second polymerase chain reaction mixture to conditions suitable for one or more polymerase chain reaction cycles, wherein the quencher molecule and the detectable label are cleaved from the one or more unitaq detection probes during the extension process, and wherein the detecting step involves detecting the cleaved detectable label; 18. The method of any one of claims 1, 4 or 17, further comprising:

26. one primary oligonucleotide primer or one secondary oligonucleotide primer further comprises a unitaq detection portion, thereby forming an extension product sequence comprising the 5' primer-specific portion, the target-specific portion, the unitaq detection portion, and the complement of the other 5' primer-specific portion, and their complements; The method comprises: providing one or more unitaq detection probes, each unitaq detection probe hybridizing to a complementary unitaq detection moiety, said detection probe comprising a quencher molecule and a detectable label separated from said quencher molecule; adding the one or more unitaq detection probes to the one or more first or second polymerase chain reaction mixtures; hybridizing the one or more unitaq detection probes to complementary unitaq detection moieties on the ligation product sequence or its complement during a polymerase chain reaction cycle after the first polymerization chain reaction, wherein during the extension process, the quencher molecule and the detectable label are cleaved from the one or more unitaq detection probes, and wherein the detecting step involves detecting the cleaved detectable label; 20. The method of any one of claims 2, 3, 5, 6, 7, or 18, further comprising:

27. 22. The method of any one of claims 1, 4, 17, 19, or 21, wherein one or both of the oligonucleotide probes of the oligonucleotide probe set contain a portion that has no nucleotide sequence mismatches or one nucleotide sequence mismatch when hybridized in a base-specific manner to the target nucleic acid sequence or bisulfite converted methylated nucleic acid sequence or its complementary sequence, but has one or more additional nucleotide sequence mismatches that prevent ligation when hybridized in a base-specific manner to a corresponding nucleotide sequence portion of the wild-type nucleic acid sequence or bisulfite converted unmethylated nucleic acid sequence or its complementary sequence.

28. the 3' portion of the first oligonucleotide probe of the oligonucleotide probe set comprises a cleavable nucleotide or nucleotide analog and a blocking group, such that the 3' end is not suitable for polymerase extension or ligation; 18. The method of any one of claims 1, 4, or 17, wherein the method further comprises, prior to the ligating step, cleaving the cleavable nucleotide or nucleotide analog of the first oligonucleotide probe when the probe is hybridized to its complementary target nucleotide sequence of the primary extension product, thereby liberating a 3' OH on the first oligonucleotide probe.

29. 29. The method of claim 28, wherein the one or more first oligonucleotide probes of the oligonucleotide probe set comprise a sequence that differs from the target nucleic acid sequence or bisulfite-converted methylated nucleic acid sequence or its complementary sequence, and the difference is located at the second or third nucleotide base from the released 3' OH terminus.

30. the second oligonucleotide probe has an identical nucleotide at its 5' end that overlaps with the 3' end of the first oligonucleotide probe, and when the first and second oligonucleotide probes of a probe set hybridize to form a junction at adjacent positions on a complementary target nucleotide sequence of a primary extension product, the overlapping identical nucleotide of the second oligonucleotide probe forms a flap at the junction with the first oligonucleotide probe; 18. The method of any one of claims 1, 4, or 17, further comprising, prior to the ligating step, cleaving the overlapping identical nucleotide of the second oligonucleotide probe with an enzyme having 5' nuclease activity, thereby liberating the 5' terminal phosphate of the second oligonucleotide probe.

31. 18. The method of any one of claims 1, 4, or 17, wherein the one or more oligonucleotide probe sets further comprise a third oligonucleotide probe having a target-specific portion, and wherein the second and third oligonucleotide probes of a probe set are configured to hybridize adjacent to each other on the target nucleotide sequence with a junction therebetween to allow ligation between the second and third oligonucleotide probes to form a ligation product sequence comprising the first, second, and third oligonucleotide probes of the probe set.

32. 32. The method of any one of claims 1 to 31, wherein the sample is selected from the group consisting of tissue, cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodily fluids, bodily secretions, bodily excretions, cell-free circulating nucleic acid, cell-free circulating tumor nucleic acid, cell-free circulating fetal nucleic acid of a pregnant woman, circulating tumor cells, tumor, tumor biopsy, and exosomes.

33. 32. The method of any one of claims 1 to 31, wherein the one or more target nucleotide sequences are low abundance nucleic acid molecules and comprise one or more nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicing, exon insertions, exon deletions, intron insertions, or other rearrangements at the genomic level, and / or methylated nucleotide bases.

34. 34. The method of claim 33, wherein the low abundance nucleic acid molecules having one or more nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicing, exon insertions, exon deletions, intron insertions, or other genomic rearrangements, and / or methylated nucleotide bases are identified and distinguished from high abundance nucleic acid molecules in the sample that have a similar nucleotide sequence to the low abundance nucleic acid molecules but do not have one or more nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicing, exon insertions, exon deletions, intron insertions, or other genomic rearrangements, and / or methylated nucleotide bases.

