A method for identifying one or more nucleic acid molecules containing a target nucleotide sequence in a sample

Abstract

The present invention relates to methods and devices for identification and quantification, including 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 insertions, exon deletions, intron insertions, or other rearrangements and / or methylated nucleotide bases at the genomic level.

Description

[0001] This application is a divisional of Chinese Patent Application No. 201580066531.3, filed October 8, 2015, entitled "Methods for Identifying and Relative Quantifying Nucleic Acid Sequence Expression, Splice Variants, Translocations, Copy Number, or Methylation Changes Using Combined Nucleases, Ligation, and Polymerase Reactions for Carryover Prevention." The original application is the national stage application of International Application No. PCT / US2015 / 054759, which claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 061,376, filed October 8, 2014, and U.S. Provisional Patent Application Serial No. 62 / 103,894, filed January 15, 2015, which are incorporated by reference herein in their entireties. TECHNICAL FIELD

[0002] The present invention relates to methods for identifying and quantifying nucleic acid sequences, expression, splice variants, translocations, copy number, and / or methylation changes using combined nuclease, ligation, and polymerase reactions for carryover prevention. BACKGROUND

[0003] Blood carries oxygen, nutrients, and physiological signals to every cell in the body, while providing immunity and protection against outside pathogens. But equally, the ability of blood to disseminate nutrients also allows disease to spread, as cancer cells metastasize to the liver, Ebola virus destroys capillaries, streptococcus pyogenes liquefies abscesses, or HIV evades detection within the unique CD4 cells that are meant to eliminate the infection.

[0004] Similar to dissemination through blood, the ubiquity of pathogens and cancer also creates opportunities for identification and early detection - allowing physicians to better treat patients and manage patient care. The development of AIDS treatment has paralleled the improvement of nucleic acid diagnostics, from the initial reverse transcription PCR assays, to protect national blood supplies, to sequencing of drug resistant variants, to RT-PCR quantification of viral load to determine treatment efficacy over time. To date, those infected have not been cured, but the nuanced diagnostic tools have guided treatment decisions, epidemiological decisions, and political decisions to contain this global infectious disease.

[0005] Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries. Cancer has now become the largest cause of death worldwide, with an estimated 8.2 million deaths from cancer in 2012. In the next two decades, cancer cases worldwide are predicted to increase by 75% and reach nearly 25 million cases. A recent report by the World Health Organization concluded that "the global war on cancer will not be won by a single treatment. Effective prevention measures are urgently needed to prevent the cancer crisis." Detecting early stage cancer in blood is the best effective prevention measure. This would save lives by enabling earlier and better treatment and reduce cancer care costs.

[0006] Plasma or serum from cancer patients contains nucleic acids released from cancer cells undergoing abnormal physiological processes. These nucleic acids have shown diagnostic utility (Diaz and Bardelli, J Clin Oncol 32:579-586 (2014); Bettegowda et al., Sci Transl Med 6:224 (2014); Newman et al., Nat Med 20:548-554 (2014); Thierry et al., Nat Med 20:430-435 (2014)). Another source of nucleic acids is within circulating tumor cells (CTCs), but early stage and most local tumors shed very few CTCs per ml to no CTCs. Normal plasma or serum contains nucleic acids released from normal cells undergoing normal physiological processes (i.e., exosome secretion, apoptosis). Additional nucleic acid release can occur under conditions of stress, inflammation, infection, or injury.

[0007] The challenge in developing reliable diagnostic and screening tests is to distinguish those markers produced by tumors that are indicative of disease (e.g., early stage cancer) from the presence of the same markers produced by normal tissue, which would result in false positive signals. There is also a need to balance the number of markers examined and the cost of the test in a determination with specificity and sensitivity. 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 differ from each other as much as they do from breast, prostrate, or other epithelial cancers (TCGA "Comprehensive Molecular Characterization of Human Colon and Rectal Cancer Nature 487:330-337 (2014)). Moreover, the few markers they share (e.g., KRAS mutations) are also present in multiple cancer types, hampering the ability to pinpoint the tissue of origin. For early stage cancer detection, nucleic acid assays should primarily serve as a screening tool, requiring a secondary diagnostic follow-up to be available (e.g., colonoscopy for colorectal cancer).

[0008] The need to reliably quantify mutations, promoter methylation, or DNA or RNA copy number from very small numbers of initial cells (i.e., from CTCs), or the added biological complexity when cancer signals are from cell-free DNA (cfDNA) in the blood and diluted by excess nucleic acid produced by normal cells or inadvertently released from normal blood cells during sample processing (Mateo et al., Genome Biol 15:448 (2014)) further complicates the problem.

[0009] Likewise, similar problems in identifying rare targets are encountered when using nucleic acid-based technologies to detect infectious diseases directly in blood. In short, pathogens can be present at 1 or fewer colony forming units (cfu) / ml and / or there are many potential pathogens and sequence variations responsible for virulence or drug resistance. While these problems are exemplified with cancer, it should be recognized that the solutions described are equally applicable to infectious diseases.

[0010] Continuous diagnostics require continuous diagnostic testing.

[0011] Most current efforts in molecular diagnostics for cancer have focused on: (i) prognostic and predictive genomics, such as identifying genetic mutations in cancer susceptibility genes such as BrCA1, BrCA2 (Ford et al. Am J Hum Genet 62:676-689 (1998)), (ii) personalized therapy, such as mutations in the EGFR gene to guide individualized drug use (Sequist and Lynch, Ann Rev Med, 59:429-442 (2008), and (iii) recurrence monitoring, such as detecting KRAS mutations in patients who have developed resistance to drug therapy (Hiley et al., Genome Biol 15:453 (2014); Amado et al., J Clin Oncol 26:1626-1634 (2008)). However, this misses much of the continuum of cancer molecular diagnostics: (i) more frequent screening of those with a family history, (ii) screening to detect early disease, and (iii) monitoring treatment efficacy. To address these three unmet needs, a new metric for blood-based testing is presented herein, called "cancer marker load," analogous to viral load.

[0012] DNA sequencing provides the ultimate ability to distinguish all nucleic acid changes associated with disease. However, the method still requires multiple up-front samples and template preparation, and is not necessarily cost effective. DNA microarrays can provide a wealth of information about multiple sequence variants, such as SNPs or different RNA expression levels, and cost less than sequencing; however, they are less suitable for obtaining highly quantitative results, nor are they suitable for detecting low abundance mutations. The other side of the coin is TaqMan TM reactions, which provide real-time quantification of known genes, but are less suitable for distinguishing multiple sequence variants or low abundance mutations.

[0013] It is critical to match each unmet diagnostic need with the appropriate diagnostic test - combining the different goals of high sensitivity (i.e., few false negatives) and high specificity (i.e., few false positives) at low cost. For example, designing TaqMan TMDirect sequencing of EGFR exons from tumor biopsies to determine treatment for non-small cell lung cancer (NSCLC) is clearly more accurate and cost effective compared to probes. The most sensitive technique for detecting point mutations, BEAMing (Dressman et al., Proc Natl Acad Sci USA 100:8817-8822 (2003)), relies on prior knowledge of the mutation sought and is thus best suited for monitoring disease recurrence, not early detection. Likewise, simple quantitative reverse transcription PCR assays for monitoring blood levels of Bcr-Abl translocation in CML patients treated with Gleevec (Jabbour et al., Cancer 112:2112-2118 (2008)) are preferred over sequencing the entire genomic DNA in 1 ml of blood (90 million cells x 3 GB = 270 million Gb raw data).

[0014] Each of the cell-free DNA (cfDNA) isolated from NSCLC patients was sequenced using 2.1 Gb to provide 10,000 fold coverage of 125 kb targeted DNA (Kandoth et al. Nature 502:333-339 (2013)). This approach correctly identified mutations present in the matched tumor, but only covered 50% of stage 1 tumors. The approach has promise for NSCLC where samples average 5 to 20 mutations / Mb, however, is not cost effective for other cancers such as breast and ovarian cancer which average less than 1 to 2 mutations / Mb. The pre-connection, amplification and / or capture steps currently required for highly accurate targeted deep sequencing are still more complex than multiplex PCR-TaqMan TM or PCR-LDR assays.

[0015] A comprehensive data analysis of over 600 colorectal cancer samples that takes into account tumor heterogeneity, tumor clusters, and biological / technical false positives (in the range of 3% to 10% per individual marker) indicates that the optimal early detection screening for colorectal cancer requires at least 5 to 6 positive markers out of 24 test 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 N.E. Genome Biol 15:452 (2014); Hiley et al., Genome Biol 15:453 (2014)); Esserman et al. Lancet Oncol 15:e234-242 (2014)). Furthermore, the marker distribution moves into different tumor clades, e.g., some tumors are heavily methylated, while others are almost not methylated and indistinguishable from age-related methylation of adjacent tissue. Therefore, a multi-dimensional approach using a combination of 3-5 groups of mutation, methylation, miRNA, mRNA, copy number, alternative splicing, or translocation markers is needed to obtain sufficient coverage of all different tumor clades. Similar to non-invasive prenatal screening for chromosomal trisomies, the actual markers scored in cancer screening are only second to those positive markers that are precisely quantified in plasma based on sequencing of cfDNA or ligation detection of random fragments of cfDNA (Benn et al., Ultrasound Obstet Gynecol. 42(1): 15-33 (2013); Chiu et al., Proc Natl Acad Sci U S A 105:20458-20463 (2008); Juneau et al., Fetal Diagn Ther. 36(4) (2014)).

[0016] Technical challenges for the development of diagnostic tests for cancer.

[0017] Diagnostic tests aimed at finding very rare or low-abundance mutant sequences face potential false-positive signals arising from (i) polymerase errors in replicating the wild-type target, (ii) DNA sequencing errors, (iii) misligations to the wild-type target, (iii) PCR products that are not target-dependent, and (iv) carry-over contamination of PCR products from previous positive samples. The profound clinical implications of a positive test result in screening for cancer require that such tests use all means possible to almost eliminate false positives.

[0018] Important to the concept of nucleic acid testing is the selective amplification or purification of the desired cancer-specific markers separate from markers that are identical or very similar to normal cells. These methods 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.

[0019] The success of PCR-LDR has been previously demonstrated using 4 primer binding regions to ensure sensitivity and specificity. The desired regions are amplified using PCR primer pairs or even PCR primer string pairs followed by amplification using orthogonal nested LDR primer pairs for detection. One advantage of using PCR-LDR is the ability to perform proportional PCR amplification of multiple fragments to enrich low copy targets followed by direct identification of cancer-specific mutations using quantitative LDR. Biofire / bioMerieux has developed a similar technology called "film array" where the initial multiplex PCR reaction products are redistributed into individual wells followed by nested real-time PCR with SYBR green dye detection.

[0020] CTC affinity purification 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. Enrichment of these tumor-specific fractions from blood enables copy number quantification and simplifies screening and validation assays.

[0021] The last method, spatial dilution of the sample, has been employed in digital PCR and a similar method to it is called BEAMing (Vogelstein and Kinzler, Proc Natl Acad Sci U S A. 96(16):9236-41 (1999); Dressman et al., Proc Natl Acad Sci USA 100:8817-8822 (2003)). The rationale of digital PCR is to overcome the limitation of enzyme discrimination ability when the sample contains very few target molecules with a known mutation containing 1,000 to 10,000-fold excess of wild-type DNA. By diluting the input DNA into 20,000 or more microdroplets or beads with less than one target molecule distributed per microdroplet, DNA can be amplified by PCR and then detected by probe hybridization or TaqMan TMReaction detection, in essence, gives a 0 / 1 digital score. The method described is currently the most sensitive for finding point mutations in plasma, but it does require prior knowledge of the mutations being scored and individual digital dilution for each mutation, which would exhaust the entire sample to score only a few mutations.

[0022] Real-time PCR and microfluidic instrumentation

[0023] Numerous PCR assays / microfabricated devices have been designed for rapid detection of pathogens and disease-associated translocations and mutations. Each assay / hardware combination has specific strengths, but when combined with the practical problem of multiple and multiplexed markers needed for cancer detection, the flexibility of PCR-LDR with microfluidics offers certain advantages.

[0024] There is a need for seamless integration of instrumentation, assay design, and microfluidic construction. Some PCR instruments use real-time fluorescence or end-point fluorescence to quantify initial template molecules by cycling chambers, wells, or droplets through different temperatures. Other instruments also include addressable microfluidic plates for real-time PCR detection. However, the high cost of the instruments and consumables limits the widespread use of these machines in clinical applications.

[0025] In a different construction, called continuous-flow PCR, the reaction mixture moves through channels arranged in a radial pattern and flows past heating elements at fixed temperatures. This construction allows the entire amplification reaction to be completed in a few minutes and is ideal for capillary separation and readout. For ligase detection reactions, readout can be achieved by using LDR-FRET or electronic detection. In LDR-FRET, one primer has a donor and the other has an acceptor group, and after ligation they form a hairpin. This allows counting of single ligation events for a highly quantitative readout of input DNA copy number. Alternatively, by appending gold nanoparticles on each primer, the ligation product will contain two nanoparticles and they can be distinguished using electronic readout.

[0026] With varying degrees of automation in mind, the methods described herein are guided by the principles of "modularity" and "scalability." First, the methods should be divided into modular steps that can be optimized initially on separate instruments. For example, the device can be composed of a first module for purification of DNA from plasma cfDNA and RNA from exosomes, a second module for multiplexed reverse transcription and / or limited amplification of various targets, and a third module for production and detection of ligation products. This modular construction allows swapping improved modules that keep up with technological developments. For a modular approach to running, it is critical that the products from one module can be seamlessly moved into the next module without the worry of leakage and leftover contamination.

[0027] Second, the modular design should be amenable to scalable manufacturing in large quantities at low cost. Manufacturing costs and how to deposit primers / reagents / samples into the device must be considered.

[0028] The present invention is directed to overcoming these and other deficiencies in the art. SUMMARY

[0029] A first aspect of the present invention relates to a method for identifying one or more nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules in the sample or other samples by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues. The method includes providing a sample containing one or more nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues; and contacting the sample with one or more enzymes capable of digesting a deoxyuracil (dU)-containing nucleic acid molecule present in the sample. One or more primary oligonucleotide primer sets are provided, each primary oligonucleotide primer set including (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a sequence 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 by the first primary oligonucleotide primer. The contacted sample is blended with the one or more primary oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a polymerase chain reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles including a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a primary extension product comprising the target nucleotide sequence or a complement thereof. The method further includes blending the primary extension product with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture. Each oligonucleotide probe set includes (a) a first oligonucleotide probe having a target nucleotide sequence-specific portion and (b) a second oligonucleotide probe having a target nucleotide sequence-specific portion, and wherein the first and second oligonucleotide probes of the probe set are configured to hybridize to the complementary target nucleotide sequence of the primary extension product in a base-specific manner. The first and second oligonucleotide probes of the one or more oligonucleotide probe sets are ligated together to form a ligation product sequence in the ligation reaction mixture, and the ligation product sequence is detected and distinguished in the sample to identify the presence of one or more nucleic acid molecules containing the target nucleotide sequence that differs from the nucleotide sequence in other 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 methylation residues.

[0030] Another aspect of the present disclosure relates to a method for identifying one or more nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules in the sample or other samples by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues. The method includes providing a sample containing one or more nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues. The method also includes providing one or more enzymes capable of digesting a deoxyuracil (dU)-containing nucleic acid molecule present in the sample; and providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set including (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a sequence 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 by the first primary oligonucleotide primer. The sample is blended with the one or more primary oligonucleotide primer sets, the one or more enzymes capable of digesting a deoxyuracil (dU)-containing nucleic acid molecule present in the sample, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a polymerase chain reaction mixture. The polymerase chain reaction mixture is subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecule present in the polymerase chain reaction mixture and one or more polymerase chain reaction cycles including a denaturation process, a hybridization process, and an extension process, thereby forming a primary extension product comprising the target nucleotide sequence or a complement thereof. The method also includes blending the primary extension product with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture, wherein each oligonucleotide probe set includes (a) a first oligonucleotide probe having a 5' primer-specific portion and a 3' target nucleotide sequence-specific portion, and (b) a second oligonucleotide probe having a 5' target nucleotide sequence-specific portion and a 3' primer-specific portion. The first and second oligonucleotide probes of the probe set are configured to hybridize to the complementary target nucleotide sequence of the primary extension product in a base-specific manner. The ligation reaction mixture is 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 to form a ligation product sequence in the ligation reaction mixture, wherein each ligation product sequence comprises 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 comprising (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer specific portion of the junction product sequence and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer specific portion of the junction product sequence; and blending the junction product sequence, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a second polymerase chain reaction mixture. The second polymerase chain reaction mixture is subjected to conditions suitable to digest deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture and one or more polymerase chain reaction cycles comprising a denaturation step, a hybridization step, and an extension step, thereby forming secondary extension products. The secondary extension products in the sample are detected and distinguished to identify the presence of one or more nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequence in other 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 methylation residues.

[0031] Another aspect of the application relates to a method for identifying one or more nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules in the sample or other samples by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues. The method further comprises providing a sample containing one or more nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules present in the sample; and 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 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 by the first primary oligonucleotide primer. The method further comprises blending the sample with 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 mixture comprising dUTP, and a DNA polymerase to form a polymerase chain reaction mixture. The polymerase chain reaction mixture is subjected to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixture and one or more polymerase chain reaction cycles comprising a denaturation process, a hybridization process, and an extension process, thereby forming a primary extension product comprising the target nucleotide sequence or a complement thereof. The primary extension product is blended with a ligase enzyme and one or more oligonucleotide probe sets to form a ligation reaction mixture, wherein each oligonucleotide probe set comprises (a) a first oligonucleotide probe having a 5' portion and a 3' target nucleotide sequence-specific portion, and (b) a second oligonucleotide probe having a 5' target nucleotide sequence-specific portion and a 3' portion, wherein the 5' portion of the first oligonucleotide probe of the probe set is complementary to a portion of the 3' portion of the second oligonucleotide probe, wherein one of the probes in the probe set comprises a portion that generates a detectable signal, and wherein the first oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize to the complementary target nucleotide sequence of the primary extension product in a base-specific manner. The method further comprises subjecting the ligation reaction mixture to one or more ligation reaction cycles, thereby ligating the first oligonucleotide probe and the second oligonucleotide probe of the one or more oligonucleotide probe sets together to form a ligation product sequence in the ligation reaction mixture, wherein each ligation product sequence comprises a 5' portion, a target-specific portion, a 3' portion, and a portion that generates a detectable signal.The 5' portion of a ligation product sequence hybridizes to its complementary 3' portion, and a signal from the portion that generates a detectable signal is detected following the hybridization. The ligation product sequence in the sample is distinguished based on the detection to identify the presence of one or more nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequences in other 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 methylation residues.

[0032] Another aspect of the application relates to a method for identifying one or more nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules in the sample or other samples by one or more methylated residues. The method includes providing a sample potentially containing one or more nucleic acid molecules comprising a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules by one or more methylated residues; and contacting the sample with one or more enzymes capable of digesting a deoxyuracil (dU)-containing nucleic acid molecule present in the sample. The method further includes contacting the sample with one or more methylation-sensitive enzymes to form a restriction enzyme reaction mixture, wherein the one or more methylation-sensitive enzymes cleave nucleic acid molecules in the sample that contain one or more unmethylated residues within at least one methylation-sensitive enzyme recognition sequence. One or more primary oligonucleotide primer sets are provided, each primary oligonucleotide primer set comprising (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a region in the target nucleotide sequence upstream of the one or more methylated residues and (b) a second primary oligonucleotide primer comprising a nucleotide sequence identical to a region in the target nucleotide sequence downstream of the one or more methylated residues. The restriction enzyme reaction mixture is blended with the one or more primary oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a primary polymerase chain reaction mixture. The method further includes subjecting the primary polymerase chain reaction mixture to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a primary extension product comprising the target nucleotide sequence or a complement thereof. One or more secondary oligonucleotide primer sets are provided, each secondary oligonucleotide primer set comprising a first nested oligonucleotide primer and a second nested oligonucleotide primer capable of hybridizing to the primary extension product. The primary extension product is blended with the one or more secondary oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a secondary polymerase chain reaction mixture, and the secondary polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a secondary extension product. The secondary extension product in the sample is detected and distinguished to identify the presence of one or more nucleic acid molecules containing a target nucleotide sequence that differs from other nucleic acid molecules in the sample by one or more methylated residues.

[0033] Another aspect of the application relates to a method for identifying one or more target ribonucleic acid molecules in a sample that differ in sequence from other 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 rearrangement at the genomic level. The method includes providing a sample containing one or more target ribonucleic acid molecules that potentially contain a sequence that differs from other ribonucleic acid molecules; and contacting the sample with one or more enzymes capable of digesting nucleic acid molecules containing dU potentially present in the sample. One or more oligonucleotide primers are provided, each primer complementary to the one or more target ribonucleic acid molecules. The contacted sample is blended with the one or more oligonucleotide primers and a reverse transcriptase to form a reverse transcription mixture, and complementary deoxyribonucleic acid (cDNA) molecules are produced in the reverse transcription mixture. Each cDNA molecule comprises a nucleotide sequence complementary to the sequence of the target ribonucleic acid molecule and contains dU. The method further includes providing one or more oligonucleotide primer sets, each primer set comprising (a) a first oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the cDNA nucleotide sequence adjacent to the complementary sequence of the sequence of the target ribonucleic acid molecule of the cDNA, and (b) a second oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the extension product formed by the first oligonucleotide primer. The reverse transcription mixture containing the cDNA molecules is blended with the one or more oligonucleotide primer sets and a polymerase to form a polymerase reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming one or more different first extension products. The method further includes providing one or more oligonucleotide probe sets. Each probe set comprises (a) a first oligonucleotide probe having a target sequence specific portion, and (b) a second oligonucleotide probe having a target sequence specific portion, wherein the first oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize in a base specific manner to the complementary portion of the first extension product corresponding to the sequence of the target ribonucleic acid molecule. The first extension product is contacted with a ligase and the one or more oligonucleotide probe sets to form a ligation reaction mixture, and the first probe and the second probe of the one or more oligonucleotide probe sets are ligated together to form a ligation product sequence in the ligation enzyme reaction mixture. The ligation product sequence in the sample is detected and distinguished, thereby identifying the presence of one or more target ribonucleic acid molecules that differ in sequence from other 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 rearrangement at the genomic level.