35. 35. The method of claim 34, wherein the copy number of one or more low abundance target nucleotide sequences is quantified relative to the copy number of a high abundance nucleic acid molecule in the sample.

36. The method of any one of claims 1 to 31, wherein the one or more target nucleotide sequences are quantified or enumerated.

37. 37. The method of claim 36, wherein the one or more target nucleotide sequences are quantified or enumerated relative to other nucleotide sequences in the sample or other samples subjected to the same subsequent step.

38. 38. The method of claim 37, wherein the relative copy number of one or more target nucleotide sequences is quantified or enumerated.

39. The method of any one of claims 1 to 31, further comprising diagnosing or predicting a disease state based on said identification.

40. The method of any one of claims 1 to 31, further comprising identifying a genotype or disease predisposition based on said identification.

41. 1. A method for diagnosing or predicting a disease state of a cell or tissue based on identifying the presence or level of a plurality of disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in a biological sample from an individual, comprising: The plurality of markers is in a set comprising between 6 and 12 markers, between 12 and 24 markers, between 24 and 36 markers, between 36 and 48 markers, between 48 and 72 markers, between 72 and 96 markers, or more than 96 markers, and each marker in a given set satisfies one of the following criteria: being present in more than 50% of diseased cell or tissue biological samples from individuals diagnosed with said disease state or above a cut-off level; is absent or below a cutoff level in more than 95% of normal cell or tissue biological samples from individuals not having the disease state; being present in more than 50% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodily fluids, bodily secretions, bodily excretions, or fractions thereof, from an individual diagnosed with said disease state, or above a cutoff level; is absent or below a cutoff level in more than 95% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals not having the disease state; is present at a z-value of greater than 1.65 in said biological sample comprising cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from an individual diagnosed with said disease state; and in said biological sample comprising cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from at least 50% of individuals diagnosed with said disease state, at least 50% of said markers of the set each comprise one or more methylated residues, and / or at least 50% of said markers of the set that are present or above a cutoff level or present at a z-score greater than 1.65 comprise one or more methylated residues; The method comprises: obtaining a biological sample comprising cell-free DNA, RNA, and / or protein from said cells or tissues and one or more other tissues or cells, said biological sample being selected from the group consisting of cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodily fluids, bodily secretions, and bodily excretions, or fractions thereof; fractionating the sample into one or more fractions, at least one of which comprises exosomes, tumor-associated vesicles, other protected, or cell-free DNA, RNA, and / or protein; subjecting the nucleic acid molecules in said one or more fractions to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues; performing at least two enrichment steps for 50% or more of the disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in said fractions and / or by performing nucleic acid amplification steps; performing one or more assays to detect and differentiate the plurality of disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers, thereby identifying their presence or levels in the sample, wherein at least two or three markers are present or above a cutoff level in a marker set comprising 6 to 12 markers, or at least three, four, or five markers are present or above a cutoff level in a marker set comprising 12 to 24 markers, or at least three, four, five, or six markers are present or above a cutoff level in a marker set comprising 24 to 36 markers, or at least three, four, five, or six markers are present or above a cutoff level in a marker set comprising 36 to 48 markers; performing said assay, wherein an individual is diagnosed or predicted with said disease state if at least 4, 5, 6, 7, or 8 markers are present or above a cutoff level, or if at least 6, 7, 8, 9, 10, 11, or 12 markers are present or above a cutoff level in a marker set comprising 48 to 72 markers, or if at least 7, 8, 9, 10, 11, 12, or 13 markers are present or above a cutoff level in a marker set comprising 72 to 96 markers, or if at least 8, 9, 10, 11, 12, 13, or "n" / 12 markers are present or above a cutoff level in a marker set comprising 96 to "n" markers, where "n" > 168. The method comprising:

42. 1. A method for diagnosing or predicting disease status of solid tissue cancer, including colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, breast lobular and ductal carcinoma, uterine endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine carcinosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, based on identifying the presence or level of a plurality of disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in a biological sample from an individual, comprising: The plurality of markers is in a set comprising 48 to 72 total cancer markers, 72 to 96 total cancer markers, or 96 or more total cancer markers, where on average more than one-quarter of such markers in a given set cover each of the major cancers tested, and where each marker in a given set for a given solid tissue cancer meets the following criteria for that solid tissue cancer: being present in more than 50% of biological samples of a given cancer tissue from individuals diagnosed with a given solid tissue cancer or above a cut-off level; being absent or below a cutoff level in more than 95% of normal tissue biological samples from individuals without that given solid tissue cancer; being present in more than 50% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals diagnosed with a given solid tissue cancer, or above a cutoff level; being absent or below a cutoff level in more than 95% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals who do not have the given solid tissue cancer; Present at a z-value of greater than 1.65 in a biological sample including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from an individual diagnosed with a given solid tissue cancer. and In said biological sample comprising cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from at least 50% of individuals diagnosed with a given solid tissue, at least 50% of said markers of the set each comprise one or more methylated residues, and / or at least 50% of said markers of the set that are present or above a cutoff level or present at a z-score greater than 1.65 comprise one or more methylated residues; The method comprises: obtaining a biological sample comprising cell-free DNA, RNA, and / or protein from said cells or tissues and one or more other tissues or cells, said biological sample being selected from the group consisting of cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodily fluids, bodily secretions, and bodily excretions, or fractions thereof; fractionating the sample into one or more fractions, at least one of which comprises exosomes, tumor-associated vesicles, other protected, or cell-free DNA, RNA, and / or protein; subjecting the nucleic acid molecules in the one or more fractions to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues; performing at least two enrichment steps for 50% or more of said given solid tissue cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in said fractionation and / or by performing a nucleic acid amplification step; performing one or more assays to detect and identify said plurality of cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers, thereby identifying their presence or levels in said sample, wherein an individual is diagnosed or predicted with said solid tissue cancer if at least four markers are present or above a cutoff level in a marker set comprising 48 to 72 total cancer markers, or at least five markers are present or above a cutoff level in a marker set comprising 72 to 96 total cancer markers, or at least 6 or "n" / 18 markers are present or above a cutoff level in a marker set comprising 96 to "n" total cancer markers (where "n" > 96 total cancer markers); The method comprising:

43. Each marker in a given set for a given solid tissue cancer satisfies the following criteria for that solid tissue cancer: being present in more than 66% of biological samples of a given cancer tissue from individuals diagnosed with a given solid tissue cancer or above a cutoff level; being absent or below a cutoff level in more than 95% of normal tissue biological samples from individuals without that given solid tissue cancer; being present in greater than 66% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals diagnosed with a given solid tissue cancer, or above a cutoff level; being absent or below a cutoff level in more than 95% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals who do not have the given solid tissue cancer; Present at a z-value of greater than 1.65 in a biological sample including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from an individual diagnosed with a given solid tissue cancer.

43. The method of claim 42, wherein the method is selected by having any one or more of:

44. 1. A method for diagnosing or predicting and identifying the disease state of a particular tissue of most likely origin(s) of the following groups of solid tissue cancers: Group 1 (colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma), Group 2 (lobular and ductal breast carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, squamous cell and adenocarcinoma of the cervix, uterine carcinosarcoma), Group 3 (lung adenocarcinoma, squamous cell lung carcinoma, squamous cell carcinoma of the head and neck), Group 4 (prostate adenocarcinoma, invasive urothelial carcinoma of the bladder), and / or Group 5 (hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma), based on identifying the presence or levels of a plurality of disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in a biological sample from an individual, the method comprising: The plurality of markers is in a set comprising 36-48 group-specific cancer markers, 48-64 group-specific cancer markers, or 64 or more group-specific cancer markers, where on average more than one-third of such markers in a given set cover each of the aforementioned cancers tested within that group, and where each marker in a given set for a given solid tissue cancer meets the following criteria for that solid tissue cancer: being present in more than 50% of biological samples of a given cancer tissue from individuals diagnosed with a given solid tissue cancer or above a cut-off level; being absent or below a cutoff level in more than 95% of normal tissue biological samples from individuals without that given solid tissue cancer; being present in more than 50% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals diagnosed with a given solid tissue cancer, or above a cutoff level; being absent or below a cutoff level in more than 95% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals who do not have the given solid tissue cancer; Present at a z-value of greater than 1.65 in a biological sample including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from an individual diagnosed with a given solid tissue cancer. and In said biological sample comprising cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof from at least 50% of individuals diagnosed with a given solid tissue cancer, at least 50% of said markers of the set each comprise one or more methylated residues, and / or at least 50% of said markers of the set that are present or above a cutoff level or present at a z-score greater than 1.65 comprise one or more methylated residues; The method comprises: obtaining said biological sample comprising cell-free DNA, RNA, and / or protein from said cell or tissue and one or more other tissues or cells, wherein said biological sample is selected from the group consisting of cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodily fluids, bodily secretions, and bodily excretions, or fractions thereof; fractionating the sample into one or more fractions, at least one of which comprises exosomes, tumor-associated vesicles, other protected, or cell-free DNA, RNA, and / or protein; subjecting the nucleic acid molecules in the one or more fractions to bisulfite treatment under conditions suitable for converting unmethylated cytosine residues to uracil residues; performing at least two enrichment steps for 50% or more of said given solid tissue cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in said fractionation and / or by performing a nucleic acid amplification step; performing one or more assays to detect and differentiate said plurality of cancer-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers, thereby identifying their presence or levels in said sample, wherein an individual is diagnosed or predicted to have solid tissue cancer if at least four markers are present or above a cutoff level in a marker set comprising 36-48 group-specific cancer markers, or if at least five markers are present or above a cutoff level in a marker set comprising 48-64 group-specific cancer markers, or if 6 or "n" / 12 markers are present or above a cutoff level in a marker set comprising 64 to "n" total cancer markers (where "n" > 64 group-specific cancer markers); The method comprising:

45. Each marker in a given set for a given solid tissue cancer satisfies the following criteria for that solid tissue cancer: being present in more than 66% of biological samples of a given cancer tissue from individuals diagnosed with a given solid tissue cancer or above a cutoff level; being absent or below a cutoff level in more than 95% of normal tissue biological samples from individuals without that given solid tissue cancer; being present in greater than 66% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals diagnosed with a given solid tissue cancer, or above a cutoff level; being absent or below a cutoff level in more than 95% of biological samples, including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from individuals who do not have the given solid tissue cancer; Present at a z-value of greater than 1.65 in a biological sample including cells, serum, blood, plasma, amniotic fluid, sputum, urine, body fluids, body secretions, body excretions, or fractions thereof, from an individual diagnosed with a given solid tissue cancer.

45. The method of claim 44, wherein the method is selected by having any one or more of:

46. 46. ​​The method of any one of claims 41 to 45, wherein the at least two enrichment steps comprise two or more of the following steps: capturing or isolating exosomes or extracellular vesicles or other protected markers, capturing or isolating platelet fractions, capturing or isolating circulating tumor cells, capturing or isolating RNA-containing complexes, capturing or isolating cfDNA nucleosomes or differentially modified cfDNA-histone complexes, capturing or isolating protein targets or protein target complexes, capturing or isolating autoantibodies, capturing or isolating cytokines, capturing or isolating methylated cfDNA, capturing or isolating marker-specific DNA, cDNA, miRNA, lncRNA, ncRNA, or mRNA, or amplified complements, by hybridization to complementary capture probes in solution, on magnetic beads, or on microarrays, using DNA polymerase, reverse transcriptase, DNA ligase, RNA ligase, DNA repair enzyme, RNase, RNase H2, endonuclease, restriction endonuclease, exonuclease, CRISPR, DNA glycosylase, or combinations thereof. and linearly or exponentially amplifying miRNA markers, non-coding RNA markers (lncRNA markers and ncRNA markers), mRNA markers, exon markers, splice variant markers, translocation markers, or copy number variation markers via polymerase extension reactions, polymerase chain reactions, bisulfite-methyl-specific polymerase chain reactions, reverse transcription reactions, bisulfite-methyl-specific ligation reactions, and / or ligation reactions, mutation markers, or bisulfite conversion DNA fragments. one or more target regions comprising a DNA methylation marker are subjected to a polymerase extension reaction, a polymerase chain reaction, a bisulfite-methyl-specific polymerase chain reaction, a reverse transcription reaction, a bisulfite-methyl-specific ligation reaction, and / or a ligation reaction using a DNA polymerase, a reverse transcriptase, a DNA ligase, an RNA ligase, a DNA repair enzyme, an RNase, an RNase H2, an endonuclease, a restriction endonuclease, an exonuclease, a CRISPR, a DNA glycosylase, or a combination thereof;Selectively amplifying, linearly or exponentially, while suppressing amplification of a target region containing the wild-type sequence or the bisulfite-converted unmethylated sequence or its complementary sequence; preferentially extending, ligating, or amplifying one or more primers or probes whose 3'-OH termini are free in an enzyme- and sequence-dependent process; using one or more blocking oligonucleotide primers containing one or more mismatched bases at the 3' terminus or containing one or more nucleotide analogs and a blocking group at the 3' terminus, under conditions that prevent polymerase extension or ligation, during said reaction in which the target-specific primer or probe is hybridized in a base-specific manner to the wild-type sequence or the bisulfite-converted unmethylated sequence or its complementary sequence;

47. 47. The method of any one of claims 41 to 46, wherein the one or more assays for detecting and distinguishing the plurality of disease-specific and / or cell / tissue-specific DNA, RNA, or protein markers comprise one or more of the following: Quantitative real-time PCR (qPCR), reverse transcriptase-polymerase chain reaction (RTPCR), bisulfite qPCR, digital PCR (dPCR), bisulfite dPCR, ligation detection, ligase chain reaction, restriction endonuclease cleavage, DNA or RNA nuclease cleavage, microarray hybridization, peptide array binding, antibody array, mass spectrometry, liquid chromatography-tandem mass spectrometry (LC-MS / MS), capillary or gel electrophoresis, chemiluminescence, fluorescent optical methods, DNA sequencing, bisulfite conversion-DNA sequencing, RNA sequencing, proximity ligation, proximity PCR, methods involving immobilization of antibody-target complexes, methods involving immobilization of aptamer-target complexes, immunoassays, methods involving Western blot assays, methods involving enzyme-linked immunosorbent assays (ELISAs), methods involving high-throughput microarray-based enzyme-linked immunosorbent assays (ELISAs), or methods involving high-throughput flow cytometry-based enzyme-linked immunosorbent assays (ELISAs).