[0034] Another aspect of the present invention relates to a method for identifying one or more target ribonucleic acid molecules in a sample that differ in sequence from other 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 rearrangement at the genomic level. The method includes providing a sample containing one or more target ribonucleic acid molecules that potentially differ in sequence from other ribonucleic acid molecules, and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. One or more oligonucleotide primers are provided, each primer complementary to the one or more target ribonucleic acid molecules, and the contacted sample is blended with the one or more oligonucleotide primers, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription mixture. Complementary deoxyribonucleic acid (cDNA) molecules are produced in the reverse transcription mixture, each cDNA molecule comprising a nucleotide sequence complementary to the target ribonucleic acid molecule and containing dU. The method further includes providing one or more oligonucleotide primer sets, each primer set comprising (a) a first oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the cDNA nucleotide sequence adjacent to the complementary sequence of the target ribonucleic acid molecule sequence of the cDNA, and (b) a second oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the extension product formed by the first oligonucleotide primer. The reverse transcription mixture containing the cDNA molecules is blended with the one or more oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a polymerase to form a polymerase reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming one or more different first extension products. The method further includes 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 oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize in a base-specific manner to the complementary portion of the first extension product corresponding to the target ribonucleic acid molecule sequence. The first extension product is contacted with a ligase and the one or more oligonucleotide probe sets to form a ligation reaction mixture, and the ligation reaction mixture is subjected to one or more ligation reaction cycles, thereby ligating the first probe and the second probe of the one or more oligonucleotide probe sets together to form a ligation product sequence in the ligation reaction mixture, wherein each ligation product sequence comprises 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 comprising (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer specific portion of the junction product sequence and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer specific portion of the junction product sequence; and blending the junction product sequence, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a second polymerase chain reaction mixture. The second polymerase chain reaction mixture is subjected to conditions suitable to digest deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture and one or more polymerase chain reaction cycles comprising a denaturation step, a hybridization step, and an extension step, thereby forming secondary extension products. The secondary extension products in the sample are detected and distinguished, thereby identifying the presence of one or more ribonucleic acid molecules that differ in sequence from other 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.

[0035] Another aspect of the present invention relates to a method for identifying one or more target ribonucleic acid molecules in a sample that differ in sequence from other 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 rearrangement at the genomic level. The method comprises providing a sample containing one or more target ribonucleic acid molecules that potentially differ in sequence from other ribonucleic acid molecules; and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The method further comprises providing one or more oligonucleotide primers, each primer complementary to the one or more target ribonucleic acid molecules, and blending the contacted sample, the one or more oligonucleotide primers, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription mixture. Complementary deoxyribonucleic acid (cDNA) molecules are produced in the reverse transcription mixture, each cDNA molecule comprising a nucleotide sequence complementary to the target ribonucleic acid molecule sequence and containing dU. The method further comprises providing one or more oligonucleotide primer sets, each primer set comprising (a) a first oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the cDNA nucleotide sequence adjacent to the complementary sequence of the target ribonucleic acid molecule sequence of the cDNA, and (b) a second oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the extension product formed by the first oligonucleotide primer. The reverse transcription mixture containing the cDNA molecules is blended with the one or more oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a polymerase to form a polymerase reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming one or more different first extension products. The method further comprises providing one or more oligonucleotide probe sets, each probe set comprising (a) a first oligonucleotide probe having a 5' portion and a 3' target nucleotide sequence specific portion, and (b) a second oligonucleotide probe having a 5' target nucleotide sequence specific portion and a 3' portion, wherein the 5' portion of the first oligonucleotide probe of the probe set is complementary to a portion of the 3' portion of the second oligonucleotide probe, wherein one of the probe sets comprises a portion that generates a detectable signal, and wherein the first oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize in a base-specific manner to the complementary portion of the first extension product corresponding to the target ribonucleic acid molecule sequence.contacting the primary extension products with a ligase and one or more sets of oligonucleotide probes to form a ligation reaction mixture, and subjecting the ligation reaction mixture to one or more ligation reaction cycles, whereby the first and second probes of the one or more sets of oligonucleotide probes are ligated together to form a ligation product sequence in the ligation reaction mixture, wherein each ligation product sequence comprises a 5' portion, a target-specific portion, a 3' portion, and a portion that generates a detectable signal. The 5' portion of the ligation product sequence hybridizes to its complementary 3' portion, and a signal from the portion that generates a detectable signal that is produced upon said hybridization is detected. The ligation product sequence in the sample is detected based on said detection to identify the presence of one or more ribonucleic acid molecules in the sample that differ in sequence from other 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.

[0036] Another aspect of the application relates to a method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence from other miRNA molecules in the sample by one or more bases. The method includes providing a sample containing one or more target miRNA molecules that potentially differ in sequence from other miRNA molecules in the sample by one or more bases; and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. One or more oligonucleotide primer sets are provided, each primer set including (a) a first oligonucleotide primer having a 5' stem-loop portion, a blocking group, an internal primer-specific portion within the loop region, and a 3' nucleotide sequence portion that is complementary to a 3' portion of the sequence of the target miRNA molecule, (b) a second oligonucleotide primer having a 3' nucleotide sequence portion that is complementary to a sequence complementary to the 5' end of the sequence of the target miRNA molecule, and a 5' primer-specific portion, (c) a third oligonucleotide primer comprising a nucleotide sequence identical to the internal primer-specific portion of the first oligonucleotide primer, and (d) a fourth oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer-specific portion of the second oligonucleotide primer. The contacted sample is blended with one or more first oligonucleotide primers of the primer set, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription reaction mixture. The first oligonucleotide primer hybridizes to the sequence of the target miRNA molecule, if present in the sample, and the reverse transcriptase extends the 3' end of the hybridized first oligonucleotide primer to produce an extended first oligonucleotide primer comprising a complement of the sequence of the target miRNA molecule. The method further includes blending the reverse transcription reaction mixture with the second, third, and fourth oligonucleotide primers of the primer set under conditions effective to hybridize one or more second oligonucleotide primers of the primer set to a region of the extended first oligonucleotide primer comprising a complement of the sequence of the target miRNA molecule and to extend to produce a primary extension product comprising the 5' primer-specific portion, a nucleotide sequence corresponding to the sequence of the target miRNA molecule, and a complement of the internal primer-specific portion to form a polymerase reaction mixture. The polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a plurality of primary extension products. The method further includes blending the plurality of primary extension products with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture.Each oligonucleotide probe set includes (a) a first oligonucleotide probe having a target sequence-specific portion, and (b) a second oligonucleotide probe having a target sequence-specific portion and a portion complementary to the primary extension product, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to the complementary portion of the primary extension product corresponding to the sequence of the target miRNA molecule. The first and second oligonucleotide probes of one or more oligonucleotide probe sets are ligated together to form a ligation product sequence in the ligation reaction mixture, and the ligation product sequence is detected and distinguished in the sample to identify one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases.

[0037] Another aspect of the application relates to a method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence from other miRNA molecules in the sample by one or more bases. The method includes providing a sample containing one or more target miRNA molecules that potentially differ in sequence from other miRNA molecules in the sample by one or more bases; and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The method further includes providing one or more oligonucleotide primer sets, each primer set including (a) a first oligonucleotide primer having a 5' stem-loop portion, a blocking group, an internal primer-specific portion within the loop region, and a 3' nucleotide sequence portion that is complementary to a 3' portion of the sequence of the target miRNA molecule, (b) a second oligonucleotide primer having a 3' nucleotide sequence portion that is complementary to a sequence complementary to the 5' end of the sequence of the target miRNA molecule, and a 5' primer-specific portion, (c) a third oligonucleotide primer comprising a nucleotide sequence identical to the internal primer-specific portion of the first oligonucleotide primer, and (d) a fourth oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer-specific portion of the second oligonucleotide primer. The contacted sample is blended with one or more first oligonucleotide primers of the primer set, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription reaction mixture, wherein the first oligonucleotide primer hybridizes to the sequence of the target miRNA molecule, if present in the sample, and the reverse transcriptase extends the 3' end of the hybridized first oligonucleotide primer to produce an extended first oligonucleotide primer comprising a complement of the sequence of the target miRNA molecule. The reverse transcription reaction mixture is blended with the second oligonucleotide primer, the third oligonucleotide primer, and the fourth oligonucleotide primer of the primer set under conditions effective to hybridize and extend one or more second oligonucleotide primers of the primer set to a region of the extended first oligonucleotide primer comprising a complement of the sequence of the target miRNA molecule to produce a primary extension product comprising the 5' primer-specific portion, a nucleotide sequence corresponding to the sequence of the target miRNA molecule, and a complement of the internal primer-specific portion to form a polymerase reaction mixture. The polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a plurality of primary extension products.oligonucleotide probe of the one or more oligonucleotide probe sets is configured to hybridize to a complementary portion of the primary extension product corresponding to a sequence of a target miRNA molecule in a base-specific manner. The ligation reaction mixture is 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 to form a ligation product sequence in the ligation reaction mixture, wherein each ligation product sequence comprises 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 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; and admixing the ligation product sequence, the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting a deoxyuracil (dU)-containing nucleic acid molecule, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a second polymerase chain reaction mixture. The second polymerase chain reaction mixture is subjected to conditions suitable for digesting the deoxyuracil (dU)-containing nucleic acid molecule present in the second polymerase chain reaction mixture, and one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming secondary extension products. The secondary extension products in the sample are detected and distinguished, thereby identifying one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases.

[0038] Another aspect of the application relates to a method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence from other miRNA molecules in the sample by one or more bases. The method includes providing a sample containing one or more target miRNA molecules that potentially differ in sequence from other miRNA molecules in the sample by one or more bases; and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The method further includes providing one or more oligonucleotide primer sets, each primer set including (a) a first oligonucleotide primer having a 5' stem-loop portion, a blocking group, an internal primer-specific portion within the loop region, and a 3' nucleotide sequence portion that is complementary to a 3' portion of the sequence of the target miRNA molecule, (b) a second oligonucleotide primer having a 3' nucleotide sequence portion that is complementary to a sequence complementary to the 5' end of the sequence of the target miRNA molecule, and a 5' primer-specific portion, (c) a third oligonucleotide primer comprising a nucleotide sequence identical to the internal primer-specific portion of the first oligonucleotide primer, and (d) a fourth oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer-specific portion of the second oligonucleotide primer. The contacted sample is blended with one or more first oligonucleotide primers of the primer set, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription reaction mixture, wherein the first oligonucleotide primer hybridizes to the sequence of the target miRNA molecule, if present in the sample, and the reverse transcriptase extends the 3' end of the hybridized first oligonucleotide primer to produce an extended first oligonucleotide primer comprising a complement of the sequence of the target miRNA molecule. The reverse transcription reaction mixture is blended with the second oligonucleotide primer, the third oligonucleotide primer, and the fourth oligonucleotide primer of the primer set under conditions effective to hybridize and extend one or more second oligonucleotide primers of the primer set to a region of the extended first oligonucleotide primer comprising a complement of the sequence of the target miRNA molecule to produce a primary extension product comprising the 5' primer-specific portion, a nucleotide sequence corresponding to the sequence of the target miRNA molecule, and a complement of the internal primer-specific portion. The method further includes subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a plurality of primary extension products.a plurality of first extension products are combined with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture, wherein each oligonucleotide probe set comprises (a) a first oligonucleotide probe having a 5' portion and a 3' target nucleotide sequence specific portion, and (b) a second oligonucleotide probe having a 5' target nucleotide sequence specific portion and a 3' portion, wherein the 5' portion of the first oligonucleotide probe of the probe set is complementary to a portion of the 3' portion of the second oligonucleotide probe, wherein one of the probes in the probe set comprises a portion that generates a detectable signal, and wherein the first oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize in a base specific manner to a complementary portion of the first extension products corresponding to a sequence of a target miRNA molecule. The ligation reaction mixture is subjected to one or more ligation reaction cycles, whereby the first oligonucleotide probe and the second oligonucleotide probe of the one or more oligonucleotide probe sets are ligated together to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises a 5' portion, a target specific portion, a 3' portion, and a portion that generates a detectable signal. The 5' portion of the ligation product sequences hybridizes to its complementary 3' portion, and the signal from the portion that generates a detectable signal produced upon said hybridization is detected. The ligation product sequences in the sample are distinguished based on said detection to identify the presence of one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases.

[0039] Another aspect of the present invention relates to a method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence from other miRNA molecules in the sample by one or more bases. The method includes providing a sample containing one or more target miRNA molecules that potentially differ in sequence from other miRNA molecules by one or more base differences; and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The contacted sample is co-mingled with a ligase and a first oligonucleotide probe comprising a 5' phosphate, a 5' stem-loop moiety, an internal primer-specific moiety within the loop region, a blocking group, and a 3' nucleotide sequence complementary to a 3' portion of a target miRNA molecule sequence to form a ligation reaction. The method further includes ligating the target miRNA molecule sequence at its 3' end to the 5' phosphate of the first oligonucleotide probe to produce a chimeric nucleic acid molecule comprising the target miRNA molecule sequence, if present in the sample, appended to the first oligonucleotide probe. One or more oligonucleotide primer sets are provided, each primer set including (a) a first oligonucleotide primer comprising a 3' nucleotide sequence complementary to a complementary sequence of a 5' end of the target miRNA molecule sequence, and a 5' primer-specific moiety, (b) a second oligonucleotide primer comprising a nucleotide sequence complementary to the internal primer-specific moiety of the first oligonucleotide probe, and (c) a third oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer-specific moiety of the first oligonucleotide primer. The chimeric nucleic acid molecule is co-mingled with one or more second oligonucleotide primers of the primer set, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription reaction mixture, wherein the one or more second oligonucleotide primers of the primer set hybridize to the internal primer-specific moiety of the chimeric nucleic acid molecule and are extended at their 3' ends to produce a complement of the chimeric nucleic acid molecule, if present in the sample. The method further includes co-mingling the reverse transcription reaction mixture with the first oligonucleotide primer and the third oligonucleotide primer of the primer set to form a polymerase reaction mixture, and subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a primary extension product. The primary extension product comprises the 5' primer-specific moiety, a nucleotide sequence corresponding to the target miRNA molecule sequence, and a complement of the internal primer-specific moiety. The primary extension product is co-mingled with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture.Each oligonucleotide probe set includes (a) a first oligonucleotide probe having a target sequence-specific portion, and (b) a second oligonucleotide probe having a target sequence-specific portion and a portion complementary to the primary extension product, wherein the first and second oligonucleotide probes of the probe set are configured to hybridize in a base-specific manner to the complementary portion of the primary extension product corresponding to the sequence of the target miRNA molecule. The first and second oligonucleotide probes of one or more oligonucleotide probe sets are ligated together to form a ligation product sequence in the ligation reaction mixture, and the ligation product sequence is detected and distinguished in the sample to identify one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases.

[0040] Another aspect of the application relates to a method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence from other miRNA molecules in the sample by one or more bases. The method includes providing a sample containing one or more miRNA molecules that potentially differ in sequence from other miRNA molecules by one or more base differences; and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The contacted sample is co-mingled with a ligase and a first oligonucleotide probe comprising a 5' phosphate, a 5' stem-loop moiety, an internal primer-specific moiety within the loop region, a blocking group, and a 3' nucleotide sequence complementary to a 3' portion of a target miRNA molecule sequence to form a ligation reaction, and the target miRNA molecule sequence is ligated at its 3' end to the 5' phosphate of the first oligonucleotide probe to produce a chimeric nucleic acid molecule comprising the target miRNA molecule sequence, if present in the sample, appended to the first oligonucleotide probe. The method further includes providing one or more oligonucleotide primer sets, each primer set comprising (a) a first oligonucleotide primer comprising a 3' nucleotide sequence complementary to a complementary sequence of a 5' end of the target miRNA molecule sequence, and a 5' primer-specific moiety, (b) a second oligonucleotide primer comprising a nucleotide sequence complementary to the internal primer-specific moiety of the first oligonucleotide probe, and (c) a third oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer-specific moiety of the first oligonucleotide primer. The chimeric nucleic acid molecule is co-mingled with the one or more second oligonucleotide primers, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription reaction mixture, wherein the one or more second oligonucleotide primers of the primer set hybridize to the internal primer-specific moiety of the chimeric nucleic acid molecule and are extended at their 3' ends to produce a complement of the chimeric nucleic acid molecule, if present in the sample. The reverse transcription reaction mixture is co-mingled with the first oligonucleotide primer and the third oligonucleotide primer of the primer set to form a polymerase reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a primary extension product comprising the 5' primer-specific moiety, a nucleotide sequence corresponding to the target miRNA molecule sequence, and a complement of the internal primer-specific moiety.The first extension product is combined with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture, wherein each oligonucleotide probe set comprises (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 first 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 to the complementary portion of the first extension product corresponding to the sequence of the target miRNA molecule. The ligation reaction mixture is 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 to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises 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 comprising (a) a first secondary oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer-specific portion of the ligation product sequences and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence complementary to the 3' primer-specific portion of the ligation product sequences. The ligation product sequences are combined with the one or more secondary oligonucleotide primer sets, one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a second polymerase chain reaction mixture. The second polymerase chain reaction mixture is subjected to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixture, and one or more polymerase chain reaction cycles comprising a denaturation step, a hybridization step, and an extension step, thereby forming secondary extension products. The secondary extension products in the sample are detected and distinguished, thereby identifying one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases.

[0041] Another aspect of the application relates to a method for identifying one or more target micro ribonucleic acid (miRNA) molecules in a sample that differ in sequence from other miRNA molecules in the sample by one or more bases. The method includes providing a sample containing one or more miRNA molecules that potentially differ in sequence from other miRNA molecules by one or more base differences; and contacting the sample with one or more enzymes capable of digesting dU-containing nucleic acid molecules potentially present in the sample. The contacted sample is co-mingled with a ligase and a first oligonucleotide probe comprising a 5' phosphate, a 5' stem-loop moiety, an internal primer-specific moiety within the loop region, a blocking group, and a 3' nucleotide sequence complementary to a 3' portion of a target miRNA molecule sequence to form a ligation reaction. The target miRNA molecule sequence is ligated at its 3' end to the 5' phosphate of the first oligonucleotide probe to produce a chimeric nucleic acid molecule comprising the target miRNA molecule sequence, if present in the sample, appended to the first oligonucleotide probe. The method further includes providing one or more oligonucleotide primer sets, each primer set comprising (a) a first oligonucleotide primer comprising a 3' nucleotide sequence complementary to a complementary sequence of a 5' end of the target miRNA molecule sequence, and a 5' primer-specific moiety, (b) a second oligonucleotide primer comprising a nucleotide sequence complementary to the internal primer-specific moiety of the first oligonucleotide probe, and (c) a third oligonucleotide primer comprising a nucleotide sequence identical to the 5' primer-specific moiety of the first oligonucleotide primer. The chimeric nucleic acid molecule is co-mingled with the one or more second oligonucleotide primers, a deoxynucleotide mixture comprising dUTP, and a reverse transcriptase to form a reverse transcription reaction mixture, wherein the one or more second oligonucleotide primers of the primer set hybridize to the internal primer-specific moiety of the chimeric nucleic acid molecule and are extended at their 3' ends to produce a complement of the chimeric nucleic acid molecule, if present in the sample. The reverse transcription reaction mixture is co-mingled with the first oligonucleotide primer and the third oligonucleotide primer of the primer set to form a polymerase reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a primary extension product comprising the 5' primer-specific moiety, a nucleotide sequence corresponding to the target miRNA molecule sequence, and a complement of the internal primer-specific moiety.The first extension product is combined with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture, wherein each oligonucleotide probe set comprises (a) a first oligonucleotide probe having a 5' portion and a 3' target nucleotide sequence specific portion, and (b) a second oligonucleotide probe having a 5' target nucleotide sequence specific portion and a 3' portion, wherein the 5' portion of the first oligonucleotide probe of the probe set is complementary to a portion of the 3' portion of the second oligonucleotide probe, wherein one of the probes in the probe set comprises a portion that generates a detectable signal, and wherein the first oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize in a base specific manner to the complementary portion of the first extension product corresponding to the sequence of the target miRNA molecule. The ligation reaction mixture is subjected to one or more ligation reaction cycles, whereby the first oligonucleotide probe and the second oligonucleotide probe of the one or more oligonucleotide probe sets are ligated together to form ligation product sequences in the ligation reaction mixture, wherein each ligation product sequence comprises a 5' portion, a target specific portion, a 3' portion, and a portion that generates a detectable signal. The 5' portion of the ligation product sequence hybridizes to its complementary 3' portion, and the signal from the portion that generates a detectable signal that is generated upon said hybridization is detected. The ligation product sequences in the sample are distinguished based on said detection to identify the presence of one or more target miRNA molecules that differ in sequence from other miRNA molecules in the sample by one or more bases.

[0042] Another aspect of the present invention relates to a device for simultaneously adding liquid to two or more wells in a row and / or column of a microtiter plate. The device has opposing top and bottom surfaces, wherein the top surface has openings to the wells and the bottom surface defines closed ends of the wells. The device includes a first layer defined by a first border and a second border, wherein a metering chamber extends between the first and second borders of the first layer and is in fluid communication with each other. The first layer is configured to fit in close proximity to the microtiter plate in an operational position, wherein the first border of the first layer is closest to the top surface of the microtiter plate and each metering chamber is in fluid communication with a single well in a row and / or column of the microtiter plate. The first layer further includes a fill chamber in fluid communication with one or more metering chambers. The device includes a second layer defined by a first border and a second border, wherein a fill port extends between the first and second borders of the second layer. The second layer is configured to fit on the first layer in an operational position, wherein the first border of the second layer is adjacent to the second border of the first layer and the fill port is aligned with the fill chamber. When the first layer, the second layer, and the microtiter plate are positioned relative to each other in their operational positions, liquid entering the device through the fill port will pass through the input chamber, the metering chamber, and into two or more wells in a row and / or column of the microtiter plate.

[0043] Another aspect of the present invention relates to a method of adding liquid to two or more wells in a row and / or column of a microtiter plate having opposing top and bottom surfaces, wherein the top surface has openings to the wells and the bottom surface defines closed ends of the wells. The method includes providing a device including a first layer having a first border and a second border, wherein a metering chamber extends between the first and second borders of the first layer and is in fluid communication with one another. The first layer of the device is configured to fit in proximity to the microtiter plate in an operative position, wherein the first border of the first layer is closest to the top surface of the microtiter plate and one metering chamber is in fluid communication with a single well in a row and / or column of the microtiter plate. The first layer further includes a fill chamber in fluid communication with one or more of the metering chambers. The device includes a second layer having a first border and a second border, wherein a fill port extends between the first and second borders of the second layer. The second layer is configured to fit over the first layer in an operative position, wherein the first border of the second layer is adjacent to the second border of the first layer and the fill port is aligned with the fill chamber. When the first layer, the second layer, and the microtiter plate are positioned relative to one another in their operative positions, liquid entering the device through the fill port will pass through the fill chamber, the metering chamber, and into two or more wells in a row and / or column of the microtiter plate. The method further includes filling the device with liquid and discharging the liquid in the device to two or more wells in a row and / or column of the microtiter plate.