48. 48. The method of any one of claims 41-47, wherein the one or more cut-off levels of the one or more assays for detecting and distinguishing the plurality of disease-specific and / or cell / tissue-specific DNA, RNA, or protein markers comprises one or more of the following calculations, comparisons, or determinations in the one or more marker assays comparing samples from the diseased versus normal individuals: a ΔCt value for the marker greater than 2, a ΔCt value for the marker greater than 4, a ratio of marker-specific signals detected greater than 1.5, a ratio of marker-specific signals detected greater than 3, a ratio of marker concentrations greater than 1.5, a ratio of marker concentrations greater than 3, the listed marker-specific signals differ by more than 20%, the listed marker-specific signals differ by more than 50%, a marker-specific signal from a given disease sample that is greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, or greater than 98% of the same marker-specific signal from a set of normal samples, or a marker-specific signal from a given disease sample that has a z-score of greater than 1.03, greater than 1.28, greater than 1.65, greater than 1.75, greater than 1.88, or greater than 2.05 compared to the same marker-specific signal from a set of normal samples.

49. 1. A two-step method for diagnosing or predicting a disease state of a cell or tissue based on identifying the presence or level of a plurality of disease-specific and / or cell / tissue-specific DNA, RNA, and / or protein markers in a biological sample from an individual, comprising: The two-step method comprises: obtaining a biological sample, said biological sample comprising exosomes, tumor-associated vesicles, markers in other protected states, cell-free DNA, RNA, and / or proteins derived from cells or tissues in a potential disease state and one or more other tissues or cells, said biological sample being selected from the group consisting of cells, serum, blood, plasma, amniotic fluid, sputum, urine, bodily fluids, bodily secretions, and bodily excretions, or fractions thereof; applying a first step to said biological samples with an overall sensitivity of greater than 80%, an overall specificity of greater than 90%, or an overall Z-score of greater than 1.28 to identify individuals who are more likely to be diagnosed or predicted with said disease state; applying a second step to biological samples from individuals identified in said first step with an overall specificity of greater than 95% or an overall Z-score of greater than 1.65 in order to diagnose or predict said disease state in said individuals; Including, The method, wherein applying the first step and / or applying the second step is performed using a method according to any one of claims 41 to 44.

50. 50. The method of any one of claims 41 to 49, wherein the disease state is solid tissue cancer comprising colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, lobular and ductal carcinoma of the breast, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine carcinosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and wherein at least 50% of the markers of the set each comprise one or more methylated cytosine residues in a CpG sequence or the complement of one or more methylated cytosine residues in a CpG sequence selected from the list of Figure 56.

51. 50. The method of any one of claims 41-49, wherein the disease state is solid tissue cancer comprising colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, lobular and ductal carcinoma of the breast, endometrial carcinoma of the uterine corpus, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine carcinosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and wherein at least 50% of the markers of the set each comprise one or more methylated residues in one or more chromosomal subregions selected from the list of Figure 57.

52. and wherein the disease state is solid tissue cancer including colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, lobular and ductal carcinoma of the breast, endometrial carcinoma of the uterine corpus, ovarian serous cystadenocarcinoma, squamous cell carcinoma and adenocarcinoma of the cervix, uterine carcinosarcoma, lung adenocarcinoma, squamous cell carcinoma of the lung, squamous cell carcinoma of the head and neck, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and wherein the one or more markers of the set are selected from the group consisting of: ID, gene ID): hsa-mir-21, MIR21, hsa-mir-182, MIR182, hsa-mir-454, MIR454, hsa-mir-96, MIR96, hsa-mir-183, MIR183, hsa-mir-549, MIR549, hsa-mir-301 ａ , MIR301A, hsa-mir-548f-1, MIR548F1, hsa-mir-301b, MIR301B, hsa-mir-103-1, MIR1031, hsa-mir-18 ａ 53. The method of any one of claims 41 to 49, comprising one or more miRNA sequences selected from the group consisting of: MIR18A, hsa-mir-147b, MIR147B, hsa-mir-4326, MIR4326, andhsa-mir-573, MIR573, or one or more lncRNA or ncRNA sequences selected from the list of Figure 53.

53. 50. The method of any one of claims 41-49, wherein the disease state is solid tissue cancer comprising colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, lobular and ductal breast carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine carcinosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and wherein the one or more markers of the set comprise one or more exonic RNA sequences selected from the list of Figure 54.