[0044] The present invention describes a variety of methods for detecting mutations, expression, splice variants, translocations, copy number, and / or methylation changes of target nucleic acid molecules using nuclease, ligase, and polymerase reactions. The present invention addresses the carryover prevention problem and enables spatial multiplexing to provide relative quantification similar to digital PCR. This technology can be used for early detection of cancer non-invasively from plasma or serum samples, non-invasive prognosis of cancer, and monitoring of cancer recurrence. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 A conditional logic tree is shown for early detection pan cancer test based on patient blood sample analysis.

[0046] Figure 2 A workflow is shown for analyzing DNA, mRNA, and miRNA from plasma cfDNA, exosomes, and CTCs.

[0047] Figure 3 A generalized analysis of DNA, mRNA, or miRNA by distribution of one sample that has been initially diluted and distributed in 24 tubes for multiplex PCR or reverse transcription PCR followed by LDR using labeled probes in a 24 x 16 row microtiter plate is shown.

[0048] Figure 4 shows the addition of 16 different tag primer sets on 24 columns of a microtiter plate of Figure 3

[0049] Figure 5 shows the hypothetical signal pattern resulting from real-time detection of each well on a microtiter plate of Figure 4

[0050] Figure 6 shows the workflow for analyzing DNA from plasma cfDNA or CTCs.

[0051] Figure 7 shows the analysis of DNA by distribution of one sample that has been initially diluted and distributed in 24 tubes for multiplex PCR or reverse transcription PCR followed by LDR using labeled probes in 24 x 16 rows of a microtiter plate.

[0052] Figure 8 shows the addition of 16 different tag primer sets on 24 columns of a microtiter plate of Figure 7

[0053] Figure 9 shows the hypothetical signal pattern resulting from real-time detection of each well on a microtiter plate of Figure 8

[0054] Figure 10 shows the workflow for analyzing mRNA or miRNA from plasma exosomes and shows the workflow for analyzing DNA, mRNA, and miRNA from plasma cfDNA, exosomes, and CTCs.

[0055] Figure 11 shows the analysis of mRNA or miRNA by distribution of one sample that has been initially serially diluted into 24 tubes for multiplex reverse transcription PCR followed by LDR using labeled probes in 24 x 16 rows of a microtiter plate.

[0056] Figure 12 shows the addition of 16 different tag primer sets on 24 columns of a microtiter plate of Figure 11

[0057] Figure 13 shows the hypothetical signal pattern resulting from real-time detection of each well on a microtiter plate of Figure 12

[0058] Figure 14 ​​​​​​This illustrates a workflow for generalized analysis of DNA, mRNA, or miRNA by means of the distribution of a single sample, which has been initially diluted in 24 tubes for multiplex PCR or reverse transcription PCR, followed by LDR using labeled probes in a 24x16 row of a microtiter plate for streptavidin-mediated capture.

[0059] Figure 15 This illustrates a generalized analysis of DNA, mRNA, or miRNA by the distribution of 24 samples, which were initially diluted and distributed in 24 tubes for multiplex PCR or reverse transcription PCR, followed by LDR using labeled probes in a 24x16 row of a microtiter plate.

[0060] Figure 16 Showing the Figure 15 Streptavidin-mediated capture of biotinylated PCR or RT-PCR amplicon in microtiter plate wells.

[0061] Figure 17 Shown in Figure 16 Sixteen different sets of tag primers were added to the 24 columns of the microtiter plate.

[0062] Figure 18 Shown by Figure 17 The hypothetical signal pattern generated by real-time LDR-FRET detection of each well on the microtiter plate.

[0063] Figure 19 The workflow for analyzing DNA distribution through a single sample, which has been initially diluted in 24 tubes for multiplex PCR, followed by LDR using labeled probes in a 24x16 row of a microtiter plate for streptavidin-mediated capture.

[0064] Figure 20 The analysis of DNA by the distribution of 24 samples, which were initially diluted and distributed in 24 tubes for multiplex PCR, followed by LDR using labeled probes in a 24x16 row of a microtiter plate.

[0065] Figure 21 Showing the Figure 20 Streptavidin-mediated capture of biotinylated PCR or RT-PCR amplicon in microtiter plate wells.

[0066] Figure 22 Shown in Figure 21 Sixteen different sets of tag primers were added to the 24 columns of the microtiter plate.

[0067] Figure 23 Shown by Figure 22The hypothetical signal pattern generated by real-time LDR-FRET detection of each well on the microtiter plate.

[0068] Figure 24 The workflow for analyzing the distribution of mRNA and miRNA through a single sample, which has been initially diluted in 24 tubes for multiplex PCR, followed by LDR using labeled probes in a 24x16 row of a microtiter plate for streptavidin-mediated capture.

[0069] Figure 25 This illustrates the analysis of mRNA or miRNA by the distribution of a sample initially serially diluted into 24 tubes for multiplex reverse transcription PCR, followed by LDR using labeled probes in a 24x16 row of a microtiter plate.

[0070] Figure 26 Showing the Figure 25 Streptavidin-mediated capture of RT-PCR amplicon in microtiter plate wells.

[0071] Figure 27 Shown in Figure 26 Sixteen different sets of tag primers were added to the 24 columns of the microtiter plate.

[0072] Figure 28 Shown by Figure 27 The hypothetical signal pattern generated by real-time LDR-FRET detection of each well on the microtiter plate.

[0073] Figure 29 Simulations of Poisson distributions for 6 to 48 molecules distributed across 24 wells are shown. The top is a table of the initial number of molecules versus number of molecules per well. Below is a histogram of #molecule(x) versus #well(y).

[0074] Figure 30 The simulation shows the Poisson distribution of 12 to 96 molecules distributed across 24 wells. The top is a table of the initial number of molecules versus the number of molecules per well. Below is a histogram of #molecule(x) versus #well(y).

[0075] Figure 31 The simulation shows the Poisson distribution of 12 to 96 molecules distributed in 48 wells. The top is a table of the initial number of molecules versus the number of molecules per well. Below is a histogram of #molecule(x) versus #well(y).

[0076] Figure 32 The simulation shows the Poisson distribution of 24 to 192 molecules distributed across 48 wells. The top is a table of the initial number of molecules versus number of molecules per well. Below is a histogram of #molecule(x) versus #well(y).

[0077] Figure 33 Simulation of Poisson distribution of 1 to 8 molecules distributed in 8 wells. Above is tabular form of starting number of molecules vs. number of molecules / well. Below is histogram of # molecules (x) vs. # wells (y).

[0078] Figure 34 Simulation of Poisson distribution of 2 to 16 molecules distributed in 8 wells. Above is tabular form of starting number of molecules vs. number of molecules / well. Below is histogram of # molecules (x) vs. # wells (y).

[0079] Figure 35 Simulation of Poisson distribution of 4 to 32 molecules distributed in 8 wells. Above is tabular form of starting number of molecules vs. number of molecules / well. Below is histogram of # molecules (x) vs. # wells (y).

[0080] Figure 36 Simulation of Poisson distribution of 8 to 64 molecules distributed in 8 wells. Above is tabular form of starting number of molecules vs. number of molecules / well. Below is histogram of # molecules (x) vs. # wells (y).

[0081] Figure 37 Simulation of Poisson distribution of 16 to 128 molecules distributed in 8 wells. Above is tabular form of starting number of molecules vs. number of molecules / well. Below is histogram of # molecules (x) vs. # wells (y).

[0082] Figure 38 PCR-LDR-qPCR carryover prevention reaction using Taqman detection for identification or relative quantification of targets and / or mutations.

[0083] Figure 39 PCR-qLDR carryover prevention reaction using FRET detection for identification or relative quantification of targets and / or mutations.

[0084] Figure 40 PCR-LDR-qPCR carryover prevention reaction using Taqman detection for identification or relative quantification of targets and / or mutations.

[0085] Figure 41 PCR-LDR-qPCR carryover prevention reaction using Taqman detection for identification or relative quantification of targets and / or mutations.

[0086] Figure 42PCR-qLDR legacy prevention reaction for identification or relative quantification of targets and / or mutations using FRET detection is shown.

[0087] Figure 43 PCR-qLDR legacy prevention reaction for identification or relative quantification of targets and / or mutations using FRET detection is shown.

[0088] Figure 44 PCR-LDR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using UniTaq detection is shown.

[0089] Figure 45 PCR-LDR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using Taqman detection is shown.

[0090] Figure 46 PCR-LDR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using UniTaq detection is shown.

[0091] Figure 47 PCR-qLDR legacy prevention reaction for identification or relative quantification of target methylation using FRET detection is shown.

[0092] Figure 48 Nuclease-Ligation-PCR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using Taqman detection is shown.

[0093] Figure 49 Nuclease-Ligation-PCR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using UniTaq detection is shown.

[0094] Figure 50 PCR-LDR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using Taqman detection is shown.

[0095] Figure 51 PCR-LDR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using UniTaq detection is shown.

[0096] Figure 52 PCR-qLDR legacy prevention reaction for identification or relative quantification of target methylation using FRET detection is shown.

[0097] Figure 53 PCR-qPCR legacy prevention reaction for identification or relative quantification of target methylation using Taqman detection is shown.

[0098] Figure 54An overview of PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying translocations at the mRNA level is shown.

[0099] Figure 55 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying translocations at the mRNA level is shown using Taqman detection.

[0100] Figure 56 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying translocations at the mRNA level is shown using UniTaq detection.

[0101] Figure 57 An overview of RT-PCR-qLDR legacy prevention reactions for identifying or relatively quantifying translocations at the mRNA level is shown using FRET detection.

[0102] Figure 58 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0103] Figure 59 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying wild type and alternatively spliced transcripts is shown using Taqman detection.

[0104] Figure 60 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying wild type and alternatively spliced transcripts is shown using UniTaq detection.

[0105] Figure 61 An overview of RT-PCR-qLDR legacy prevention reactions for identifying or relatively quantifying wild type and alternatively spliced transcripts is shown using FRET detection.

[0106] Figure 62 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying low levels of alternatively spliced transcripts is shown using Taqman detection.

[0107] Figure 63 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying low levels of alternatively spliced transcripts is shown using UniTaq detection.

[0108] Figure 64 An overview of RT-PCR-qLDR legacy prevention reactions for identifying or relatively quantifying low levels of alternatively spliced transcripts is shown using FRET detection.

[0109] Figure 65An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0110] Figure 66 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0111] Figure 67 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0112] Figure 68 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0113] Figure 69 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0114] Figure 70 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0115] Figure 71 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0116] Figure 72 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0117] Figure 73 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0118] Figure 74 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0119] Figure 75 An overview of RT-PCR-LDR-qPCR legacy prevention reactions for identifying or relatively quantifying alternative splicing is shown.

[0120] Figure 76 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying low levels of alternatively spliced (exon deletion) transcripts using Taqman detection is shown.

[0121] Figure 77 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying low levels of alternatively spliced (exon deletion) transcripts using UniTaq detection is shown.

[0122] Figure 78 RT-PCR-qLDR legacy prevention reaction for identifying or relatively quantifying low levels of alternatively spliced (exon deletion) transcripts using FRET detection is shown.

[0123] Figure 79 Overview of RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying intron insertion alternative splicing is shown.

[0124] Figure 80 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying wild type and alternatively spliced (intron insertion) transcripts using Taqman detection is shown.

[0125] Figure 81 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying wild type and alternatively spliced (intron insertion) transcripts using UniTaq detection is shown.

[0126] Figure 82 RT-PCR-qLDR legacy prevention reaction for identifying or relatively quantifying wild type and alternatively spliced (intron insertion) transcripts using FRET detection is shown.

[0127] Figure 83 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying low levels of alternatively spliced (intron insertion) transcripts using Taqman detection is shown.

[0128] Figure 84 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying low levels of alternatively spliced (intron insertion) transcripts using UniTaq detection is shown.

[0129] Figure 85 RT-PCR-qLDR legacy prevention reaction for identifying or relatively quantifying low levels of alternatively spliced (intron insertion) transcripts using FRET detection is shown.

[0130] Figure 86 PCR-LDR-qPCR legacy prevention reaction for counting DNA copy number shown using Taqman detection.

[0131] Figure 87 PCR-LDR-qPCR legacy prevention reaction for counting DNA copy number shown using UniTaq detection.

[0132] Figure 88 PCR-qLDR legacy prevention reaction for counting DNA copy number shown using FRET detection.

[0133] Figure 89 RT-PCR-LDR-qPCR legacy prevention reaction for counting RNA copy number shown using Taqman detection.

[0134] Figure 90 RT-PCR-LDR-qPCR legacy prevention reaction for counting RNA copy number shown using UniTaq detection.

[0135] Figure 91 RT-PCR-qLDR legacy prevention reaction for counting RNA copy number shown using FRET detection.

[0136] Figure 92 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relative quantification of miRNA shown using Taqman detection.

[0137] Figure 93 RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relative quantification of miRNA shown using UniTaq detection.

[0138] Figure 94 RT-PCR-qLDR legacy prevention reaction for identifying or relative quantification of miRNA shown using FRET detection.

[0139] Figure 95 Ligation-RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relative quantification of miRNA shown using Taqman detection.

[0140] Figure 96 Ligation-RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relative quantification of miRNA shown using UniTaq detection.

[0141] Figure 97 Ligation-RT-PCR-qLDR legacy prevention reaction for identifying or relative quantification of miRNA shown using FRET detection.

[0142] Figure 98 A ligation-RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying miRNAs using Taqman detection is shown.

[0143] Figure 99 A ligation-RT-PCR-LDR-qPCR legacy prevention reaction for identifying or relatively quantifying miRNAs using UniTaq detection is shown.

[0144] Figure 100 A ligation-RT-PCR-qLDR legacy prevention reaction for identifying or relatively quantifying miRNAs using FRET detection is shown.

[0145] Figure 101 The dimensions of one version of a commercially available 384-well microtiter plate are shown.

[0146] Figure 102 The dimensions of another version of a commercially available 384-well microtiter plate are shown.

[0147] Figure 103 A top view and a side view of a typical 384-well microtiter plate configuration are shown.

[0148] Figure 104 A perspective view of a typical 384-well microtiter plate configuration is shown.

[0149] Figure 105 A top view and a side view of the middle layer of a sample dispersion device positioned over several wells of a microtiter plate are shown.

[0150] Figure 106 An exploded perspective view of the middle layer of a sample dispersion device positioned over several wells of a microtiter plate is shown.

[0151] Figure 107 A top view and a side view of the first layer and the middle layer of a sample dispersion device positioned over several wells of a microtiter plate are shown.

[0152] Figure 108 An exploded perspective view of the first layer and the middle layer of a sample dispersion device positioned over several wells of a microtiter plate is shown.

[0153] Figure 109 A top view and a side view of the third layer, the first layer, and the middle layer of a sample dispersion device positioned over several wells of a microtiter plate are shown.

[0154] Figure 110 An exploded perspective view of the third layer, the first layer, and the middle layer of a sample dispersion device positioned over several wells of a microtiter plate are shown.

[0155] Figure 111 Top and side views of the second, third, first, and intermediate layers of a sample dispersion device positioned over several wells of a microtiter plate.

[0156] Figure 112 Exploded perspective views of the second, third, first, and intermediate layers of a sample dispersion device positioned over several wells of a microtiter plate.

[0157] Figure 113 Top and side views of the first and intermediate layers of a sample dispersion device that pressure fills the channels and metering chambers of each row of a microtiter plate using an alternative fill port.

[0158] Figure 114 Exploded perspective views of the first and intermediate layers of a sample dispersion device that pressure fills the channels and metering chambers of each row of a microtiter plate using an alternative fill port.

[0159] Figure 115 Top and side views of the third, first, and intermediate layers of a sample dispersion device that pressure fills the channels and metering chambers of each row of a microtiter plate using an alternative fill port.

[0160] Figure 116 Exploded perspective views of the third, first, and intermediate layers of a sample dispersion device that pressure fills the channels and metering chambers of each row of a microtiter plate using an alternative fill port.

[0161] Figure 117 Top and side views of the second, third, first, and intermediate layers of a sample dispersion device that pressure fills the channels and metering chambers of each row of a microtiter plate using an alternative fill port.

[0162] Figure 118 Exploded perspective views of the second, third, first, and intermediate layers of a sample dispersion device that pressure fills the channels and metering chambers of each row of a microtiter plate using an alternative fill port.

[0163] Figure 119 Top and side views of the intermediate layer of the outlet side of a sample dispersion device that independently addresses each row of a microtiter plate.

[0164] Figure 120 Exploded perspective views of the intermediate layer of the outlet side of a sample dispersion device that independently addresses each row of a microtiter plate.

[0165] Figure 121Top and side views of the first and middle layers of the outlet side of the sample dispersion device showing each row of an individually addressed microtiter plate.

[0166] Figure 122 Exploded perspective view of the third, first, and middle layers of the outlet side of the sample dispersion device showing each row of an individually addressed microtiter plate.

[0167] Figure 123 Top and side views of the third, first, and middle layers of the outlet side of the sample dispersion device showing each row of an individually addressed microtiter plate.

[0168] Figure 124 Exploded perspective view of the third, first, and middle layers of the outlet side of the sample dispersion device showing each row of an individually addressed microtiter plate.

[0169] Figure 125 Top and side views of the second, third, first, and middle layers of the outlet side of the sample dispersion device showing each row of an individually addressed microtiter plate.

[0170] Figure 126 Exploded perspective view of the second, third, first, and middle layers of the outlet side of the sample dispersion device showing each row of an individually addressed microtiter plate.

[0171] Figure 127 Top and side views of the middle layer of the sample dispersion device showing each row of an individually addressed microtiter plate from both sides.

[0172] Figure 128 Exploded perspective view of the middle layer of the sample dispersion device showing each row of an individually addressed microtiter plate from both sides.

[0173] Figure 129 Top and side views of the first and middle layers of the sample dispersion device showing each row of an individually addressed microtiter plate from both sides.

[0174] Figure 130 Exploded perspective view of the first and middle layers of the sample dispersion device showing each row of an individually addressed microtiter plate from both sides.

[0175] Figure 131 Top and side views of the third, first, and middle layers of the sample dispersion device showing each row of an individually addressed microtiter plate from both sides.

[0176] Figure 132 Exploded perspective view of the third, first, and middle layers of the sample dispersion device showing each row of an individually addressed microtiter plate from both sides.

[0177] Figure 133Top and side views of the second layer, third layer, first layer, and middle layer of a sample dispersion device showing each row of a microtiter plate addressed independently from both sides.

[0178] Figure 134 Exploded perspective view of the second layer, third layer, first layer, and middle layer of a sample dispersion device showing each row of a microtiter plate addressed independently from both sides.

[0179] Figure 135 Top and side views of the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0180] Figure 136 Exploded perspective view of the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0181] Figure 137 Top and side views of the first region of the first layer and the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0182] Figure 138 Exploded perspective view of the first region of the first layer and the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0183] Figure 139 Top and side views of the first region and the second region of the first layer and the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0184] Figure 140 Exploded perspective view of the first region and the second region of the first layer and the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0185] Figure 141 Top and side views of the third layer, the first layer (having a first region and a second region), and the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0186] Figure 142 Exploded perspective view of the third layer, the first layer (having a first region and a second region), and the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0187] Figure 143 Top and side views of the second layer, the third layer, the first layer (having a first region and a second region), and the middle layer of a sample dispersion device showing each row and each column of a microtiter plate addressed independently.

[0188] Figure 144Expedited perspective view of the second, third, first (with first and second regions) and intermediate layers of a sample dispersion device showing each row and column of an independently addressed microtiter plate.

[0189] Figure 145 PCR-LDR-qPCR legacy prevention reaction using Taqman readout for identification or relative quantification of low level mutations is shown.

[0190] Figure 146 PCR-LDR-qPCR legacy prevention reaction using UniTaq readout for identification or relative quantification of low level mutations is shown.

[0191] Figure 147 PCR-qLDR legacy prevention reaction using FRET readout for identification or relative quantification of low level mutations is shown.

[0192] Figure 148 PCR-LDR-qPCR legacy prevention reaction using Taqman readout for identification or relative quantification of low level mutations is shown.

[0193] Figure 149 PCR-LDR-qPCR legacy prevention reaction using UniTaq readout for identification or relative quantification of low level mutations is shown.

[0194] Figure 150 PCR-qLDR legacy prevention reaction using FRET readout for identification or relative quantification of low level mutations is shown.

[0195] Figure 151 PCR-LDR-qPCR legacy prevention reaction using Taqman readout for identification or relative quantification of low level target methylation is shown.

[0196] Figure 152 PCR-LDR-qPCR legacy prevention reaction using UniTaq readout for identification or relative quantification of low level target methylation is shown.

[0197] Figure 153 PCR-qLDR legacy prevention reaction using FRET readout for identification or relative quantification of low level target methylation is shown.

[0198] Figure 154 Loop-PCR-LDR-qPCR legacy prevention reaction using Taqman readout for identification or relative quantification of low level targets and / or mutations is shown.

[0199] Figure 155Loop-PCR-qLDR legacy prevention reaction using FRET reader for identification or relative quantification of low level mutations.

[0200] Figure 156 Loop-PCR-LDR-qPCR legacy prevention reaction using Taqman reader for identification or relative quantification of low level target methylation.

[0201] Figure 157 Loop-PCR-qLDR legacy prevention reaction using FRET reader for identification or relative quantification of low level target methylation.

[0202] Figure 158 PCR-qPCR legacy prevention reaction using Taqman reader for identification or relative quantification of low level target methylation.

[0203] Figure 159 Real-time PCR amplification plot obtained in a PCR-LDR-qPCR experiment for detecting BRAF V600E mutation in the presence of excess wild-type DNA.

[0204] Figure 160 Real-time PCR amplification plot obtained in a pixel PCR-LDR-qPCR experiment for counting single molecules of BRAF V600E mutation.

[0205] Figure 161 Real-time PCR amplification plot obtained in a pixel PCR-LDR-qPCR experiment for counting single molecules of BRAF V600E mutation in the presence of excess wild-type DNA from plasma.

[0206] Figure 162 Real-time PCR amplification plot obtained in a PCR-LDR-qPCR experiment for detecting TP53 R248Q mutation in the presence of excess wild-type DNA.