54. 50. The method of any one of claims 41-49, wherein the disease state is solid tissue cancer, including colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, lobular and ductal carcinoma of the breast, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine carcinosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and wherein the set of one or more markers comprises one or more mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products selected from the list of Figure 55 or from the group consisting of: (Protein Name, UniProt ID) Uncharacterized protein C19orf48, Q6RUI8, protein FAM72B, Q86X60, protein FAM72D, Q6L9T8, hydroxyacylglutathione hydrolase-like protein, Q6PII5, putative methyltransferase NSUN5, Q96P11, RNA pseudouridylate synthase domain-containing protein 1, Q9UJJ7, collagen triple helix repeat-containing protein 1 , Q96CG8, interleukin-11, P20809, stromelysin-2, P09238, matrix metalloproteinase-9, P14780, podocan-like protein 1, Q6PEZ8, putative peptide YY-2, Q9NRI6, osteopontin, P10451, sulfhydryl oxidase 2, Q6ZRP7, glypican-2, Q8N158, macrophage migration inhibitory factor, P14174, peptidyl-prolyl cis-trans isomerase A, P62937, and calreticulin, P27797.

55. 50. The method of any one of claims 41-49, wherein the disease state is solid tissue cancer including colorectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, lobular and ductal carcinoma of the breast, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine carcinosarcoma, lung adenocarcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, prostate adenocarcinoma, invasive bladder urothelial carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and wherein the set of one or more markers comprises one or more mutations, insertions, deletions, copy number alterations, or expression alterations of a gene selected from the group consisting of TP53 (tumor protein p53), TTN (titin), MUC16 (mucin 16), and KRAS (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog).

56. 50. The method of any one of claims 41 to 49, wherein the disease state is colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, and at least 50% of the markers of the set each comprise one or more methylated cytosine residues of a CpG sequence or the complement of one or more methylated cytosine residues of a CpG sequence selected from the list of Figure 44 or Figure 59.

57. 50. The method of any one of claims 41-49, wherein the disease state is colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, and at least 50% of the markers of the set comprise one or more methylated residues in one or more chromosomal subregions selected from the list of Figure 45 or Figure 60, respectively.

58. 50. The method of any one of claims 41-49, wherein the disease state is colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, and the set of one or more markers comprises one or more miRNA sequences selected from the list in Figure 39, hsa-mir-624, MIR624, or one or more lncRNA or ncRNA sequences selected from the list in Figure 40 or the group consisting of: [Gene ID, coordinates (GRCh38)] ENSEMBL ID: LINC01558, chr6: 167784537-167796859 and ENSG00000146521.

8.

59. 50. The method of any one of claims 41-49, wherein the disease state is colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, and the one or more markers of the set comprise one or more exon RNA sequences selected from the list in Figure 41 or Figure 58.

60. 50. The method of any one of claims 41-49, wherein the disease state is colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, and the set of one or more markers comprises one or more mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products selected from the list of Figure 42, Figure 43, or the group consisting of: (gene symbol, chromosome band, gene title, UniProt ID) SELE, 1q22-q25, selectin E, P16581, OTUD4, 4q31.21, OTU domain containing 4, Q01804, BPI, 20q11.23, bactericidal / permeability enhancing protein, P17213, ASB4, 7q21-q22, ankyrin repeat and SOCS box containing 4, Q9Y574, C6orf123, 6q27, chromosome 6 open reading frame 123, Q9Y6Z2, KPNA3, 13q14.3, karyopherin α3 (importin α4), O00505, and NUP98, 11p15, nucleoporin 98 kDa, P52948, or (protein name, UniProt ID) Bactericidal permeability-increasing protein (BPI) (CAP57), P17213.

61. and wherein the disease state is colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, or esophageal cancer, and the set of one or more markers is APC (APC, regulator of WNT signaling pathway), ATM (ATM serine / threonine kinase), CSMD1 (CUB and Sushi multiple domain 1), DNAH11 (dynein axoneme heavy chain 11), DST (dystonin), EP400 (E1A binding protein p400), FAT3 (FAT atypical cadherin 3), FAT4 (FAT atypical cadherin 4), FLG (filaggrin), GLI3 (GLI family zinc finger 3), KRAS (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), LRP1B (LDL receptor-related protein 1), or 50. The method of any one of claims 41-49, comprising one or more mutations, insertions, deletions, copy number changes, or expression alterations in a gene selected from the group consisting of PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), RYR2 (ryanodine receptor 2), SYNE1 (spectrin repeat-containing nuclear membrane protein 1), TP53 (tumor protein 53), TTN (titin), and UNC13C (unc-13 homolog C).

62. 50. The method of any one of claims 41-49, wherein the disease state is lobular and ductal carcinoma of the breast, endometrial carcinoma of the uterine corpus, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, and wherein at least 50% of the markers of the set each comprise one or more methylated cytosine residues in a CpG sequence or the complement of one or more methylated cytosine residues in a CpG sequence selected from the list of Figure 61.

63. 50. The method of any one of claims 41-49, wherein the disease state is lobular and ductal carcinoma of the breast, endometrial carcinoma of the uterine corpus, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, and wherein at least 50% of the markers of the set each comprise one or more methylated residues in one or more chromosomal subregions selected from the list of Figure 62.