[0207] Figure 163 Real-time PCR amplification plot obtained in a PCR-LDR-qPCR experiment for detecting TP53 R248Q mutation in the presence of excess wild-type DNA.

[0208] Figure 164 Real-time PCR amplification plot obtained in a pixel PCR-LDR-qPCR experiment for counting single molecules of TP53 R248Q mutation in the presence of excess wild-type DNA.

[0209] Figure 165Real-time PCR amplification plot showing results obtained in a pixel PCR-LDR-qPCR experiment for counting single molecules of TP53 R248Q mutation in the presence of excess wild-type DNA from plasma.

[0210] Figure 166 Real-time PCR amplification plot showing results obtained in a PCR-LDR-qPCR experiment for detecting KRAS G12C mutation in the presence of excess wild-type DNA.

[0211] Figure 167 Real-time PCR amplification plot showing results obtained in a PCR-LDR-qPCR experiment for detecting KRAS G12S mutation in the presence of excess wild-type DNA.

[0212] Figure 168 Real-time PCR amplification plot showing results obtained in a pixel PCR-LDR-qPCR experiment for counting single molecules of KRAS G12C mutation in the presence of excess wild-type DNA from plasma.

[0213] Figure 169 Real-time PCR amplification plot showing results obtained in a PCR-LDR-qPCR experiment for detecting KRAS G12D mutation in the presence of excess wild-type DNA.

[0214] Figure 170 Real-time PCR amplification plot showing results obtained in a PCR-LDR-qPCR experiment for detecting KRAS G12A mutation in the presence of excess wild-type DNA.

[0215] Figure 171 Real-time PCR amplification plot showing results obtained in a PCR-LDR-qPCR experiment for detecting KRAS G12V mutation in the presence of excess wild-type DNA.

[0216] Figure 172 Real-time PCR amplification plot showing results obtained in a pixel PCR-LDR-qPCR experiment for counting single molecules of KRAS G12V mutation in the presence of excess wild-type DNA in plasma.

[0217] Figure 173 Real-time PCR amplification plot showing results obtained in a PCR-LDR-qPCR experiment for detecting the presence or absence of vimentin gene methylation.

[0218] Figure 174 Real-time PCR amplification plot showing results obtained in a pixel PCR-LDR-qPCR experiment for counting single molecules of methylated DNA in the presence of excess unmethylated DNA (hgDNA).

[0219] Figure 175 Real-time PCR amplification plot obtained in an experiment for detecting methylation of VIM S3 top strand using Taqman probe pattern "A" is shown.

[0220] Figure 176 Real-time PCR amplification plot obtained in an experiment for detecting methylation of VIM S3 bottom strand using Taqman probe pattern "A" is shown.

[0221] Figure 177 Real-time PCR amplification plot obtained in an experiment for detecting methylation of VIM S3 top strand using Taqman probe pattern "B" is shown.

[0222] Figure 178 Real-time PCR amplification plot obtained in an experiment for detecting methylation of VIM S3 bottom strand using Taqman probe pattern "B" is shown. DETAILED DESCRIPTION

[0223] Universal design for early detection of disease using "disease marker load"

[0224] The most cost effective early disease detection test can combine an initial multiplexed amplification and ligation assay to determine "disease load". For cancer detection, this would achieve >95% sensitivity with >97% specificity for all cancers (pan-tumorology). Figure 1 A flow chart showing the process for cancer tumor load determination is shown in FIG. 2. An initial multiplexed PCR / LDR screening assay scored for mutations, methylation, miRNA, mRNA, alternative splicing, and translocations identifies those samples that are positive for >5 of 24-48 markers. The putatively positive samples are then assayed with additional tissue specific markers using a "pixel" PCR / LDR assay to confirm the initial results and identify the tissue of origin. The physician can then order targeted sequencing to further guide treatment decisions for the patient.

[0225] The present invention relates to a universal diagnostic method that attempts to combine the best features of digital polymerase chain reaction (PCR), ligation detection reaction (LDR) with quantitative detection of multiple disease markers (e.g. cancer markers). A series of assay configurations and devices includes three modules for PCR-LDR quantification of low abundance disease markers from blood. Each module can be independently optimized and can be manual, semi-automated, or fully automated. The design enables integration of the modules together such that any module can be independently optimized to bring improved performance to the overall assay.

[0226] The first series of assay designs is based on initial multiplex PCR or RT-PCR amplification followed by multiplex LDR using LDR probes with unique sequence tags containing primer specific portions. The products are distributed and subjected to real-time PCR TM with TaqMan probes with target orientation added in each row, allowing real-time PCR

[0227] The first module takes the input blood sample and separates the plasma from the red blood cells (RBCs) and white blood cells (WBCs). It further separates the plasma to remove any residual cells. In addition, a cell fraction is isolated that contains all WBCs and circulating tumor cells (CTCs) and some RBCs. Exosomes are isolated or affinity captured from the plasma. The module (i) purifies RNA from the WBC and CTC fraction, (ii) purifies miRNA and RNA from the exosomes, and (iii) purifies cell-free DNA (cfDNA) from the plasma.

[0228] The second module is capable of distributing the above components into 24 or 48 chambers or wells to enable spatial multiplexing of multiplex PCR or RT-PCR amplification of target genes, promoters, miRNA or mRNA regions in proportion. These include: (i) specific splice variants or gene fusion mRNAs in the CTC containing WBC fraction, (ii) specific miRNAs from exosomes, (iii) specific mRNAs from exosomes, (iv) specific cancer gene DNA regions from cfDNA, and (v) specific (methylation) promoter regions from cfDNA.

[0229] The third module is capable of spatially distributing the above products into the wells of microtiter plates (e.g. in a 24 x 16 or 48 x 32 configuration). This module is capable of detecting and counting the LDR products using real-time PCR to provide quantitative results for each disease marker.

[0230] The first and second modules can be configured to simultaneously process multiple samples for a screening assay mode, where the LDR products containing sequence tags are read out using a real-time PCR reader to provide relative quantitative results (see Figure 2 ). In this configuration, the DNA and RNA isolated from various blood fractions from 24 individual samples are subjected to multiplex PCR-LDR and RT-PCR-LDR and then distributed along the columns, e.g. 16 wells, in a microtiter plate as shown in Figure 3 The tag primer sets for TaqMan TM probes are added in each row, allowing real-time PCR Figure 4 Figure 5 ​). In this illustration, samples #2 and #15 have strong signals at >5 locations, so are considered potentially positive (awaiting additional verification as described more below), while sample #8 with 4 weak signals should also be checked further.

[0231] Two modules can also be configured to process a single sample, where spatial multiplexing enables "pixel" PCR / LDR, where LDR products are able to count original target molecules. This is similar to digital PCR, but at a higher level of multiplexing (see Figure 6 ). In this configuration, DNA and RNA from a single sample are distributed into 24 chambers prior to multiplexed PCR-LDR, as shown in Figure 7 . In this embodiment, some chambers have one or no target molecules. After multiplexing, LDR products are distributed along columns, e.g., 16 wells, in a microtiter plate Figure 7 . Tag primer sets for TaqMan TM probes with target orientation or UniTaq primer sets are added in each row (see Figure 8 ), allowing real-time PCR (see Figure 9 ). The results are interpreted based on Poisson distribution of Ct values representing integer multiples (i.e., 0, 1, 2, etc.) of single molecules in the original mixture. Figure 29 and 30 show Poisson distributions of 6 to 48 and 12 to 96 molecules in 24 wells, respectively, and Figure 31 and 32 show Poisson distributions of 12 to 96 and 24 to 192 molecules in 48 wells, respectively. Figure 9 Row A shows (17:0; 6:1; 1:2), which corresponds to 8 molecules. Figure 9 Row K shows (7:0; 10:1; 5:2; 2:4), which corresponds to about 30 molecules.

[0232] Different forms of dilution and distribution can be used to count a wider range of molecules, as shown in Figure 10-13 for miRNA or mRNA quantification. Here, the original sample is distributed into 8 chambers, diluted 10-fold and distributed into another 8 chambers, etc. (see Figure 11 ). The samples are subjected to multiplexed RT-PCR and LDR, and LDR products are individually distributed along columns Figure 11 . Tag primer sets for TaqMan TM probes with target orientation or UniTaq primer sets are added in each row (see Figure 12 ), allowing real-time PCR and detection (see Figure 13). For the 24 chamber example, this can be quantified over 3 orders of magnitude, but on 48 chambers, it can cover 6 orders of magnitude difference. Figure 33-37 Poisson distribution of 1 to 128 molecules in 8 wells is shown. In Figure 13 In the example shown in row G, the first two dilutions produce higher signals than the last 8 wells (1 :0; 3:1 ; 3:2; 1 :4), which corresponds to about 14-16 x 100 x 1.25 = 1,750 to 2,000 molecules. In contrast, Figure 13 Row N of the same design produces signals only in the first 8 wells (4:0; 3:1 ; 1 :2) which corresponds to about 5-6 x 1.25 = 6 to 8 molecules.

[0233] A second series of assay designs is based on initial multiplex PCR or RT-PCR amplification, followed by distribution and capture of the PCR amplified targets on the wells of a microtiter plate. A single cycle of LDR is able to capture the LDR products on the correct targets on a solid support, while washing away the mismatched adapters. The LDR products are quantified by LDR-FRET, real-time PCR or other reporter systems.

[0234] The first module takes an input blood sample and isolates CTCs (if present), separates the plasma from the blood cells, separates the exosomes from the plasma, and then (i) purifies DNA and RNA (if present) from the CTCs, (ii) purifies miRNA and RNA from the exosomes, and (iii) purifies cfDNA from the plasma.

[0235] The second module is able to distribute the above components into 24 or 48 chambers or wells to enable spatial multiplexing of multiplex PCR or RT-PCR amplification of target genes, promoters, miRNAs or mRNA regions in proportion. These include: (i) specific chromosomal regions from CTCs for copy number counting, (ii) specific splice variants or gene fusion mRNAs from CTCs, (iii) specific miRNAs from exosomes, (iv) specific mRNAs from exosomes, (v) specific cancer gene DNA regions from cfDNA, and (vi) specific (methylation) promoter regions from cfDNA.

[0236] The third module is able to spatially distribute the above products along columns, e.g., 16 wells in a microtiter plate, and then capture the amplified targets on a solid support, e.g., in a 24 x 16 or 48 x 32 configuration. This module is able to capture the LDR products on a solid support, and then detect and count the LDR products to provide quantitative results for each marker.

[0237] The first and second modules can be configured to process multiple samples simultaneously for a screening assay mode, where the LDR products provide relative quantitative results analogous to real-time PCR readouts (see Figure 14 ). In this configuration, DNA and RNA isolated from various blood fractions from 24 individual samples are subjected to multiplex PCR and RT-PCR, then distributed along columns, e.g., 16 wells, in a microtiter plate, and captured on a solid support (see Figure 15 and 16 ). LDR probes are added in each row, and ligation on the correct target captures the product, while washing away unreacted primers ( Figure 17 ). The LDR-FRET results shown in Figure 18 are shown in their respective wells, but in this module, the products can also be denatured and counted using capillary electrophoresis techniques to provide quantitative results. In this illustration ( Figure 18 ), samples #2 and 15 have strong signals at >5 locations, so are presumptive positives, while sample 8 with 4 weak signals should also be checked

[0238] The two modules can also be configured to process a single sample, where spatial multiplexing enables "pixel" PCR / LDR, where the LDR products are capable of counting the original target molecules, analogous to digital PCR, but at a higher multiplexing level ( Figure 19 ). In this configuration, DNA and RNA from a single sample are distributed into 24 chambers prior to multiplex PCR ( Figure 20 ), such that some chambers have one or no target molecules. Amplicons are distributed along columns, e.g., 16 wells, in a microtiter plate, and captured on a solid support ( Figure 20 and 21 ). LDR probe addition and LDR product detection are as described above ( Figure 22 and 23 ). The results are interpreted based on the Poisson distribution of LDR values representing integer multiples (i.e., 0, 1, 2, etc.) of the single molecules in the original mixture. Figure 29 and 30 show Poisson distributions of 6-48 and 12-96 molecules in 24 wells, respectively, and Figure 31 and 32 show Poisson distributions of 12-96 and 24-192 molecules in 48 wells, respectively. Figure 23 , row A shows (#addressed:#initial target molecules) as (17:0; 6:1; 1:2), which corresponds to 8 molecules. Figure 23 , row K shows (7:0; 10:1; 5:2; 2:4), which corresponds to about 30 molecules.

[0239] Different forms of dilution and distribution can be used to count a wider range of molecules, such as Figure 24-28 The quantification is indicated for miRNA or mRNA. In this paper, the initial sample was distributed into 8 chambers, diluted 10-fold, and distributed into another 8 chambers, etc. Figure 25 Perform multiplex RT-PCR on the sample and distribute it along the column. Capture the RT-PCR products on a solid support. Figure 26 And add the LDR primer set to each line (see...) Figure 27 For a 24-compartment example, this approach quantifies on three orders of magnitude, but on a 48-compartment example, it covers six orders of magnitude of variation (see [link to relevant documentation]). Figure 33-37 (Poisson distribution). Figure 28 In the example shown, row G, the first two dilutions produced higher signals than the last eight wells (1:0; 3:1; 3:2; 1:4), corresponding to approximately 14-16 x 100 x 1.25 = 1,750 to 2,000 molecules. In contrast, Figure 28 Row N generates a signal only in the first 8 pore distributions (4:0; 3:1; 1:2), corresponding to approximately 5-6 x 1.25 = 6 to 8 molecules.

[0240] False positives and residual prevention

[0241] Distinguishing true signals generated by desired disease-specific nucleic acid differences from false signals generated by normal nucleic acids present in a sample, and from false signals generated in the absence of disease-specific nucleic acid differences (i.e., somatic mutations), presents a technical challenge.

[0242] The following section presents several solutions to these challenges, but they all share a common theme.

[0243] The first topic is multiplexing. PCR works optimally when primer concentrations are relatively high, ranging from 50 nM to 500 nM, limiting multiplexing. Furthermore, the more PCR primer pairs added, the exponentially increases the likelihood of amplifying incorrect products or generating primer dimers. In contrast, for LDR probes, low concentrations in the order of 4 nM to 20 nM are used, and the requirement for ligation events through adjacent hybridization on the target limits probe dimerization. Using low concentrations of gene-specific PCR primers or LDR probes containing a universal primer sequence "tail" allows for the subsequent addition of higher concentrations of universal primers to achieve proportional amplification of the initial PCR or LDR product. Another way to avoid or minimize spurious PCR amplicon or primer dimers is to use PCR primers containing several additional bases and a blocking group that is released only upon hybridization with the target via cleavage by a nuclease to form a free 3'OH, such as a ribonucleotide base as the blocking group and RNase H2 as the cleavage nuclease.

[0244] The second theme is signal fluctuation caused by low input of target nucleic acids. Typically, the target nucleic acids are derived from several cells, captured in CTC format or from tumor cells that undergo apoptosis and release their DNA in small fragments (140-160 bp) into the serum. In such cases, it is preferable to perform a certain level of proportional amplification to avoid complete loss of signal or reporting inaccurate copy numbers due to fluctuations when distributing small numbers of starting molecules into individual wells (for real-time or droplet PCR quantification). As long as these initial universal amplifications are kept at a reasonable level (approximately 12 to 20 cycles), the risk of carryover contamination during the process of opening tubes and distributing amplicons for subsequent detection / quantification (using real-time or droplet PCR quantification) is minimized.

[0245] The third theme is target-independent signal, also known as "no template control" (NTC). This arises from polymerase or ligase reactions that occur in the absence of the correct target. A portion of this signal can be minimized by judicious primer design. For ligation reactions, the 5'→3' nuclease activity of the polymerase can be used to release the 5' phosphate of the downstream ligation primer (only when hybridized to the target), so the ligation reaction is adapted to ligate. Additional specificity for distinguishing low levels of mutation presence can be achieved by (i) using an upstream LDR probe containing a mismatch at the 2nd or 3rd position from the 3' OH, (ii) using an LDR probe against the wild-type sequence that (optionally) ligates but does not undergo additional amplification, and (iii) using an upstream LDR probe containing several additional bases and a blocking group that is released by cleavage with a nuclease only when hybridized to the complementary target, forming a free 3' OH (e.g., RNase H2 and ribonucleotide bases).

[0246] The fourth theme is inhibited (reduced) amplification or inaccurate (false) amplification caused by unused primers in the reaction. One way to eliminate such unused primers is to capture the genomic or target or amplified target DNA on a solid support, hybridize and ligate the ligation probes, and then remove the unhybridized probes or products. An alternative solution includes pre-amplification followed by a subsequent nested LDR and / or PCR step, such that there is a secondary selection in the process.

[0247] A fifth theme is carryover prevention. Carryover signals can be eliminated by incorporating standard uracils during the universal amplification step and using UDG (and optionally AP endonuclease) in the pre-amplification check procedure. Incorporation of uracils in conjunction with carryover prevention is a key to the method of the present application, as described in more detail below. Initial PCR amplification is performed using incorporation of uracils. LDR reaction is performed using LDR probes that lack uracils. Thus, when LDR products are subjected to real-time PCR amplification, the addition of UDG destroys the initial PCR products but not the LDR products. Furthermore, because LDR is a linear process and the tag primers use sequences that are not present in the human genome, carryover of LDR products back to the original PCR does not generate target-independent amplification. Additional solutions to provide carryover prevention in the case of methylation targets include using restriction endonucleases prior to amplification or after bisulfite treatment, if the latter approach is used, as described below.

[0248] Methods of identifying disease markers

[0249] A first aspect of the invention relates to a method for identifying one or more nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequence of other nucleic acid molecules in the sample or other samples by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues. The method includes providing a sample that potentially contains one or more nucleic acid molecules that contain a target nucleotide sequence that differs from the nucleotide sequence of other nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and / or one or more methylation residues; and contacting the sample with one or more enzymes capable of digesting a deoxyuracil (dU)-containing nucleic acid molecule present in the sample. One or more primary oligonucleotide primer sets are provided, each primary oligonucleotide primer set including (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a sequence 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 by the first primary oligonucleotide primer. The contacted sample is blended with the one or more primary oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a polymerase chain reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles including a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a primary extension product comprising the target nucleotide sequence or a complement thereof. The method further includes blending the primary extension product with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture. Each oligonucleotide probe set includes (a) a first oligonucleotide probe having a target nucleotide sequence-specific portion and (b) a second oligonucleotide probe having a target nucleotide sequence-specific portion, wherein the first oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize to the complementary target nucleotide sequence of the primary extension product adjacent to each other in a base-specific manner, with a junction between the first oligonucleotide probe and the second oligonucleotide probe. The first oligonucleotide probe and the second oligonucleotide probe of the one or more oligonucleotide probe sets are ligated together to form a ligation product sequence in the ligation reaction mixture, and the ligation product sequence is detected and distinguished in the sample to identify the presence of one or more nucleic acid molecules that contain the target nucleotide sequence that differs from the nucleotide sequence of other 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 methylation residues.

[0250] Figure 38-44 Various embodiments of this aspect of the invention are illustrated.

[0251] Figure 38(Step AF) illustrates an exemplary PCR-LDR-qPCR legacy prevention reaction for detecting mutations in genomic DNA or cell-free DNA (cfDNA). The method begins with the isolation of genomic DNA or cell-free DNA (cfDNA), as shown in Step A. Figure 38 As shown in step B, the DNA sample is treated with an enzyme capable of digesting nucleic acid molecules containing deoxyuridine (dU) that may be present in the sample. Suitable enzymes include, but are not limited to, E. coli uracil DNA glycosylase (UDG), Antarctic thermosensitive UDG, or human single-stranded selective monofunctional uracil DNA glycosylase (hSMUG1). The sample is then subjected to an amplification reaction, such as polymerase chain reaction (PCR), to amplify the target region containing the mutation. The amplification reaction is performed using locus-specific primers and a mixture of deoxynucleotides containing dUTP. In one embodiment, finite-cycle amplification (12-20 cycles) is performed to maintain the relative ratio of different amplicones produced. In another embodiment, 20-40 cycles are used to amplify the target region. Figure 38 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0252] like Figure 38 As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and a ligase (solid circle) covalently seals the two oligonucleotides together when hybridizing with their complementary sequence. The upstream oligonucleotide probe contains a 5' primer-specific portion (Ai), and the downstream oligonucleotide probe contains a 3' primer-specific portion (Ci') that allows for subsequent amplification of the ligation product. After ligation, the ligation product is aliquoted into individual wells containing one or more tag-specific primer pairs, each pair containing matched primers Ai and Ci, treated with UDG or a similar enzyme to remove amplification products or contaminants containing dU, and then subjected to PCR amplification and detection. Figure 38 As shown in steps E and F, the traditional TaqMan can be used. TM The detection assay is used to detect the linkage products (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 herein by reference in their entirety). For the use of TaqMan... TM The detection method involves combining an oligonucleotide probe spanning the ligation junction with primers suitable for hybridization to the primer-specific portion of the ligation product for amplification and detection. TaqMan TM The probe contains a fluorescent reporter group (F1) at one end and a quencher molecule (Q) at the other end. In a complete probe, the two are brought close enough together that the quencher molecule quenches the fluorescence of the reporter group. During amplification, TaqMan... TMThe probe and upstream primer hybridize to the complementary region of their ligation product. The 5'→3' nuclease activity of the polymerase extends the hybridization primer and releases TaqMan. TM The fluorescent group of the probe generates a detectable signal. Figure 38 (Step F). During the amplification reaction, dUTP is used to generate a product containing dU, which can then be destroyed using UDG for legacy prevention.

[0253] Figure 39 An exemplary PCR-qLDR legacy prevention reaction for detecting mutations in genomic DNA or cell-free DNA (cfDNA) is shown. The method begins with the isolation of genomic DNA or cell-free DNA (cfDNA), as shown in step A. Figure 39 As shown in step B, the DNA sample is treated with a deoxyuridine (dU) digestive enzyme (such as UDG) to digest any dU-containing nucleic acid molecules that may be present in the sample, followed by an amplification reaction, such as polymerase chain reaction (PCR), to amplify the target region containing the mutation. The amplification reaction is performed using a locus-specific primer and a mixture of deoxynucleotides containing dUTP. In one embodiment, a limited number of cycles of amplification (12-20 cycles) are performed to maintain a relative ratio of different amplicones produced. In another embodiment, 20-40 cycles are used to amplify the target region. In this embodiment, the locus-specific primer also contains a 5' primer region, such as a universal primer region, which allows for subsequent universal PCR amplification using biotin-labeled primers to attach 5' biotin to the amplified product containing the target region. Figure 39 (Step B).

[0254] like Figure 39 As shown in step C, the amplification product is incorporated with dU, thus allowing for legacy prevention, and is captured on a solid support via an additional 5' biotin moiety. The target mutation is detected using a mutation-specific ligation probe, such as... Figure 39 As shown in step D. In this embodiment, the first ligation probe contains a 3' target-specific region and a 5' tail sequence having a donor or acceptor portion, and the second ligation probe in the probe set contains a 5' target-specific region and a 3' tail sequence having a acceptor or donor portion. The 5' and 3' tail sequences of the ligation probes in the probe set are complementary to each other, and the acceptor and donor groups are capable of generating a detectable signal through fluorescence resonance energy transfer (FRET) that occurs when they are in close proximity to each other. After ligation, the unligated oligonucleotide probes are washed away, and the ligation product is denatured from the immobilized amplification product. After denaturation ( Figure 39 In step E), the complementary 5' and 3' tail sequences of the ligation product hybridize with each other, thereby bringing the donor and acceptor groups into close proximity to generate a detectable FRET signal.

[0255] Alternative forms of FRET detection can be used to distinguish the ligation products formed according to this aspect of the invention. For example, the upstream probe may contain a fluorescent reporter group at the 5' end, followed by a tail sequence portion, a quencher group (e.g., ZEN), and a target-specific portion, such as... Figure 39 As shown in step F. In the single-stranded form, the fluorescent group is quenched by the Zen group. After connecting the upstream and downstream ligation probes and denaturing the resulting ligation product, the complementary 5' and 3' tails of the ligation product hybridize to form a short double-stranded portion. In this formation, the reporter group is no longer quenched by the quenching group, and a detectable signal is generated.

[0256] Figure 39 A surface-coated method for capturing extension products on a solid support using a biotinylated universal primer-streptavidin coating is illustrated. This capture can occur before or after the ligation step. Other methods for ligating products to a solid support include covalently attaching a portion or majority of the universal primer to the solid support prior to PCR amplification.

[0257] In addition to using biotin-streptavidin to capture polymerase extension products, primers can be designed to include a capture sequence at the 5' end, a polymerase extension blocking group, and a universal or target-specific portion at the 3' end. After amplification, the 5' capture sequence portion of the product is single-stranded, and if it is long and / or GC-rich, it can be captured on a complementary sequence under conditions that cause denaturation of the uncaptured strand or removal by cleavage, such as cleavage with a λ exonuclease. The capture step can be enhanced by using PNA, LNA, or other nucleotide analogs within the primer, capture probe sequence, or both.

[0258] In another embodiment, primers can be covalently attached to a solid surface using the following: dibenzocyclooctyl (DBCO) for copper-free click chemistry (with azide); 5-octadiynyl dU for click chemistry (with azide); amino-modifying C6 dT (for peptide bonds); or azide for click chemistry with olefin DBCO.

[0259] Figure 40 This illustrates another exemplary PCR-LDR-qPCR legacy prevention response for detecting mutations. Genomic DNA or cfDNA is isolated ( Figure 40 Step A) involves treating the isolated DNA sample with UDG to digest any dU-containing nucleic acid molecules that may be present in the sample. In this embodiment, initial amplification is performed using locus-specific PCR primers containing a cleavable blocking group at its 3' end. The blocking group prevents non-target-specific polymerase extension and amplification. Figure 40As shown in step B, the suitable blocking group is an RNA base (r) that is cleaved by RNase-H (star symbol) only when the primer hybridizes to its complementary sequence (see, 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), which is incorporated herein by reference in its entirety). Cleavage of the RNA base releases a 3'OH group suitable for extension by polymerase.

[0260] After the primer blocking groups are cleaved, the target region is amplified and the PCR product contains dU, thus allowing for residual prevention (…). Figure 40 (Step C). Then, the target-specific oligonucleotide probe containing primer tags (Ai and Ci') hybridizes with the amplification product in a base-specific manner, and when hybridization occurs with its complementary sequence, a ligase (solid circle) covalently seals the two oligonucleotides together. Figure 40 (Step D). Use matching primer pairs Ai and Ci and TaqMan. TM The probe detects the connection products, the TaqMan TM The probe spans across as Figure 38 The connection points described in (see) Figure 40 (Steps EF).

[0261] Figure 41 This illustrates another exemplary PCR-LDR-qPCR legacy prevention response for detecting mutations. Genomic DNA or cfDNA is isolated ( Figure 41 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 41 (Step B) Amplify the target region using locus-specific primers and a mixture of deoxynucleotides containing dUTP. In one embodiment, finite-cycle amplification (12-20 cycles) is performed to maintain the relative ratio of the different amplicons produced. In another embodiment, 20-40 cycles are used to amplify the target region. Figure 41 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0262] like Figure 41As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridized with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. In this embodiment, the upstream oligonucleotide probe with a sequence specific for detecting the target mutation also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product, while the upstream oligonucleotide probe with a sequence specific for detecting the wild-type (non-mutated) nucleic acid sequence does not contain a 5' primer-specific portion. The downstream oligonucleotide probe with 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 with a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Following target-specific hybridization, RNase H (star symbol) removes the RNA group to generate a 3'OH group that can be linked. Figure 41 (Step D). After ligation, matching primer pairs Ai and Ci and TaqMan can be used. TM The TaqMan probe or other suitable means known in the art are used to detect the connection products. TM The probe spans across as Figure 38 The connection points described in (see) Figure 41 Step EG).

[0263] Figure 42 This illustrates another exemplary PCR-qLDR legacy prevention response for detecting mutations. Genomic DNA or cfDNA is isolated ( Figure 42 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 42 (Step B). The target region is amplified using a mixture of locus-specific primers and deoxynucleotides containing dUTP. In this embodiment, the locus-specific primers also contain a 5' primer region, such as a universal primer region. Such sequences can be amplified by subsequent universal PCR using biotin-labeled primers to attach 5' biotin to the amplification product containing the target region. Figure 42 (Step B). The biotinylated PCR product is immobilized onto a solid support and the target mutation is detected using a mutation-specific ligation probe, such as... Figure 42As shown in step D. In this embodiment, the ligation probe having a ligation pair capable of detecting mutant nucleic acid sequences (but not wild-type sequences) contains complementary tail sequences and contains acceptor or donor groups, which are capable of generating a detectable signal through FRET that occurs when they are in close proximity to each other, as described above. Figure 39 As described above, as shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. After target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a ligation-enabled 3'OH group (r). Figure 42 Step D). After the connection ( Figure 42 Step E) involves the complementary 5' and 3' ends of the ligation product hybridizing to each other, thereby bringing their respective donor and acceptor portions into close proximity to generate a detectable FRET signal. Figure 42 Step F).

[0264] Figure 43 This illustrates another exemplary PCR-qLDR legacy prevention response for detecting mutations. Genomic DNA or cfDNA is isolated ( Figure 43 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 43 (Step B). The target region is amplified using a mixture of locus-specific primers and deoxynucleotides containing dUTP. In this embodiment, the locus-specific primers also contain a 5' primer region, such as a universal primer region. These regions can be amplified by subsequent universal PCR using biotin-labeled primers to attach 5' biotin to the amplification product containing the target region. Figure 43 (Step B). The biotinylated PCR product is immobilized onto a solid support and the target mutation is detected using a mutation-specific ligation probe, such as... Figure 43 As shown in step D. In this embodiment, the oligonucleotide probes of the probe set are designed such that the 3' base of the first oligonucleotide probe overlaps with the adjacent 5' base of the second oligonucleotide probe, which is complementary to the target nucleic acid molecule, as shown in step D. Figure 43As shown in step D. The overlapping nucleotides are referred to as "flaps". When the overlapping flanking nucleotide of the second oligonucleotide probe is complementary to the target nucleic acid molecule sequence and is the same sequence as the terminating 3' nucleotide of the first oligonucleotide probe, the phosphodiester bond upstream of the flanking nucleotide of the second oligonucleotide probe is discriminatively cleaved by an enzyme with flanking endonuclease (FEN) or 5' nuclease activity (e.g., the 5'-3' exonuclease of Taq polymerase). This specific FEN activity generates a ligable 5' phosphate terminus on the second oligonucleotide probe, which is precisely positioned along the adjacent 3' OH of the first oligonucleotide probe. Very high target detection specificity and sensitivity are achieved because of (a) target-specific annealing through adjacent oligonucleotide probes, (b) selective generation of the 5' phosphate only when the cleaved flanking nucleotide matches the template, and (c) the addition of a ligase that discriminates against non-Watson-Crick pairing of the 3' base of the first oligonucleotide probe. According to this embodiment, the oligonucleotide probe used for ligation also contains complementary tail sequences and, respectively, acceptor or donor groups, which are capable of generating a detectable signal through FRET that occurs when they are in close proximity to each other, as described above. Figure 39 As mentioned above. After the connection ( Figure 43 Step E) involves the complementary 5' and 3' ends of the ligation product hybridizing to each other, thereby bringing their respective donor and acceptor portions into close proximity to generate a detectable FRET signal. Figure 43 Step F).

[0265] Figure 44 This illustrates another PCR-LDR-qPCR legacy prevention reaction for detecting mutations. Genomic DNA or cfDNA is isolated ( Figure 44 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 44 (Step B) Amplification of the target region is performed using locus-specific primers and a mixture of deoxynucleotides containing dUTP. In this embodiment, the ligation probe is designed to contain UniTaq primers and tag sequences to facilitate detection. The UniTaq system is fully described in Spier's U.S. Patent Application Publication No. 2011 / 0212846, which is incorporated herein by reference in its entirety. The UniTaq system involves the use of three unique "tag" sequences, wherein at least one unique tag sequence (Ai) is present in the first oligonucleotide probe, and a second unique tag sequence and a third unique tag sequence (Bi' and Ci') are present in the second oligonucleotide probe, as shown below. Figure 44shown in Step D. After ligation of the set of ligation probes, the resulting ligation product contains the Ai sequence - target-specific sequence - Bi’ sequence - Ci’ sequence. The essence of the UniTaq method is that both oligonucleotide probes in the set of ligation probes need to be correct to obtain a positive signal, which thus allows for highly multiplexed nucleic acid detection. For example, and as described herein, this is achieved by requiring that two parts, the two of the tag, hybridize to each other.

[0266] Prior to detection of the ligation product, the sample is treated with UDG to destroy the original target amplicon, thus allowing only the true ligation product to be detected. For detection, the ligation product containing Ai (first primer-specific portion), Bi’ (UniTaq detection portion), and Ci’ (second primer-specific portion) is primed on both strands using a first oligonucleotide primer with the same nucleotide sequence as Ai and a second oligonucleotide primer complementary to Ci’ (i.e., Ci). The first oligonucleotide primer also contains at one end a UniTaq detection probe (Bi) with a detectable label Fl and at the other end a quencher molecule (Q) (Fl-Bi-Q-Ai). Optionally, positioned in close proximity to the quencher is a polymerase blocking unit, such as HEG, THF, Sp-18, ZEN, or any other blocker known in the art that is sufficient to stop polymerase extension. PCR amplification results in a double-stranded product, as shown in Step E. Figure 44 Step F). In this example, the polymerase blocking unit prevents the polymerase from copying the 5’ portion of the first universal primer (Bi), such that the bottom strand of the product cannot form a hairpin when it becomes single-stranded. Formation of this hairpin anneals the 3’ end of the stem to the amplicon, such that polymerase extension of this 3’ end terminates the PCR reaction.

[0267] The double-stranded PCR product is denatured, and when the temperature is subsequently lowered, 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 G). In addition, in this step, the second oligonucleotide primer anneals to the 5’-primer-specific portion of the hairpin-like product (Ci’). After extension of the second universal primer in Step G, 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. Figure 44

[0268] Figure 145 Another exemplary PCR-LDR-qPCR legacy prevention reaction for detecting low levels of mutations is shown. Genomic DNA or cfDNA is isolated (Step A), and the isolated DNA sample is treated with UDG to digest any dU-containing nucleic acid molecules that can be present in the sample (Step B). Figure 145 Figure 145 ​​(Step B). The target region is selectively amplified using a mutant-selective upstream primer, a locus-specific downstream primer, and a mixture of deoxynucleotides containing dUTPs. As shown in this figure, another layer of selectivity can be added to the method by including a cleavable blocking group (Blk 3', e.g., a C3 spacer) and a mutant-specific RNA base (mr) in the upstream mutant-specific primer. After target-specific hybridization, RNase H (star symbol) removes the RNA base to release a 3'OH group suitable for polymerase extension. Figure 145 (Step B). RNase H preferentially cleaves RNA bases when the primers are a perfect match for mutant DNA, but is less likely to cleave RNA bases when hybridizing with wild-type DNA. Once cleavage occurs, the polymerase faithfully extends the released 3'OH and copies either the mutant or wild-type bases of the target. Therefore, unlike allelic-specific PCR, PCR primers do not amplify primer-derived mutations. Instead, this PCR selectively amplifies the mutant target relative to the wild-type target in each amplification cycle by copying bases via repeated cycles of hybridization, cleavage, extension, and denaturation. Optional primers with wild-type sequences lack RNA bases and remain blocking, thus further reducing the amplification of wild-type sequences. Optionally, samples are aliquoted into 24, 48, or 96 wells prior to PCR. Figure 145 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0269] like Figure 145 As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridized with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. In this embodiment, the upstream oligonucleotide probe with a sequence specific for detecting the target mutation also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product, while the upstream oligonucleotide probe with a sequence specific for detecting the wild-type (non-mutated) nucleic acid sequence does not contain a 5' primer-specific portion. The downstream oligonucleotide probe with 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 with a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Following target-specific hybridization, RNase H (star symbol) removes RNA bases to generate a 3'OH group that can be linked. Figure 145(Step D). After ligation, matching primer pairs Ai and Ci and TaqMan can be used. TM The TaqMan probe or other suitable means known in the art are used to detect the connection products. TM The probe spans across as Figure 38 The connection points described in (see) Figure 145 Step EG).

[0270] Figure 146 This illustrates another exemplary PCR-LDR-qPCR legacy prevention response for detecting low-level mutations. Genomic DNA or cfDNA is isolated ( Figure 146 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 146 (Step B). The target region is selectively amplified using a mutant-selective upstream primer, a locus-specific downstream primer, and a mixture of deoxynucleotides containing dUTP. As shown in this figure, another layer of selectivity can be added to the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and a mutant-specific RNA base (mr) in the upstream mutant-specific primer. After target-specific hybridization, RNase H (star symbol) removes the RNA base to release a 3'OH group suitable for polymerase extension (…). Figure 146 (Step B). RNase H preferentially cleaves RNA bases when the primers are a perfect match for mutant DNA, but is less likely to cleave RNA bases when hybridizing with wild-type DNA. Once cleavage occurs, the polymerase faithfully extends the released 3'OH and copies either the mutant or wild-type bases of the target. Therefore, unlike allelic-specific PCR, PCR primers do not amplify primer-derived mutations. Instead, this PCR selectively amplifies mutant targets relative to wild-type targets in each amplification cycle by copying bases via repeated cycles of hybridization, cleavage, extension, and denaturation. Optional primers with wild-type sequences lack RNA bases and remain blocking, thus further reducing the amplification of wild-type sequences. Optionally, samples are aliquoted into 12, 24, 48, or 96 wells prior to PCR. Figure 146 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0271] like Figure 146As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridized with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. In this embodiment, the upstream oligonucleotide probe with a sequence specific for detecting the target mutation also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product, while the upstream oligonucleotide probe with a sequence specific for detecting the wild-type (non-mutated) nucleic acid sequence does not contain a 5' primer-specific portion. The downstream oligonucleotide probe with 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 probe with a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Following target-specific hybridization, RNase H (star symbol) removes RNA bases to generate a 3'OH group that can be linked. Figure 146 (Step D). After ligation, the ligation product is amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and as described above for... Figure 44 As described (see Figure 146 Detection can be performed using steps EH or other suitable means known in the art.

[0272] Figure 147 This illustrates another exemplary PCR-qLDR legacy prevention response for detecting low-level mutations. Genomic DNA or cfDNA is isolated ( Figure 147 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 147 Step B). The target region is selectively amplified using a mutant-selective upstream primer, a locus-specific downstream primer, and a mixture of deoxynucleotides containing dUTPs. As shown in Step B of this figure, another layer of selectivity can be added to the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and a mutant-specific RNA base (mr) in the upstream mutant-specific primer. After target-specific hybridization, RNase H (star symbol) removes the RNA base to release a 3'OH group suitable for polymerase extension (…). Figure 147, step B). RNase H will preferentially cleave RNA bases when the primer is perfectly matched to the mutant DNA, but is unlikely to cleave RNA bases when hybridized to wild-type DNA. Once the cleavage reaction occurs, the polymerase faithfully extends the released 3' OH and copies the mutant or wild-type bases of the target. Thus, unlike allele-specific PCR, the PCR primer does not propagate the primer-derived mutation. Rather, this PCR selectively amplifies the mutant target over the wild-type target during each amplification cycle by copying the bases through repeated cycles of hybridization, cleavage, elongation, and denaturation. The optional primer with the wild-type sequence lacks the RNA base and remains blocked, thus further reducing amplification of the wild-type sequence. In this embodiment, the downstream locus-specific primer also contains a 5' primer region, such as a universal primer region, enabling universal PCR amplification using biotin-labeled primers to append 5' biotin to the amplification product containing the region of interest Figure 146 , step B). Optionally, the sample is aliquoted into 12, 24, 48, or 96 wells prior to PCR. The biotinylated PCR product is immobilized to a solid support and the target mutation is detected using a mutation-specific ligation probe, as Figure 147 shown in step D. In this embodiment, the ligation probe with the pair of ligation probes capable of detecting the mutant nucleic acid sequence (but not the wild-type sequence) contains a complementary tail sequence and contains a receptor group or a donor group, respectively, that are capable of generating a detectable signal through FRET that occurs upon close proximity to each other, as described above for Figure 39 . As shown in this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Upon target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a 3' OH group that can be ligated Figure 147 , step D). Following ligation Figure 147 , step E), the complementary 5' and 3' tail ends of the ligation product hybridize to each other, bringing their respective donor and receptor moieties into close proximity to each other to generate a detectable FRET signal Figure 147 , step F).

[0273] Figure 148 shows another exemplary PCR-LDR-qPCR legacy prevention reaction for detecting low-level mutations. Genomic DNA or cfDNA is isolated Figure 148 , step A), and the isolated DNA sample is treated with UDG to digest any dU-containing nucleic acid molecules that can be present in the sample Figure 148(Step B). The target region is selectively amplified using a locus-specific upstream primer, a locus-specific downstream primer, a blocking LNA or PNA probe containing a wild-type sequence, and a mixture of deoxynucleotides containing dUTP. In this embodiment, another layer of selective binding to the method can be added by including a cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream primer. After target-specific hybridization, RNase H (star symbol) removes the RNA base to release several bases upstream of the mutation and a 3'OH group suitable for polymerase extension (r). Figure 148 (Step B). 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 not for mutant DNA, thereby inhibiting the amplification of wild-type DNA in each round of PCR. Optionally, the samples are aliquoted into 12, 24, 48, or 96 wells before PCR. Figure 148 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0274] like Figure 148 As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridized with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. In this embodiment, the upstream oligonucleotide probe, having a sequence specific for detecting the target mutation, also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product. Again, during the PCR amplification step, after enrichment of the mutant sequence, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits ligation with the wild-type target sequence (if present). The downstream oligonucleotide probe, having 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 having a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Following target-specific hybridization, RNase H (star symbol) removes RNA bases to generate a 3'OH group that can be linked. Figure 148 (Step D). After ligation, matching primer pairs Ai and Ci and TaqMan can be used. TM The TaqMan probe or other suitable means known in the art are used to detect the connection products. TM The probe crosses the above-mentioned targets Figure 38 The connection point (see above) Figure 148Step EG).

[0275] Figure 149 This illustrates another exemplary PCR-LDR-qPCR legacy prevention response for detecting low-level mutations. Genomic DNA or cfDNA is isolated ( Figure 149 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 149 Step B). The upstream locus-specific primers are designed to be several bases upstream of the mutation and contain a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r). After target-specific hybridization, RNase H (star symbol) removes the RNA base to release a 3' OH group suitable for polymerase elongation. Figure 149 (Step B). 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 not for mutant DNA, thereby inhibiting the amplification of wild-type DNA in each round of PCR. Optionally, the samples are aliquoted into 12, 24, 48, or 96 wells before PCR. Figure 148 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0276] like Figure 149 As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridized with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. In this embodiment, the upstream oligonucleotide probe, having a sequence specific for detecting the target mutation, also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product. Again, during the PCR amplification step, after enrichment of the mutant sequence, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits ligation with the wild-type target sequence (if present). The downstream oligonucleotide probe, having 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 probe having a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Following target-specific hybridization, RNase H (star symbol) removes RNA bases to generate a 3'OH group that can be linked. Figure 149 (Step D). After ligation, the ligation product is amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and as described above for...Figure 44 As described (see Figure 149 Detection can be performed using steps EH or other suitable means known in the art.

[0277] Figure 150 This illustrates another exemplary PCR-qLDR legacy prevention response for detecting low-level mutations. Genomic DNA or cfDNA is isolated ( Figure 150 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 150 Step B). The upstream locus-specific primers are designed to be several bases upstream of the mutation and contain a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r). After target-specific hybridization, RNase H (star symbol) removes the RNA base to release a 3' OH group suitable for polymerase elongation. Figure 150 (Step B). 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 not for mutant DNA, thereby inhibiting the amplification of wild-type DNA in each round of PCR. In this embodiment, the downstream locus-specific primer also contains a 5' primer region, such as a universal primer region, thereby enabling universal PCR amplification using biotin-labeled primers to attach 5' biotin to the amplification product containing the target region (…). Figure 150 (Step B). Optionally, samples are aliquoted into 12, 24, 48, or 96 wells prior to PCR. The biotinylated PCR product is immobilized on a solid support and the target mutation is detected using a mutation-specific ligation probe, such as... Figure 150 As shown in step D. Again, during the PCR amplification step, after enriching the mutant sequence, the presence of a blocking LNA or PNA probe containing the wild-type sequence inhibits ligation with the wild-type target sequence (if present). In this embodiment, the ligation probe having a ligation pair capable of detecting the mutant nucleic acid sequence (but not the wild-type sequence) contains complementary tail sequences and, respectively, a acceptor group or a donor group, which can generate a detectable signal through FRET that occurs when they are in close proximity to each other, as described above. Figure 39 As shown in this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. After target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a ligation-enabled 3'OH group (r). Figure 150 Step D). After the connection ( Figure 150, step E), the complementary 5' and 3' tails of the ligation product hybridize to each other, thereby bringing their respective donor and acceptor moieties into close proximity to generate a detectable FRET signal Figure 150

[0278] In another embodiment of this aspect of the application, the first primary oligonucleotide primer of the primary oligonucleotide primer set comprises a 5' portion having a nucleotide sequence identical to the portion of the nucleotide sequence in the wild-type nucleic acid molecule to which the primary oligonucleotide primer hybridizes, but having one or more nucleotide sequence mismatches to the corresponding portion of the nucleotide sequence in the target nucleic acid molecule.