64. 50. The method of any one of claims 41 to 49, wherein the disease state is lobular and ductal carcinoma of the breast, endometrial carcinoma of the uterine corpus, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, and wherein the set of one or more markers comprises one or more miRNA sequences selected from the group consisting of the following (mir ID, gene ID): hsa-mir-1265, MIR1265.

65. 50. The method of any one of claims 41 to 49, wherein the disease state is lobular and ductal breast carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, and the set of one or more markers comprises one or more exonic RNA sequences selected from the group consisting of (exon position, gene): chr2:179209013-179209087:+,OSBPL6, chr2:179251788-179251866:+,OSBPL6, and chr2:179253736-179253880:+,OSBPL6.

66. 50. The method of any one of claims 41-49, wherein the disease state is lobular and ductal carcinoma of the breast, endometrial carcinoma of the endometrium, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, and the set of one or more markers comprises one or more mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products selected from the group consisting of: (Gene Symbol, Chromosome Band, Gene Title, UniProt ID) RSPO2, 8q23.1, R-spondin 2, Q6UXX9, KLC4, 6p21.1, Kinesin light chain 4, Q9NSK0, and GLRX, 5q14, Glutaredoxin (thioltransferase), P35754, or (Protein Name, UniProt ID) R-spondin-2 (tectal plate specific spondin-2) (hRspo2), Q6UXX9.

67. 50. The method of any one of claims 41-49, wherein the disease state is lobular and ductal breast carcinoma, endometrial carcinoma, ovarian serous cystadenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, or uterine carcinosarcoma, and the set of one or more markers comprises one or more mutations, insertions, deletions, copy number changes, or expression changes in a gene selected from the group consisting of PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) and TTN (titin).

68. 50. The method of any one of claims 41 to 49, wherein the disease state is lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, and at least 50% of the markers of the set each comprise one or more methylated cytosine residues in a CpG sequence, or the complement of one or more methylated cytosine residues in a CpG sequence selected from the list of Figure 63.

69. 50. The method of any one of claims 41-49, wherein the disease state is lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, and at least 50% of the markers of the set each comprise one or more methylated residues in one or more chromosomal subregions selected from the list of Figure 64.

70. 50. The method of any one of claims 41 to 49, wherein the disease state is lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, and the set of one or more markers comprises one or more miRNA sequences selected from (mir ID, gene ID) hsa-mir-28, MIR28.

71. and wherein the disease state is lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, and the set of one or more markers is (exon position, gene): chr2: chr1: 93307721-93309752: -, FAM69A, chr1: 93312740-93312916: -, FAM69A, chr1: 93316405-93316512: -, FAM69A, chr1: 93341853-93342152: -, FAM69A, chr1: 93426933-93427079: -, FAM69A, chr7: 402215 54-40221627:+, C7orf10, chr7:40234539-40234659:+, C7orf10, chr8:22265823-22266009:+, SLC39A14, chr8:22272293-22272415:+, SLC39A14, chr14:39509936-39510091:-, SEC23A, and chr14:39511990-39512076:-, SEC23A.

72. 50. The method of any one of claims 41-49, wherein the disease state is lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, and the set of one or more markers comprises one or more mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products selected from the group consisting of: (gene symbol, chromosome band, gene title, UniProt ID) STRN3, 14q13-q21, striatin, calmodulin-binding protein 3, Q13033, LRRC17, 7q22.1, leucine-rich repeat-containing 17, Q8N6Y2, FAM69A, 1p22, family with sequence similarity 69, member A, Q5T7M9, ATF2, 2q32, activating transcription factor 2, P15336, BHMT, 5q14.1, betaine-homocysteine ​​S-methyltransferase, Q93088, ODZ3 / TENM3, 4q34.3-q35.1, teneurin transmembrane protein 3, Q9P273, and ZFHX4, 8q21.11, zinc finger homeobox 4, Q86UP3, or (Protein Name, UniProt ID) Leucine-rich repeat-containing protein 17 (p37NB), Q8N6Y2.

73. and wherein the disease state is lung adenocarcinoma, lung squamous cell carcinoma, or head and neck squamous cell carcinoma, and the set of one or more markers is selected from the group consisting of CSMD3 (CUB and Sushi multiple domain 3), DNAH5 (dynein axoneme heavy chain 5), FAT1 (FAT atypical cadherin 1), FLG (filaggrin), KRAS (Ki-ras2, Kirsten rat sarcoma viral oncogene homolog), LRP1B (LDL receptor-related protein 1B), MUC16 (mucin 16, cell surface associated), PCLO (piccolo presynaptic cytosolic protein), PK 50. The method of any one of claims 41 to 49, comprising one or more mutations, insertions, deletions, deletions, copy number changes, or expression changes in a gene selected from the group consisting of HD1L1 (PKHD1-like 1), RELN (reelin), RYR2 (ryanodine receptor 2), SI (sucrase-isomaltase), SYNE1 (spectrin repeat-containing nuclear membrane protein 1), TP53 (tumor protein p53), TTN (titin), USH2A (usshalin), and XIRP2 (xin actin-binding repeat-containing 2).