[0279] According to this embodiment, a polymerase is provided which lacks 5' nuclease, 3' nuclease and strand displacement activity. Optionally, the primary oligonucleotide primer can also contain a cleavable nucleotide or nucleotide analog which is cleaved during the hybridization step of the PCR to release a free 3' OH end on the oligonucleotide primer prior to the extension process. The polymerase chain reaction mixture is subjected to one or more additional polymerase chain reaction cycles comprising a denaturation process in which the extension products from the reaction are separated from each other and a hybridization process in which the first primary oligonucleotide primer hybridizes to the extension product generated from the second primary oligonucleotide primer. The extension product generated from the second primary oligonucleotide is capable of forming an intramolecular loop-hairpin between the 3' end within the extension product and a complementary sequence, the loop-hairpin (i) comprising a mismatch at or near the 3' end which inhibits self-extension in the case of hybridization to a mutant target sequence or (ii) comprising a match at the 3' end which enhances self-extension in the case of self-hybridization to a wild-type target sequence. The second primary oligonucleotide primer hybridizes to the extension product generated from the first primary oligonucleotide primer. The extension product of the first primary primer forms an intramolecular loop-hairpin between the 5' portion within the extension product and a complementary sequence. In the extension step of the PCR, the first primary oligonucleotide primer (i) preferentially extends on the extension product comprising the mutant target sequence, thereby preferentially forming a primary extension product comprising the mutant target nucleotide sequence or its complement, or (ii) inhibits the formation of a primary extension product comprising the wild-type target nucleotide sequence or its complement due to prior self-hybridization and self-extension on the target. The second primary oligonucleotide primer extends on the extension product independent of the target sequence, wherein the mutant sequence is preferentially amplified due to the different primary extension products generated from the hybridization of the first primary oligonucleotide primer to the target or a copy thereof, thereby enriching the mutant sequence extension product and its complement during the primary polymerase chain reaction.

[0280] Figure 154 and 155 The above-described embodiments of this aspect of the application are illustrated. As Figure 154 ​As shown, genomic DNA or cfDNA is isolated ( Figure 154 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 154 Step B). The target region is selectively amplified using a locus-specific upstream primer containing a 5' portion, the 5' portion having the same nucleotide sequence as the nucleotide sequence portion of the wild-type nucleic acid molecule that hybridizes with the primer, allowing the extension product to form a loop hairpin. In other words, the 5' portion of the upstream primer contains a nucleotide sequence that is the same as or complementary to the sequence portion within the antisense wild-type DNA strand. The amplification reaction also contains a locus-specific downstream primer and a mixture of deoxynucleotides containing dUTPs. As shown in Step B of this figure, another layer of selectivity can be selectively incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream mutation-specific primer. After target-specific hybridization, RNase H (star symbol) removes the RNA base to release a 3' OH group suitable for polymerase extension (r). Figure 154 (Step B). Optionally, the samples are aliquoted into 12, 24, 48, or 96 wells before PCR. Figure 154 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention. PCR is performed using a polymerase lacking 5' nuclease, 3' nuclease, and strand displacement activity. Figure 154 Step D further illustrates that in subsequent rounds of PCR, (i) the denatured wild-type base strand forms a loop-hairpin at the 3' end with a perfectly matched base and extended by polymerase, (ii) the denatured mutant base strand forms a loop-hairpin at the 3' end with at least one mismatched base and is not normally extended by polymerase, and (iii) the denatured top strand forms a loop-hairpin on the 5' side, which denatures during the extension step of PCR at 72°C. Figure 154Step E further illustrates that: (i) after loop-hairpin extension on wild-type DNA, the extended hairpin sequence remains unchanged at 72°C and prevents the upstream primer from producing a full-length top strand. However, the loop-hairpin sequence (ii) of the mutant DNA does not extend due to a 3' mismatch and thus denatures at 72°C, enabling the upstream primer to produce a full-length top strand. Similarly, the top strand product (iii) denatures at 72°C, allowing the polymerase to produce a full-length bottom strand. The difference in loop-hairpin extension preference of the upstream primer in the presence of wild-type (i) and mutant (ii) templates results in the preferential removal of the wild-type product in each amplification cycle, and thus preferential amplification of the mutant DNA. The difference in extension efficiency of the 3' end of the loop hairpin when hybridizing with mutant relative to wild-type DNA can be further increased by designing the upstream 5' portion to contain a mismatch with the wild-type DNA at the 2nd or 3rd position from the end. The extension product from the base primer produces only one mismatch at the 2nd or 3rd position starting from the 3' end when it self-hybridizes with the wild-type sequence, which is easily extended by polymerase. However, when it self-hybridizes with the mutant sequence, it produces two mismatches at the 3' end, which are not extended by polymerase.

[0281] like Figure 154 As shown in step G, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridized with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. In this embodiment, the upstream oligonucleotide probe with a sequence specific for detecting the target mutation also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product, while the upstream oligonucleotide probe with a sequence specific for detecting the wild-type (non-mutated) nucleic acid sequence does not contain a 5' primer-specific portion. The downstream oligonucleotide probe with 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 with a sequence specific for detecting the mutation, allows for subsequent amplification and detection of only the mutant ligation product. As shown in step F of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Following target-specific hybridization, RNase H (star symbol) removes RNA bases to generate a 3'OH group that can be linked. Figure 38 (Step H). After ligation, matching primer pairs Ai and Ci and TaqMan can be used. TM The TaqMan probe or other suitable means known in the art are used to detect the connection products. TM probe spans like Figure 154 The connection points described in (see) Figure 155 (Step HJ).

[0282] Figure 155 This illustrates another exemplary PCR-LDR legacy prevention response for detecting mutations. Genomic DNA or cfDNA is isolated ( Figure 155 Step A), and the isolated DNA sample is treated with UDG to digest any nucleic acid molecules containing dU that may be present in the sample. Figure 155 Step B). The target region is selectively amplified using the following: (i) a locus-specific upstream primer that also contains a 5' sequence portion complementary to the wild-type sequence of the top strand, thereby allowing loop-hairpin formation after extension; (ii) a locus-specific downstream primer; and (iii) a mixture of deoxynucleotides containing dUTP. As shown in Step B of this figure, another layer of selectivity can be added to the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream mutation-specific primer. After target-specific hybridization, RNase H (star symbol) removes the RNA base to release a 3'OH group suitable for polymerase extension ( Figure 155 (Step B). Optionally, the samples are aliquoted into 12, 24, 48, or 96 wells before PCR. Figure 155 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention. PCR is performed using a polymerase lacking 5' nuclease, 3' nuclease, and strand displacement activity. Figure 155 Step D further illustrates that, in subsequent rounds of PCR: (i) denatured wild-type base strands form loop-hairpins at the 3' end that are perfectly matched and extended by polymerase; (ii) denatured mutant base strands form loop-hairpins at the 3' end that are normally not extended by polymerase; and (iii) denatured top strands form loop-hairpins on the 5' side, which are denatured during the extension step of PCR at 72°C. Figure 155 Step E further illustrates that after loop-hairpin extension on wild-type DNA, (i) the extended hairpin sequence remains unchanged at 72°C and prevents the upstream primer from producing a full-length top strand. However, the loop-hairpin sequence (ii) of the mutant DNA does not extend due to a 3' mismatched base and thus denatures at 72°C, enabling the upstream primer to produce a full-length top strand. Similarly, the top strand product (iii) denatures at 72°C, allowing the polymerase to produce a full-length bottom strand. The difference in loop-hairpin extension preference of the upstream primer in the presence of wild-type (i) and mutant (ii) templates results in the preferential removal of the wild-type product in each amplification cycle, and thus preferential amplification of the mutant DNA.

[0283] In this embodiment, the downstream locus-specific primer also contains a 5' primer region, such as a universal primer region, which is capable of universal PCR amplification using a biotin-labeled primer to append a 5' biotin to the amplification product containing the region of interest Figure 155 , step B). The biotinylated PCR product is immobilized to a solid support and the mutation-specific ligation probe is used to detect the mutation of interest, as Figure 39 shown in step G. In this embodiment, the ligation probe with the ligation pair capable of detecting the mutant nucleic acid sequence (but not the wild-type sequence) contains a complementary tail sequence and contains a receptor group or a donor group, respectively, which are capable of producing a detectable signal by FRET occurring upon close proximity to each other, as described above for Figure 155 . As shown in this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (such as a C3 spacer) and an RNA base (r) in the upstream ligation probe. Upon target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a 3' OH group that can be ligated Figure 155 , step G). Upon ligation Figure 155 , step H), the complementary 5' and 3' tail ends of the ligation product hybridize to each other, bringing their respective donor and receptor moieties into close proximity to each other to generate a detectable FRET signal Figures 45-53 , step I).

[0284] The ligation reactions used in the methods of the present application are well known in the art. Ligases suitable for ligating the oligonucleotide probes of the probe set together (optionally after 3' ribose and blocking group on the first oligonucleotide probe, or 5' flap cleavage 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. In accordance with the present application, the nuclease ligation methods of the present application can be performed by employing an oligonucleotide ligation assay (OLA) reaction (see Landegren et al., "A Ligase-Mediated Gene Detection Technique," Science 241 : 1077-80 (1988); Landegren et al., "DNA Diagnostics— Molecular Techniques and Automation," Science 242: 229-37 (1988); and U.S. Patent No. 4,988,617 to Landegren et al.), a ligation detection reaction (LDR) using a set of complementary oligonucleotide probes (see, e.g., Barany et al., WO 90 / 17239, which is incorporated by reference in its entirety), or a ligation chain reaction (LCR) using two sets of complementary oligonucleotide probes (see, e.g., Barany et al., WO 90 / 17239, which is incorporated by reference in its entirety).

[0285] 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 phospho-sugar-backbone oligonucleotides, nucleotide analogs, and mixtures thereof.

[0286] The hybridization step in the ligase detection reaction (preferably a thermal hybridization treatment) distinguishes between nucleotide sequences based on the discriminating 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 rearrangement. Such sequence differences involving more than one base can also be detected. Preferably, the oligonucleotide probe set has substantially the same length so that it hybridizes to the target nucleotide sequences under substantially similar hybridization conditions.

[0287] The ligase discrimination ability can be further enhanced by employing various probe design features. For example, an intentional mismatch or nucleotide analog (e.g., inosine, nitroindole, or nitropyrrole) can be incorporated into the first oligonucleotide probe at the 2nd or 3rd base from the 3' junction end, such that hybridization at the 3' end is slightly destabilized if there is a perfect match at the 3' end, but significantly destabilized if there is a mismatch at the 3' end. This design reduces inappropriate false ligation when mutant probes hybridize to wild-type targets. Alternatively, an RNA base cleaved by an RNase 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), which is incorporated by reference in its entirety, describes the use of an RNA base proximal to the 3' end of an oligonucleotide probe with a 3'-blocked end, and cleavage of the RNA base with RNase H2, resulting in a PCR extendable and ligatable 3'-OH. This method can be used to generate a ligatable 3' OH or 5'-P or both, provided that a ligase that can ligate a 5'-RNA base is utilized.

[0288] Other possible modifications include abasic sites, such as internal abasic furan or oxo-G. These unusual "bases" are removed by specific enzymes to generate a ligatable 3'-OH or 5'P site. After annealing of the ligation oligonucleotide to the target nucleic acid, endonuclease IV, Tth Endo IV (NEB) removes the abasic residue, rather than from single-stranded DNA. Similarly, oxo-G with Fpg or inosine / uracil with EndoV or thymidine glycol with EndoVIII can be used.

[0289] Connection discrimination ability can also be enhanced by using the coupled nuclease-ligase reaction described in Barany et al. WO 2013 / 123220, which is incorporated by reference herein in its entirety. In this embodiment, the first oligonucleotide probe has a ligatable 3' OH group, while the second oligonucleotide probe has a non-ligatable 5' end (i.e., an oligonucleotide probe that does not have a 5' phosphate). The oligonucleotide probes of the probe set are designed such that the most 3' base of the first oligonucleotide probe overlaps the most 5' base of the second oligonucleotide probe that is immediately adjacent to the complement of the target nucleic acid molecule. The overlapping nucleotides are referred to as "flanking." When the flanking overlapping nucleotide of the second oligonucleotide probe is complementary to the sequence of the target nucleic acid molecule and is the same sequence as the terminal 3' nucleotide of the first oligonucleotide probe, the phosphodiester bond immediately upstream of the flanking nucleotide of the second oligonucleotide probe is discriminately cleaved by an enzyme having flanking endonuclease (FEN) or 5' nuclease activity. This specific FEN activity creates a new ligatable 5' phosphate end on the second oligonucleotide probe that is precisely positioned next to the adjacent 3' OH of the first oligonucleotide probe to allow ligation of the two probes to occur. According to this embodiment, flanking endonucleases or 5' nucleases suitable for cleaving the 5' flanking of the second oligonucleotide probe prior to ligation include, but are not limited to, polymerases with 5' nuclease activity such as E. coli DNA polymerase and polymerases from Taq and Thermus thermophilus, as well as T4 RNase H and TaqExo.

[0290] For insertions or deletions, incorporation of a matching base or nucleotide analog (e.g., -amino-dA or 5-propynyl-dC) at the 2nd or 3rd position from the junction in the first oligonucleotide probe improves stability and can improve discrimination of such frameshift mutations from the wild type sequence. For insertions, use of one or more phosphorothioate-modified nucleotides downstream of the desired cleavable phosphate bond of the second oligonucleotide probe will prevent inappropriate cleavage by 5' nuclease when the probe is hybridized to wild type DNA and thereby reduce false positive ligation on wild type targets. Likewise, for deletions, use of one or more phosphorothioate-modified nucleotides upstream of the desired cleavable phosphate bond of the second oligonucleotide probe will prevent inappropriate cleavage by 5' nuclease when the probe is hybridized to wild type DNA and thereby reduce false positive ligation on wild type targets.

[0291] One or more target nucleic acid sequences that differ from other nucleic acid sequences in the sample by one or more methylation residues can also be identified using the methods of the present application. According to this aspect of the present application, the methods further comprise contacting the sample with at least a first methylation-sensitive enzyme to form a restriction enzyme reaction mixture prior to forming the polymerase chain reaction mixture. According to this aspect of the present application, the first primary oligonucleotide primer comprises a nucleotide sequence that is complementary to a region in the target nucleotide sequence that is upstream of the one or more methylation residues, and the second primary oligonucleotide primer comprises the same nucleotide sequence as the region in the target nucleotide sequence that is downstream of the one or more methylation residues.

[0292] 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. According to this embodiment, the detecting comprises detecting one or more nucleic acid molecules that contain the target nucleotide sequence, wherein the nucleic acid molecules initially contain one or more methylation residues.

[0293] According to this and all aspects of the present application, a "methylation-sensitive enzyme" is an endonuclease that will not cleave or reduce the cleavage efficiency of a recognition sequence in a nucleic acid molecule when the cognate recognition sequence in the nucleic acid molecule contains a methylation residue (i.e., the endonuclease is sensitive to the presence of a methylation residue within its recognition sequence). A "methylation-sensitive enzyme recognition sequence" is a cognate recognition sequence for a methylation-sensitive enzyme. In some embodiments, the methylation residue is a 5-methyl-C within a sequence CpG (i.e., 5-methyl-CpG). A non-limiting list of methylation-sensitive restriction endonucleases suitable for use in the methods of the present application includes, but is not limited to, Acil, HinPll, Hpy99l, HpyCH4IV, BstUI, HpaII, Hhal, or any combination thereof.

[0294] The methods of the present application can also comprise subjecting the restriction enzyme reaction mixture to bisulfite treatment under conditions suitable to convert unmethylated cytosine residues to uracil residues prior to forming the polymerase chain reaction mixture. In this embodiment, the first primary oligonucleotide primer of the primary oligonucleotide primer set comprises a nucleotide sequence that is complementary to the bisulfite-treated target nucleotide sequence that contains one or more methylation-unclipped restriction sites, and the second primary oligonucleotide primer of the provided primary oligonucleotide primer set comprises a nucleotide sequence that is complementary to a portion of the extension product formed by the first oligonucleotide primer.

[0295] The method of the application can also include providing one or more second methylation-sensitive enzymes that cleave nucleic acid molecules containing unmethylated residues within methylation-sensitive enzyme recognition sequences. The at least one second methylation-sensitive enzyme is blended with the polymerase chain reaction mixture comprising the bisulfite-treated restriction enzyme reaction mixture to form a second restriction enzyme reaction mixture, wherein the second methylation-sensitive enzyme cleaves nucleic acid molecules potentially present in the sample that contain one or more unmethylated residues within the recognition sequence of the at least one methylation-sensitive enzyme during the hybridization treatment.

[0296] In one embodiment of this aspect of the application, one or both of the primary oligonucleotide primers of the primary oligonucleotide primer set has a 3' portion having a cleavable nucleotide or nucleotide analog and a blocking group such that the 3' end of the one or more primers is not suitable for polymerase extension. According to this embodiment, the method further includes cleaving the cleavable nucleotide or nucleotide analog of the one or both oligonucleotide primers during the hybridization treatment, thereby releasing a free 3' OH end on the one or both oligonucleotide primers prior to the extension treatment.

[0297] In one embodiment of this aspect of the application, the method further includes providing one or more blocking oligonucleotides capable of hybridizing to regions of the bisulfite-treated target nucleotide sequence that contain unmethylated residues. The mixture is contacted with the one or more blocking oligonucleotides prior to subjecting the polymerase chain reaction mixture comprising the bisulfite-treated restriction enzyme reaction mixture to one or more cycles of polymerase chain reaction. The one or more blocking oligonucleotides hybridize to the complementary target nucleic acid sequence during the hybridization treatment and prevent extension of the primary oligonucleotide primers during the extension treatment.

[0298] Figure 45 Various embodiments of the method of the application for detecting target nucleic acid molecules containing one or more methylated residues are shown.

[0299] Figure 45 The first step of the PCR-LDR-qPCR reaction depicted in the middle comprises isolation of genomic DNA or cfDNA. Optionally, methylated DNA can be enriched using methylation-specific antibodies. The sample is then treated with a methylation-sensitive restriction endonuclease such as Bsh1236I (CG^CG) and / or HinP1I (G^CGC) and UNG (37°C, 30-60 min) to completely digest unmethylated DNA and prevent carryover Figure 45 , step A). As Figure 45As shown in step B, the target methylated region is amplified by PCR using locus-specific primers in the presence of dUTP. In one embodiment, limited-cycle amplification (12-20 cycles) is performed to maintain the relative ratio of different amplicons produced. In another embodiment, 20-40 cycles are used to amplify the target region. The PCR product incorporates dU, thereby allowing for residual prevention (…). Figure 45 Step C), and the lack of a methyl group in the product provides additional protection. For example... Figure 45 As shown in step D, the methyl region-specific linked oligonucleotide probe contains a mixture suitable for use with tag primer pairs Ai and Ci' and TaqMan. TM The tag primer-specific portion (Ai, Ci') for subsequent PCR amplification of the probe.

[0300] After the ligation reaction, the sample containing the ligation product was aliquoted into different wells for detection. The treatment using UDG destroyed the original target amplicon (…). Figure 45 Step E) thus allows only the true LDR product to be amplified and detected. In this implementation, conventional TaqMan is used as described above. TM Detection and assay to detect linker products ( Figure 46 (Steps EF).

[0301] Figure 46 Another exemplary PCR-LDR-qPCR legacy prevention reaction for detecting methylation is shown. In this embodiment, genomic DNA or cfDNA is isolated and treated with methyl-sensitive restriction endonucleases such as Bsh1236I (CG^CG) and / or HinP1I (G^CGC) and UNG (37°C, 30-60 min) to completely digest unmethylated DNA and prevent legacy ( Figure 46 Step A). Figure 46 As shown in step B, the target methylated region is amplified by PCR using locus-specific primers in the presence of dUTP. In one embodiment, limited-cycle amplification (12-20 cycles) is performed to maintain the relative ratio of different amplicon produced. In another embodiment, 20-40 cycles are used to amplify the target region. The primers contain identical 8-11-base tails to prevent primer dimerization. The PCR product contains dU, thereby allowing for residual preventative (…). Figure 46 Step C).

[0302] In this implementation, the oligonucleotide probe is designed to contain UniTaq primers and a tag sequence, as described above, to facilitate UniTaq detection. Therefore, after the ligation reaction and treatment with UDG for legacy prevention, the ligation product is amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci), as shown below. Figure 46shown in steps E and F and the amplified products are detected as described above. Figure 47 shown in step G and as described above.

[0303] Figure 47 Another exemplary PCR-qLDR legacy prevention reaction for detecting methylation is shown. In this embodiment, genomic DNA or cfDNA is isolated and treated with a methylation sensitive restriction endonuclease such as Bsh1236I (CG^CG) and / or HinP1I (G^CGC) and UNG (37°C, 30-60 min) to completely digest unmethylated DNA and prevent legacy Figure 47 , step A). As Figure 47 shown in step B, the target methylation region is amplified using PCR in the presence of dUTP using locus specific primers. In this embodiment, the locus specific primers also contain a 5' primer region, such as a universal primer region, that enables subsequent universal PCR amplification using biotin labeled primers to append a 5' biotin to the amplified product containing the target region Figure 47 , step B). The biotinylated PCR products are immobilized to a solid support and the target mutation is detected using mutation specific ligation probes as Figure 39 shown in step D. According to this embodiment, the oligonucleotide probes used for ligation contain complementary tail sequences and either contain an acceptor group or a donor group, respectively, that are capable of generating a detectable signal by FRET that occurs upon close proximity to each other as described above for Figure 47 . Following ligation Figure 47 , step D), the complementary 5' and 3' tail ends of the ligation products hybridize to each other, bringing their respective donor and acceptor moieties in close proximity to each other to generate a detectable FRET signal Figure 48 , step E).

[0304] Figure 48 A nuclease-ligation-PCR-qPCR legacy prevention reaction for detecting methylation is shown. In this embodiment, genomic DNA or cfDNA is isolated and treated with HaeIII (GG^CC) methylation sensitive restriction endonuclease such as Bsh1236I (CG^CG) and / or HinP1I (G^CGC) and UNG (37°C, 30-60 min) to completely digest unmethylated DNA and prevent legacy Figure 48 , step A). As Figure 48 shown in step B, a hairpin oligonucleotide containing a tag sequence (Ai') is ligated to the newly released phosphate of the digested target DNA in the sample template strand. As Figure 48In Step C, the sample is treated with HinP1I and Bsh1236I at 37°C. The methyl-sensitive restriction enzymes are then heat-inactivated, while the Taq polymerase is activated for subsequent PCR amplification using locus-specific primers containing the Ci tag sequence. As described above, the primers can contain a cleavable blocking group (e.g., C3 spacer) and an RNA base (r) that is removed by RNase H2 (asterisk) prior to amplification only when the primer is bound to the complementary target sequence. The unblocked primer is extended with the polymerase, and the 5' nuclease activity of the polymerase digests the 5' portion of the attached hairpin to generate a product that is complementary to the target containing the Ci and Ai' sequences. The unattached hairpin oligonucleotide is extended on itself.

[0305] As Figure 48 In Step D, the target methyl-containing region is amplified using PCR with dUTP and tag primers Ai and Ci. Limited cycle amplification (12-20) is performed to maintain the relative ratios of different amplicons. The PCR product incorporates dU, allowing for strand-displacement prevention Figure 49 , Step E), and the product lacks methylation, providing additional protection. As described above, the amplification products are aliquoted into separate wells for TaqMan TM detection using locus-specific primers and TaqMan TM probes.

[0306] Figure 48 A nuclease-ligation-PCR-qPCR strand-displacement prevention reaction for detecting methylation is shown. As shown and described above with respect to Figure 49 Steps A-D, PCR products containing the target original methylation residues and dU are generated. In this embodiment, subsequent amplification using UniTaq primers and tag sequences is used to detect the target methylation residues. As Figure 49 shown in Step E, the PCR products containing dU for strand-displacement prevention are aliquoted into separate wells and amplified using locus-specific primers ending in Aj and Bj-Cj, and UniTaq-specific primers (Fl-Bj-Q-Aj and Cj). The resulting double-stranded DNA products are shown in Figure 49 Step F. As Figure 50 shown in Step G, after a denaturation step, the temperature is cooled to allow the formation of a hairpin between Bj and Bj'. The 5'→3' nuclease activity of the Taq polymerase (filled square) extends the primer Ci and releases the fluorescent group to generate a signal.

[0307] Figure 50A PCR-LDR-qPCR carryover prevention reaction for detecting methylation is shown. In this embodiment, genomic DNA or cfDNA is isolated and treated with a methylation sensitive restriction endonuclease such as Bsh1236I (CG^CG) and UNG (37°C, 30-60 min) to completely digest unmethylated DNA and prevent carryover Figure 50 , step A). The digested DNA is subjected to bisulfite treatment to convert unmethylated dC residues to uracil (dU), rendering the double stranded DNA non-complementary. As Figure 50 shown in B, locus specific primers are hybridized in the presence of BstU1 (CG^CG) (solid triangles) that cleaves carryover DNA containing unmethylated residues. The locus specific primers contain a cleavable blocker at their 3’ end to prevent non-target specific extension. Once hybridized to their complementary target sequence, the blocking group (C3 spacer) and RNA base are removed with RNase H2 (star symbol). PCR is used to amplify the target methylated region in the presence of dUTP. Limited cycle amplification (12-20) is performed to maintain the relative ratios of different amplicons, and the digested sample is optionally aliquoted into 12, 24, 48, or 96 wells prior to PCR. As shown in this embodiment, a blocking oligonucleotide (thick black line) can be used to limit amplification of wild type DNA.

[0308] The amplification product contains dU and lacks methylation, allowing carryover prevention as Figure 50 shown in C. As Figure 50 shown in D, a methylation region specific ligation oligonucleotide probe containing primer specific sequences (Ai, Ci’) suitable for subsequent PCR amplification is hybridized to the target region of interest. A ligase (solid circle) covalently seals the two oligonucleotides together to form a ligation product containing upstream and downstream primer specific portions and a portion corresponding to the target methylation region. The ligation product is aliquoted into individual wells for detection using a matching primer pair Ai and Ci and a TaqMan TM probe spanning the ligation junction as Figure 50 shown in E-F. The sample mixture is treated with UDG to effect carryover prevention and remove the original target amplicon Figure 51 , step E), such that only the true LDR product is amplified and detected.

[0309] Figure 50 Another PCR-LDR-qPCR carryover prevention reaction for detecting methylation is shown. As above with respect to Figure 51As shown and described, PCR products containing target original methylation residues and dU are generated after steps A-C. In this embodiment, the methylation region specific ligation oligos contain UniTaq detection primer specific sequences (Ai and Ci') and tag sequences (Bi') for subsequent PCR amplification detection. The ligation products are amplified and detected using UniTaq specific primers (F1-Bi-Q-Ai, Ci) as described above Figure 52 , steps E-G).

[0310] Figure 50 Another PCR-qLDR legacy prevention reaction for detecting methylation is shown. Similar to the embodiments shown in Figure 52 and 51 , genomic DNA or cfDNA is isolated and treated with a methylation sensitive restriction endonuclease, such as Bsh1236I (CG^CG) and UNG (37°C, 30-60 min) to completely digest unmethylated DNA and prevent legacy Figure 52 , step A). The digested DNA is subjected to bisulfite treatment to convert unmethylated residues to uracil, rendering the double stranded DNA non-complementary. As shown in Figure 52 B, locus specific primers containing a 3' cleavable blocking group are hybridized in the presence of BstU1 (CG^CG) (solid triangles) that cleaves legacy DNA containing unmethylated residues. Once the primers are hybridized to their complementary target sequences, the blocking group is removed and the target methylation containing region is amplified using PCR in the presence of dUTP. In this embodiment, the locus specific primers contain a universal tail (with identical 8-11 bases to prevent primer dimerization), which enables subsequent universal primer amplification to append a 5' biotin group. Blocking oligo primers can be used during amplification to limit the formation of wild type amplicons.

[0311] The amplification products are captured on a solid support via the 5' biotin group Figure 52 , step C). As shown in Figure 52 D, methylation region specific ligation oligo probes are used to form ligation products, where the downstream probe contains a 5' acceptor group and a sequence tail complementary to the 3' sequence tail of the upstream ligation probe. The upstream ligation probe also contains a 3' donor group, such that upon formation of the ligation product, the 5' and 3' complementary regions of the product hybridize, bringing the acceptor and donor groups in close proximity to each other to generate a FRET signal for detection of the target methylation residue Figure 151 step E).

[0312] Figure 151 Another exemplary PCR-LDR-qPCR legacy prevention reaction for detecting low level target methylation is shown. Genomic DNA or cfDNA is isolated (Figure 151 Step A), and the isolated DNA sample is treated with methyl-sensitive restriction endonucleases such as Bsh1236I (CG^CG) and UNG to completely digest unmethylated DNA and prevent residual DNA. Figure 151 Step A). ​​The digested DNA is treated with bisulfite to convert unmethylated residues to uracil, thereby making the double-stranded DNA non-complementary. The target region is selectively amplified using a locus-specific upstream primer, a locus-specific downstream primer, a blocking LNA or PNA probe containing the bisulfite-converted unmethylated sequence or its complement, and a mixture of deoxynucleotides containing dUTP. In this embodiment, another layer of selective binding to the method can be achieved by including a cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in both the upstream and downstream primers. After target-specific hybridization, RNase H (star symbol) removes the RNA base to release several bases upstream of the methylation site of the upstream primer and a 3'OH group suitable for polymerase extension ( Figure 151 (Step B). A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complementary sequence) that partially overlaps with the upstream PCR primer preferentially competes with the upstream primer and with respect to the bisulfite-converted methylated DNA for binding to the bisulfite-converted unmethylated sequence, thereby inhibiting the amplification of the bisulfite-converted unmethylated DNA in each round of PCR. Optionally, the samples are aliquoted into 12, 24, 48, or 96 wells prior to PCR. Figure 151 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0313] like Figure 151As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridized with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. In this embodiment, the upstream oligonucleotide probe having specificity for detecting the target bisulfite-converted methylated target sequence also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product. Again, during the PCR amplification step, after enrichment of the bisulfite-converted methylated target sequence, the presence of a blocking LNA or PNA probe containing the bisulfite-converted unmethylated sequence (or its complementary sequence) inhibits hybridization of the upstream ligation probe with the bisulfite-converted unmethylated target sequence (if present). The downstream oligonucleotide probe having a sequence common to both the bisulfite-converted unmethylated target sequence and the bisulfite-converted methylated target sequence contains a 3' primer-specific portion (Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream probe having specificity for detecting the bisulfite-converted methylated target sequence. The ligation of upstream and downstream oligonucleotide probes allows for subsequent amplification and detection of only the ligated target sequence product after bisulfite conversion. As shown in step D of this figure, another layer of specificity is incorporated into the method by including a cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. After target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a ligable 3'OH group (r). Figure 38 (Step D). After ligation, matching primer pairs Ai and Ci and TaqMan can be used. TM The TaqMan probe or other suitable means known in the art are used to detect the connection products. TM The probe crosses the above-mentioned targets Figure 151 The connection point (see above) Figure 152 Step EG).

[0314] Figure 152 This illustrates another exemplary PCR-LDR-qPCR legacy prevention reaction for detecting low levels of target methylation. Genomic DNA or cfDNA is isolated. Figure 152 Step A), and the isolated DNA sample is treated with methyl-sensitive restriction endonucleases such as Bsh1236I (CG^CG) and UNG to completely digest unmethylated DNA and prevent residual DNA. Figure 152Step A). ​​The digested DNA is treated with bisulfite to convert unmethylated residues to uracil, thus making the double-stranded DNA non-complementary. Upstream and downstream locus-specific primers are designed to contain a cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r). After target-specific hybridization, RNase H (star symbol) removes the RNA base to release the 3' OH and is adapted for polymerase extension ( Figure 152 (Step B). A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complementary sequence) that partially overlaps with the upstream PCR primer preferentially competes with the upstream primer and with respect to the bisulfite-converted methylated target sequence for binding to the bisulfite-converted unmethylated target sequence, thereby inhibiting the amplification of the bisulfite-converted unmethylated target sequence in each round of PCR. Optionally, the samples are aliquoted into 12, 24, 48, or 96 wells prior to PCR. Figure 152 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention.

[0315] like Figure 152 As shown in step D, the target-specific oligonucleotide probe hybridizes with the amplification product, and when hybridization occurs with its complementary sequence, the ligase (solid circle) covalently seals the two oligonucleotides together. Again, during the PCR amplification step, after enrichment of the bisulfite-converted methylated target sequence, the presence of a blocking LNA or PNA probe containing the bisulfite-converted unmethylated sequence (or its complementary sequence) inhibits hybridization with the bisulfite-converted unmethylated target sequence (if present). In this embodiment, the upstream oligonucleotide probe, specifically designed for detecting the target bisulfite-converted methylated target sequence, also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product. Downstream oligonucleotide probes containing a sequence common to both the unmethylated target sequence converted to bisulfite and the methylated target sequence converted to bisulfite contain a 3' primer-specific portion (Bi'-Ci'). This 3' primer-specific portion, together with the 5' primer-specific portion (Ai) of the upstream probe containing a sequence specifically for detecting the methylated target sequence converted to bisulfite, allows for subsequent amplification and detection of ligation products containing only the methylated target sequence converted to bisulfite. As shown in step D of this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. After target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a ligation-capable 3'OH group (r). Figure 44 (Step D). After ligation, the ligation product is amplified using UniTaq-specific primers (i.e., F1-Bi-Q-Ai, Ci) and as described above for...Figure 152 Detection can be performed as described above or using other suitable means known in the art (see [link]). Figure 153 Step EH).

[0316] Figure 153 This illustrates another exemplary PCR-qLDR legacy prevention reaction for detecting low levels of target methylation. Genomic DNA or cfDNA is isolated. Figure 153 Step A), and the isolated DNA sample is treated with methyl-sensitive restriction endonucleases such as Bsh1236I (CG^CG) and UNG to completely digest unmethylated DNA and prevent residual DNA. Figure 153 Step A). ​​The digested DNA is treated with bisulfite to convert unmethylated residues to uracil, thus making the double-stranded DNA non-complementary. Upstream and downstream locus-specific primers are designed to contain a cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r). After target-specific hybridization, RNase H (star symbol) removes the RNA base to release the 3' OH and is adapted for polymerase extension ( Figure 153 (Step B). A blocking LNA or PNA probe containing a bisulfite-converted unmethylated sequence (or its complementary sequence) that partially overlaps with the upstream PCR primer preferentially competes with the upstream primer for binding to the bisulfite-converted unmethylated target sequence, thereby inhibiting the amplification of the bisulfite-converted unmethylated target sequence in each round of PCR. In this embodiment, the downstream locus-specific primer also contains a 5' primer region, such as a universal primer region, which enables universal PCR amplification using biotin-labeled primers to attach 5' biotin to the amplification product containing the target region. Figure 153 (Step B). Optionally, the samples are aliquoted into 12, 24, 48, or 96 wells before PCR. Figure 153 As shown in step C, the amplification product contains dU, which allows for subsequent processing with UDG or similar enzymes to achieve legacy prevention. The biotinylated PCR product is immobilized onto a solid support, and the target methylated target sequence converted from bisulfite is detected using a ligation probe specific to the target bisulfite-converted methylation sequence, such as... Figure 39as described above for the method of Figure 1. As shown in this figure, another layer of specificity can be incorporated into the method by including a 3' cleavable blocking group (e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. Upon target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a 3' OH group that can be ligated (step D). Following ligation (step E), the complementary 5' and 3' tail ends of the ligation product hybridize to each other, thereby bringing their respective donor and acceptor moieties into close proximity to generate a detectable FRET signal (step F). Figure 153 Figure 153 Figure 153 Figure 156

[0317] In another embodiment of this aspect of the application, the first primary oligonucleotide primer of the primary oligonucleotide primer set comprises a 5' portion having a nucleotide sequence identical to the portion of the nucleotide sequence of the primary oligonucleotide primer that hybridizes to the unmethylated target sequence in the bisulfite-treated sample, but having one or more nucleotide sequence mismatches to the corresponding portion of the nucleotide sequence in the methylated target sequence in the bisulfite-treated sample.

[0318] ​​​​According to this embodiment, the DNA polymerase is one that lacks 5' nuclease, 3' nuclease and strand displacement activity. Optionally, the primary oligonucleotide primer further contains a cleavable nucleotide or nucleotide analog that is cleaved during the hybridization step of the polymerase chain reaction to release a free 3' OH end on the oligonucleotide primer, suitable for extension. The polymerase chain reaction mixture is subjected to one or more additional polymerase chain reaction cycles comprising a denaturation step in which the extension products from the reaction are separated from each other and a hybridization step in which the first primary oligonucleotide primer hybridizes to the extension product produced from the second primary oligonucleotide primer. The extension product produced from the second primary primer forms an intramolecular loop-hairpin between the 3' end within the extension product and the complementary sequence, which (i) contains one or more mismatches at or near the 3' end that inhibit self-extension in the presence of hybridization to the bisulfite treated methylated sequence or (ii) contains a match at the 3' end that enhances self-extension in the presence of self-hybridization to the bisulfite treated unmethylated target sequence. The second primary oligonucleotide primer hybridizes to the extension product produced from the first primary oligonucleotide primer. The extension product produced from the first primary oligonucleotide primer forms an intramolecular loop-hairpin between the 5' portion within the extension product and the complementary sequence. In the extension step of the PCR, the first primary oligonucleotide primer (i) preferentially extends on the extension product containing the bisulfite treated methylated target sequence, thereby preferentially forming a primary extension product containing the bisulfite treated methylated target nucleotide sequence or the complement thereof or (ii) inhibits the formation of a primary extension product containing the bisulfite treated unmethylated target nucleotide sequence or the complement thereof due to prior self-hybridization and self-extension on the target. The second primary oligonucleotide primer (iii) extends on the extension product that is independent of the target sequence, wherein the bisulfite treated methylated sequence is preferentially amplified due to the different primary extension product produced from the hybridization of the first primary oligonucleotide primer to the target or a copy thereof, thereby enriching the bisulfite treated methylated sequence extension product and the complement thereof during the primary polymerase chain reaction.

[0319] Figure 157 and Figure 156 This embodiment of the application is illustrated. As shown in Figure 156 Genomic DNA or cfDNA is isolated and the sample is treated with a methylation sensitive restriction endonuclease, such as Bsh1236I (CG^CG) and UNG (37°C, 30-60 minutes) to completely digest unmethylated DNA and prevent carryover contamination of the sample with unmethylated DNA. Figure 156, step A). The digested DNA is subjected to bisulfite treatment to convert unmethylated dC residues to uracil (dU), thus rendering the double stranded DNA non-complementary. The region of interest is selectively amplified using (i) a locus-specific upstream primer also comprising a 5' sequence portion that is complementary to the bisulfite treated unmethylated sequence of the top strand, thus allowing the formation of a loop-hairpin after extension, (ii) a locus-specific downstream primer, and (iii) a deoxynucleotide mix comprising dUTP. As shown in this figure step B, another layer of selectivity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g. C3 spacer) and an RNA base (r) in the upstream mutation-specific primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base to release a 3' OH group suitable for polymerase extension Figure 156 , step B). Optionally, the digested sample is aliquoted into 12, 24, 48 or 96 wells prior to PCR. As shown in step C, the amplification product contains dU, which allows for subsequent treatment with UDG or similar enzyme to achieve carryover prevention. PCR is performed using a polymerase lacking 5' nuclease, 3' nuclease and strand displacement activity. Figure 156 Step C shows that the amplification product contains dU, which allows for subsequent treatment with UDG or similar enzyme to achieve carryover prevention. PCR is performed using a polymerase lacking 5' nuclease, 3' nuclease and strand displacement activity. Figure 156 Step D further shows that in the subsequent round of amplification: (i) the denatured bisulfite treated unmethylated bottom strand forms a loop-hairpin with perfect match at the 3' end, which is extended by the polymerase, (ii) the denatured bisulfite treated methylated bottom strand forms a loop-hairpin with two or more mismatches, which is typically not extended by the polymerase, and (iii) the denatured top strand forms a loop-hairpin at its 5' side, which is denatured during the extension step of PCR at 72°C. Figure 156 Step E further shows that upon loop-hairpin extension on bisulfite treated unmethylated DNA: (i) the extended hairpin sequence is not denatured at 72°C and prevents the upstream primer from producing a full-length top strand. However, the loop-hairpin sequence of bisulfite treated methylated DNA (ii) is not extended due to the two or more mismatched bases and is thereby denatured at 72°C, enabling the upstream primer to produce a full-length top strand. Likewise, the top strand product (iii) is denatured at 72°C, allowing the polymerase to produce a full-length bottom strand. The difference in loop-hairpin extension bias of the upstream primer in the presence of bisulfite treated unmethylated (i) and bisulfite treated methylated (ii) templates results in preferential removal of bisulfite treated unmethylated amplification products during each amplification cycle and thereby enables preferential amplification of bisulfite treated methylated DNA.

[0320] As shown in step F, the amplification product contains dU, which allows for subsequent treatment with UDG or similar enzyme to achieve carryover prevention. PCR is performed using a polymerase lacking 5' nuclease, 3' nuclease and strand displacement activity. Figure 38As shown in step G, an oligonucleotide probe specific to the bisulfite-treated methylated target sequence hybridizes with the amplification product, and a ligase (solid circle) covalently seals the two oligonucleotides together when hybridizing with its complementary sequence. In this embodiment, the upstream oligonucleotide probe specific for detecting the target bisulfite-treated methylated sequence also contains a 5' primer-specific portion (Ai) to facilitate subsequent detection of the ligation product, while the optional upstream oligonucleotide probe specific for detecting the unmethylated nucleic acid sequence treated with bisulfite does not contain a 5' primer-specific portion. The downstream oligonucleotide probe specific for detecting the bisulfite-treated methylated sequence contains a 3' primer-specific portion (Ci'), which, together with the 5' primer-specific portion (Ai) of the upstream probe specific for detecting the target bisulfite-treated methylated sequence, allows for subsequent amplification and detection of only mutant ligation products. As shown in this figure, another layer of specificity can be added to the method by including a cleavable blocking group (Blk 3', e.g., a C3 spacer) and an RNA base (r) in the upstream ligation probe. After target-specific hybridization, RNase H (star symbol) removes the RNA base to generate a ligation-enabled 3'OH group (r). Figure 156 (Step H). After ligation, matching primer pairs Ai and Ci and TaqMan can be used. TM The TaqMan probe or other suitable means known in the art are used to detect the connection products. TM The probe spans across as Figure 157 The connection points described in (see) Figure 157 (Step HJ).

[0321] Figure 157 This illustrates another PCR-qLDR legacy prevention reaction for detecting methylation. In this embodiment, genomic DNA or cfDNA is isolated and treated with a methyl-sensitive restriction endonuclease such as Bsh1236I (CG^CG) and UNG (37°C, 30–60 min) to completely digest unmethylated DNA and prevent legacy (…). Figure 157, step A). The digested DNA is subjected to bisulfite treatment to convert unmethylated dC residues to uracil (dU), rendering the double stranded DNA non-complementary. The region of interest is selectively amplified using (i) a locus-specific upstream primer also comprising a 5' portion having a sequence complementary to the bisulfite treated unmethylated sequence of the top strand to allow formation of a loop-hairpin after extension, (ii) a locus-specific downstream primer, and (iii) a deoxynucleotide mix comprising dUTP. As shown in this figure, another layer of selectivity can be incorporated into the method by including a 3' cleavable blocking group (Blk 3', e.g. C3 spacer) and an RNA base (r) in the upstream primer. Upon target-specific hybridization, RNase H (asterisk) removes the RNA base to release a 3' OH group suitable for polymerase extension Figure 157 , step B). Optionally, the digested sample is aliquoted into 12, 24, 48 or 96 wells prior to PCR. As shown in step C, the amplification product contains dU, which allows for subsequent treatment with UDG or similar enzyme to achieve carryover prevention. PCR is performed using a polymerase lacking 5' nuclease, 3' nuclease and strand displacement activity. Figure 157 As shown in step C, the amplification product contains dU, which allows for subsequent treatment with UDG or similar enzyme to achieve carryover prevention. PCR is performed using a polymerase lacking 5' nuclease, 3' nuclease and strand displacement activity. Figure 157 As shown in step D, in the subsequent round of amplification, (i) the denatured bisulfite treated unmethylated bottom strand forms a loop-hairpin with perfect match at the 3' end, which is extended by the polymerase, (ii) the denatured bisulfite treated methylated bottom strand forms a loop-hairpin with two or more mismatches, which is typically not extended by the polymerase, and (iii) the denatured top strand forms a loop-hairpin at its 5' side, which is denatured during the extension step of PCR at 72°C. Figure 150 As shown in step E, upon loop-hairpin extension on bisulfite treated unmethylated DNA, (i) the extended hairpin sequence is not denatured at 72°C and prevents the upstream primer from producing a full-length top strand. However, the loop-hairpin sequence of bisulfite treated methylated DNA (ii) is not extended due to the two or more mismatched bases and is thereby denatured at 72°C, enabling the upstream primer to produce a full-length top strand. Likewise, the top strand product is denatured at 72°C, allowing the polymerase to produce a full-length bottom strand (iii). The difference in loop-hairpin extension bias of the upstream primer in the presence of (i) bisulfite treated unmethylated and (ii) bisulfite treated methylated templates results in preferential removal of bisulfite treated unmethylated amplification products during each amplification cycle and thereby enables preferential amplification of bisulfite treated methylated DNA.