74. 50. The method of any one of claims 41 to 49, wherein the disease state is prostate adenocarcinoma or invasive bladder urothelial carcinoma, and at least 50% of the markers of the set each comprise one or more methylated cytosine residues of a CpG sequence, or the complement of one or more methylated cytosine residues of a CpG sequence selected from the list of Figure 65.

75. 50. The method of any one of claims 41-49, wherein the disease state is prostate adenocarcinoma or invasive bladder urothelial carcinoma, and at least 50% of the markers of the set each comprise one or more methylated residues in one or more chromosomal subregions selected from the list of Figure 66.

76. 50. The method of any one of claims 41 to 49, wherein the disease state is prostate adenocarcinoma or invasive bladder urothelial carcinoma, and the set of one or more markers comprises one or more miRNA sequences selected from the group consisting of the following (mir ID, gene ID): hsa-mir-491, MIR491 and hsa-mir-1468, MIR1468, or one or more lncRNA or ncRNA sequences selected from the group consisting of the following [Gene ID, coordinates (GRCh38), ENSEMBL ID]: AC007383.3, chr2:206084605-206086564, ENSG00000227946.1, and LINC00324, chr17:8220642-8224043, ENSG00000178977.

3.

77. 50. The method of any one of claims 41 to 49, wherein the disease state is prostate adenocarcinoma or invasive bladder urothelial carcinoma, and the one or more markers of the set comprise one or more exonic RNA sequences selected from: (exon position, gene) chr21:45555942-45556055:+,C21orf33.

78. 50. The method of any one of claims 41 to 49, wherein the disease state is prostate adenocarcinoma or invasive bladder urothelial carcinoma, and the set of one or more markers comprises one or more mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies to protein products selected from: (Gene Symbol, Chromosome Band, Gene Title, UniProt ID) PMM1, 22q13, Phosphomannomutase 1, Q92871.

79. 50. The method of any one of claims 41-49, wherein the disease state is prostate adenocarcinoma or invasive bladder urothelial carcinoma, and the set of one or more markers comprises one or more mutations, insertions, deletions, copy number changes, or expression changes of a gene selected from the group consisting of BAGE2 (BAGE family member 2), DNM1P47 (dynamin 1 pseudogene 47), FRG1BP (regional gene 1 family member B, pseudogene), KRAS (Ki-ras2, Kirsten rat sarcoma viral oncogene homolog), RP11-156P1.3, TTN (titin), and TUBB8P7 (tubulin beta 8 class VIII pseudogene 7).

80. 50. The method of any one of claims 41-49, wherein the disease state is hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and at least 50% of the markers of the set each comprise one or more methylated cytosine residues of a CpG sequence, or the complement of one or more methylated cytosine residues of a CpG sequence selected from the list of Figure 70.

81. 50. The method of any one of claims 41-49, wherein the disease state is hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and at least 50% of the markers of the set each comprise one or more methylated residues in one or more chromosomal subregions selected from the list of Figure 71.

82. 50. The method of any one of claims 41-49, wherein the disease state is hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and the set of one or more markers comprises one or more miRNA sequences selected from: (mir ID, gene ID) hsa-mir-132, MIR132, or one or more lncRNA or ncRNA sequences selected from the list in Figure 67.

83. 50. The method of any one of claims 41-49, wherein the disease state is hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and the one or more markers of the set comprise one or more exon RNA sequences selected from the list in Figure 68.

84. The disease state is hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and the set of one or more markers is selected from the list in FIG. 69 or the following (protein name, UniProt 50. The method of any one of claims 41-49, comprising measuring one or more mRNA sequences, protein expression levels, protein product concentrations, cytokines, or autoantibodies against protein products selected from the group consisting of: Gelsolin (AGEL) (actin depolymerizing factor) (ADF) (brevin), P06396, Proneuregulin-2, O14511, CD59 glycoprotein (1F5 antigen) (20 kDa homologous restriction factor) (HRF-20) (HRF20) (MAC-inhibitory protein) (MAC-IP) (MEM43 antigen) (membrane attack complex inhibitor) (MACIF) (membrane inhibitor of reactive lysis) (MIRL) (protectin) (CD antigen CD59), P13987, and divergent protein kinase 2B (deficient in autism-related protein 1), Q9H7Y0.

85. 50. The method of any one of claims 41-49, wherein the disease state is hepatocellular carcinoma, pancreatic ductal adenocarcinoma, or gallbladder adenocarcinoma, and the set of one or more markers comprises one or more mutations, insertions, deletions, copy number changes, or expression alterations in a gene selected from the group consisting of KRAS (Ki-ras2, Kirsten rat sarcoma viral oncogene homolog), MUC16 (mucin 16, cell surface associated), MUC4 (mucin 4, cell surface associated), TP53 (tumor protein p53), and TTN (titin).

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