[0322] In this embodiment, the downstream locus-specific primer also contains a 5' primer region, such as a universal primer region, which is capable of universal PCR amplification using a biotin-labeled primer to append a 5' biotin to the amplification product containing the region of interest (Step B). The biotinylated PCR product is immobilized to a solid support and the target bisulfite-treated methylated sequence is detected using a ligation probe specific for the bisulfite-treated methylated target sequence, such as Figure 39 Step G. In this embodiment, the ligation probe with the pair of ligation probes capable of detecting bisulfite-treated methylated nucleic acid sequences (but not bisulfite-treated unmethylated sequences) contains a complementary tail sequence and contains a receptor group or a donor group, respectively, which are capable of producing a detectable signal by FRET occurring upon close proximity to each other, as described above for Figure 150 Step G. In this embodiment, the ligation probe with the pair of ligation probes capable of detecting bisulfite-treated methylated nucleic acid sequences (but not bisulfite-treated unmethylated sequences) contains a complementary tail sequence and contains a receptor group or a donor group, respectively, which are capable of producing a detectable signal by FRET occurring upon close proximity to each other, as described above for Figure 157 Step G. In this embodiment, the ligation probe with the pair of ligation probes capable of detecting bisulfite-treated methylated nucleic acid sequences (but not bisulfite-treated unmethylated sequences) contains a complementary tail sequence and contains a receptor group or a donor group, respectively, which are capable of producing a detectable signal by FRET occurring upon close proximity to each other, as described above for Figure 157 Step G). Upon ligation (Step H), the complementary 5' and 3' tail ends of the ligation product hybridize to each other, bringing their respective donor and receptor moieties into close proximity to each other to produce a detectable FRET signal (Step I). Figure 158 Figure 158

[0323] ​​Another aspect of the application relates to a method for identifying one or more nucleic acid molecules in a sample that contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules in the sample or other samples by one or more methylated residues. The method includes providing a sample containing one or more nucleic acid molecules that potentially contain a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules by one or more methylated residues; and contacting the sample with one or more enzymes capable of digesting a deoxyuracil (dU)-containing nucleic acid molecule present in the sample. The method further includes contacting the sample with one or more methylation-sensitive enzymes to form a restriction enzyme reaction mixture, wherein the one or more methylation-sensitive enzymes cleave nucleic acid molecules in the sample that contain one or more unmethylated residues within at least one methylation-sensitive enzyme recognition sequence. One or more primary oligonucleotide primer sets are provided, each primary oligonucleotide primer set including (a) a first primary oligonucleotide primer comprising a nucleotide sequence complementary to a region in the target nucleotide sequence upstream of the one or more methylated residues and (b) a second primary oligonucleotide primer comprising a nucleotide sequence identical to a region in the target nucleotide sequence downstream of the one or more methylated residues. The restriction enzyme reaction mixture is blended with the one or more primary oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a primary polymerase chain reaction mixture. The method further includes subjecting the primary polymerase chain reaction mixture to one or more polymerase chain reaction cycles including a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a primary extension product comprising the target nucleotide sequence or a complement thereof. One or more secondary oligonucleotide primer sets are provided, each secondary oligonucleotide primer set including a first nested oligonucleotide primer and a second nested oligonucleotide primer capable of hybridizing to the primary extension product. The primary extension product is blended with the one or more secondary oligonucleotide primer sets, a deoxynucleotide mixture comprising dUTP, and a DNA polymerase to form a secondary polymerase chain reaction mixture, and the secondary polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles including a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming a secondary extension product. The secondary extension product in the sample is detected and distinguished to identify the presence of one or more nucleic acid molecules containing a target nucleotide sequence that differs from the nucleotide sequence in other nucleic acid molecules in the sample by one or more methylated residues.

[0324] According to this aspect of the application, the secondary polymerase chain reaction mixture can further comprise one or more oligonucleotide detection probes, such as TaqMan TMOligonucleotide detection probes. Detection probes hybridize to a target nucleotide sequence or its complement within the primary extension product and have a quencher molecule and a detectable label separated from but in close proximity to each other such that the quencher molecule quenches the detectable label. During the hybridization step of the secondary polymerase chain reaction process, one or more oligonucleotide detection probes hybridize to complementary portions of the primary extension product and the quencher molecule and detectable label are subsequently cleaved from the one or more oligonucleotide detection probes during the extension step. Upon cleavage, the detectable label is separated from the quencher such that the detectable label is detected.

[0325] In one embodiment, one or both of the primary oligonucleotide primers of the primary oligonucleotide primer set can optionally have a 3' portion comprising a cleavable nucleotide or nucleotide analog and a blocking group such that the 3' end of the one or more primers is not suitable for polymerase extension until the cleavable nucleotide or nucleotide analog is cleaved. Upon cleavage, the free 3' OH end on the one or both of the primary oligonucleotide primers is released, after which primer extension is allowed.

[0326] In another embodiment, the primary oligonucleotide primers of the primary oligonucleotide primer set comprise identical or substantially identical 5' nucleotide sequence portions that are between about 6 and 20 bases in length. According to this embodiment, the desired extension products produced are of sufficient length such that the primary primers preferentially hybridize thereto. However, when undesired primer dimer products are formed, the products will form hairpins by their complementary 5' ends themselves and are not suitable for continued amplification.

[0327] Figure 158 An exemplary PCR-PCR carryover prevention reaction for detecting methylation according to this aspect of the application is shown. In this embodiment, genomic DNA or cfDNA is isolated and treated with a methylation sensitive restriction endonuclease such as Bsh1236I (CG^CG) and / or HinP1I (G^CGC) and UNG to completely digest unmethylated DNA and prevent carryover Figure 158 , step A). As Figure 53 As shown in step B, the target methylation region is amplified using PCR in the presence of dUTP using locus specific primers. In one embodiment, limited cycle amplification (12-20 cycles) is performed to maintain the relative ratios of different amplicons produced. In another embodiment, 20-40 cycles are used to amplify the target region. The primers contain identical 8-11 base tails to prevent primer dimer. The PCR products contain dU, allowing carryover Figure 53, step C). Optionally, the sample is aliquoted into 12, 24, 48 or 96 wells prior to PCR. Nested or semi-nested locus-specific primers and internal conventional TaqMan TM Detection assay amplifies methyl-containing regions. The PCR product incorporates dU, allowing carryover prevention.

[0328] Figure 53 Another PCR-qPCR carryover prevention reaction for detection of methylation is shown. Similar to other embodiments of the application, genomic DNA or cfDNA is isolated and treated with a methylation-sensitive restriction endonuclease, such as Bsh1236I (CG^CG) and UNG (37°C, 30-60 min) to completely digest unmethylated DNA and prevent carryover Figure 53 , step A). The digested DNA is subjected to bisulfite treatment to convert unmethylated residues to uracil, rendering the double-stranded DNA non-complementary. As Figure 53 Hybridization of locus-specific primers containing a 3' cleavable blocking group in the presence of BstU1 (CG^CG) (solid triangles) that cleaves carryover DNA containing unmethylated residues is shown in step B. Once the primers are hybridized to their complementary target sequences, the blocking group is removed. In this embodiment, PCR is used to amplify the target methyl-containing regions in the presence of dNTPs. In this embodiment, a blocking oligo primer is used during amplification to limit the formation of wild-type amplicons. As Figures 54-85 The PCR product is unmethylated, providing carryover prevention, as shown in step C.

[0329] As Figure 54 The PCR product is aliquoted into individual wells for TaqMan TM detection using locus-specific primers and TaqMan TM probes (black bars), as shown in steps D and E. The locus-specific primers are optionally nested or semi-nested with the primary set of locus-specific primers. Optionally, a blocking oligo (thick black bar) can also be incorporated in this reaction to limit the formation of wild-type amplicons. In this embodiment, the TaqMan TM reaction is performed in the presence of dUTP to allow carryover prevention.

[0330] Another aspect of the application relates to a method for identifying one or more ribonucleic acid molecules in a sample that contain a target ribonucleotide sequence that differs from ribonucleotide sequences in other 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 includes providing a sample containing one or more ribonucleic acid molecules that potentially contain a target ribonucleotide sequence that differs from ribonucleotide sequences in other ribonucleic acid molecules; and contacting the sample with one or more enzymes capable of digesting nucleic acid molecules containing dU potentially present in the sample. One or more oligonucleotide primers are provided, each primer complementary to one or more ribonucleic acid molecules containing the target ribonucleotide sequence. The contacted sample is blended with the one or more oligonucleotide primers and a reverse transcriptase to form a reverse transcription mixture, and complementary deoxyribonucleic acid (cDNA) molecules are produced in the reverse transcription mixture. Each cDNA molecule comprises a nucleotide sequence complementary to the target ribonucleotide sequence and contains dU. The method further includes providing one or more oligonucleotide primer sets, each primer set comprising (a) a first oligonucleotide primer comprising a nucleotide sequence complementary to a portion of the cDNA nucleotide sequence adjacent to the complementary sequence of the target ribonucleotide sequence of the cDNA, and (b) a second oligonucleotide primer comprising a nucleotide sequence complementary to a portion of an extension product formed by the first oligonucleotide primer. The reverse transcription mixture containing the cDNA molecules is blended with the one or more oligonucleotide primer sets and a polymerase to form a polymerase reaction mixture, and the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming one or more different first extension products. The method further includes providing one or more oligonucleotide probe sets. Each probe set comprises (a) a first oligonucleotide probe having a target sequence specific portion and (b) a second oligonucleotide probe having a target sequence specific portion, wherein the first oligonucleotide probe and the second oligonucleotide probe of the probe set are configured to hybridize to one another adjacent to the complementary first extension product in a base-specific manner, with a junction between the first oligonucleotide probe and the second oligonucleotide probe. The first extension product is contacted with a ligase and the one or more oligonucleotide probe sets to form a ligation reaction mixture, and the first probe and the second probe of the one or more oligonucleotide probe sets are ligated together to form a ligation product sequence in the ligation reaction mixture.The ligation product sequences in the sample are detected and differentiated to identify the presence of one or more ribonucleic acid molecules whose target ribonucleic acid sequences differ from those in other 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.

[0331] Figure 54 Various embodiments of this aspect of the invention are shown.

[0332] Figure 54 This provides an overview of the RT-PCR-LDR-qPCR legacy prevention response used to detect translocations at the mRNA level. Figure 54 Step A illustrates the translocation between two genes at the DNA level. Examples of different fusion sites between exons 1-b (mRNA fusion 1), 2-b (mRNA fusion 2), and 3-b (mRNA fusion 3) in the mRNA are shown. Figure 54 Step B). The method includes isolating mRNA from whole blood cells, exogenous cells, or circulating tumor cells (CTC), and generating cDNA using reverse transcriptase and primers complementary to exon b. The generated cDNA is amplified by PCR using forward primers for exons 1, 2, and 3 and primers for exon b. Figure 54 (Step B). Primers amplify the smallest fragments containing exon junction regions, independent of translocation breakpoints. Figure 54 Step C shows the various products formed during PCR amplification.

[0333] LDR is performed using exon-junction-specific ligation oligonucleotide probes. The ligation probes can be designed with primers (Ai, Ci) and TaqMan suitable for use. TM The probe uses the tag primer-specific portion (e.g., Ai, Ci') for subsequent detection. Figure 54 (Steps CD, left figure). Alternatively, the ligation probe can be designed to contain a UniTaq primer-specific portion (Ai, Ci') and a tag-specific portion (Bi'). Figure 55 (Steps CD, right figure). After the formation of exon junction-specific ligation products, PCR amplification and detection of the ligation products are performed. Figure 55 Step D). When using tag-specific primers (Ai, Ci) to amplify LDR products, each TaqMan... TM The probe spans the junction and can be scored individually. When UniTaq-specific primers (F1-Bi-Q-Ai, Ci) are used to amplify LDR products, the same primer set scores a given translocation independently of specific exon junctions.

[0334] Figure 55 An RT-PCR-LDR-qPCR Legacy Prevention reaction for detecting translocations at the mRNA level is shown. In this embodiment, mRNA is isolated (step A) and treated with UDG to effect Legacy Prevention (step B). cDNA is generated using a 3' transcript specific primer and reverse transcriptase in the presence of dUTP. Taq polymerase is activated for limited cycle PCR amplification (12-20) to maintain the relative ratios of different amplicons (step B). The primers contain identical 8-11 base tails to prevent primer dimerization. The PCR product incorporates dUTP, allowing for Legacy Prevention (step C). Figure 55 Figure 55 Figure 55 As shown in step D, exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. Ligation enzymes covalently seal the two oligonucleotides together (step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q) that span the ligation junction (step E). The samples are treated with UDG to effect Legacy Prevention, which also destroys the original target amplicon (step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional Legacy Prevention. Figure 55

[0335] As shown in step D, exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. Ligation enzymes covalently seal the two oligonucleotides together (step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q) that span the ligation junction (step E). The samples are treated with UDG to effect Legacy Prevention, which also destroys the original target amplicon (step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional Legacy Prevention. Figure 55 Figure 55 Figure 56 An alternative RT-PCR-LDR-qPCR Legacy Prevention reaction for detecting translocations at the mRNA level is shown. In this embodiment, mRNA is isolated (step A) and treated with UDG to effect Legacy Prevention (step B). cDNA is generated using a 3' transcript specific primer and reverse transcriptase in the presence of dUTP. Taq polymerase is activated for limited cycle PCR amplification (12-20) to maintain the relative ratios of different amplicons (step B). The primers contain identical 8-11 base tails to prevent primer dimerization. The PCR product incorporates dU, allowing for Legacy Prevention (step C). Figure 56

[0336] As shown in step D, exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. Ligation enzymes covalently seal the two oligonucleotides together (step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q) that span the ligation junction (step E). The samples are treated with UDG to effect Legacy Prevention, which also destroys the original target amplicon (step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional Legacy Prevention. Figure 56 Figure 56 Figure 56 As shown in step D, exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. Ligation enzymes covalently seal the two oligonucleotides together (step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q) that span the ligation junction (step E). The samples are treated with UDG to effect Legacy Prevention, which also destroys the original target amplicon (step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional Legacy Prevention. Figure 56 Figure 56 As shown in step D, exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. Ligation enzymes covalently seal the two oligonucleotides together (step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q) that span the ligation junction (step E). The samples are treated with UDG to effect Legacy Prevention, which also destroys the original target amplicon (step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional Legacy Prevention.

[0337] As shown in step D, exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. Ligation enzymes covalently seal the two oligonucleotides together (step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q) that span the ligation junction (step E). The samples are treated with UDG to effect Legacy Prevention, which also destroys the original target amplicon (step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional Legacy Prevention. Figure 56As shown in step D, an exon junction-specific oligonucleotide probe containing UniTaq primer-specific portions (Ai, Ci') and tag portions (Bi') suitable for subsequent PCR amplification and detection hybridizes with the nucleic acid sequence corresponding to the target mRNA molecule to be detected. Figure 56 Step D). After ligating the oligonucleotide probe, the sample is subjected to UDG treatment to remove the original target amplicons, thereby allowing selective amplification and detection of the ligation product using UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above. Figure 57 Steps EF) facilitate the detection of mRNA translocations and mRNA fusions. Figure 57 As shown in steps E and F, the amplified ligation product is incorporated with dUTP to allow for future legacy prevention.

[0338] Figure 57 An example of a residual preventative response to RT-PCR-qLDR for detecting translocations at the mRNA level is shown. In this embodiment, mRNA ( Figure 57 Step A), and it is processed with UDG to achieve legacy prevention ( Figure 57 Step B). cDNA is generated using 3' transcript-specific primers and reverse transcriptase in the presence of dUTP. Taq polymerase is activated for finite-cycle PCR amplification (12-20 cycles) to maintain the relative ratio of different amplicons. Figure 57 (Step B). The primers contain the same 8-11 base tail to prevent primer dimerization, and a universal primer-specific portion to enable subsequent universal PCR amplification using biotin-labeled primers to attach 5' biotin to the amplification product containing the target region. The PCR product incorporates dU, thus allowing for residual prevention (…). Figure 57 Step C). The biotinylated PCR product is immobilized onto a solid support and the target region is detected using an exon junction-specific ligation probe, such as... Figure 57 As shown in step D. In this embodiment, the exon junction-specific ligation probes of the ligation pair contain complementary tail sequences and, respectively, acceptor or donor groups, which are capable of generating a detectable signal via FRET that occurs when they are in close proximity to each other, as described above. Therefore, after ligation ( Figure 58 Step D) involves the complementary 5' and 3' ends of the ligation product hybridizing with each other, thereby bringing their respective donor and acceptor portions into close proximity to generate a detectable FRET signal. Figure 58 Step E).

[0339] Figure 58 An overview of the residual preventative response of RT-PCR-LDR-qPCR for detecting alternative splicing is presented. Figure 58Step A is a graphical representation of a gene with 5 exons shown at the DNA level, and Figure 58 Step B shows examples of normal (1-2-3a-4) and alternatively spliced (1-2-3b-4) variant mRNAs. The method includes isolation of mRNA from whole blood cells, exosomes or CTCs, and production of cDNA using reverse transcriptase and a primer complementary to exon 4, as Figure 58 shown in Step B. The cDNA is PCR amplified using an exon 4 primer and a forward primer to exon 2 to produce amplicons for the normal and alternatively spliced variants, if present. As Figure 58 shown in Step C, exon junction specific ligation oligonucleotide probes containing a tag-primer sequence (Ai, Ci'; left panel) or UniTaq primer and tag sequence (Ai, Bi'-Ci'; right panel) hybridize to their corresponding target sequences in the PCR product and, if perfect complementarity exists at the junction, a ligase covalently seals the two oligonucleotides together. As described above, a tag specific primer (Ai, Ci) and TaqMan TM probe (F1-Q or F2-Q, Figure 59 Step D, left panel) or UniTaq primer (F1-Bi-Q-Ai, F2-Bi-Q-Ai, Ci, Figure 59 Step D, right panel) amplify and detect the ligation product.

[0340] Figure 59 RT-PCR-LDR-qPCR Legacy Prevention Reaction for quantifying wild type and alternatively spliced mRNA transcripts is shown. Figure 59 Step A shows wild type transcripts containing exon 3a (top) and alternatively spliced transcripts containing exon 3b to be detected (bottom). The method includes isolation of mRNA and its treatment with UDG to effect Legacy Prevention Figure 59 , Step B). cDNA is produced using 3' transcript specific primers and reverse transcriptase in the presence of dUTP. Taq polymerase is activated for limited cycle PCR amplification (12-20) to maintain the relative ratios of different amplicons Figure 59 , Step B). The primers contain identical 8-11 base tails to prevent primer dimerization. The PCR product incorporates dU, allowing Legacy Prevention Figure 59 , Step C). As Figure 59 Step D, shown in Step D, exon junction specific ligation oligonucleotide probes containing a tag-primer specific portion (Ai, Ci') suitable for subsequent PCR amplification hybridize to their corresponding target sequences in a base specific manner. A ligase covalently seals the two oligonucleotides together Figure 59Step D), and the ligation products are aliquoted into individual wells to use tag primers (Ai, Ci) and TaqMan across the ligation junctions. TM Detection is performed using probes (F1-Q and F2-Q). Figure 60 Steps EF). Using real-time PCR and TaqMan to detect different markers. TM Probes are used to quantify and differentiate between wild-type and alternative splicing variants. Samples are treated with UDG for legacy prevention, which also destroys the original target amplicon. Figure 60 (Step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probe amplify the LDR product, thus providing additional legacy protection.

[0341] Figure 59 This demonstrates another RT-PCR-LDR-qPCR legacy preventative response for quantifying wild-type and alternatively spliced ​​mRNA transcripts. Figure 60 Step A shows the wild-type transcript containing exon 3a (top) and the alternatively spliced ​​transcript containing the exon 3b to be detected (bottom). Steps A and D of the method are essentially the same as those for... Figure 61 The similarities are the same, except that the exon junction-specific ligation probe is designed to contain UniTaq primer sequences (Ai, Ci') and a UniTaq tag sequence (Bi'). Therefore, in this embodiment, the ligation products corresponding to wild-type and alternative splicing variants are subsequently amplified, detected, and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci), as described above and in... Figure 61 As shown in step EG.

[0342] Figure 61 The RT-PCR-qLDR legacy prevention response for quantifying wild-type and alternatively spliced ​​mRNA transcripts is shown. Figure 61 Step A shows a wild-type transcript containing exon 3a (top) and an alternatively spliced ​​transcript containing exon 3b to be detected (bottom). The method includes isolating the mRNA and treating it with UDG to achieve legacy prevention. Figure 61 Step B). cDNA is generated using 3' transcript-specific primers and reverse transcriptase in the presence of dUTP. The primers contain identical 8-11 base tails to prevent primer dimerization, and a universal primer-specific portion to enable subsequent universal PCR amplification using biotin-labeled primers to attach 5' biotin to the amplification product containing the target region. The PCR product incorporates dU, thus allowing for residual prevention (…). Figure 61, step C). The biotinylated PCR product is immobilized to a solid support and the region of interest is detected using exon junction specific ligation probes, as Figure 61 shown in step D. In this embodiment, the exon junction specific ligation probes of the ligation pair contain complementary tail sequences and contain either an acceptor group or a donor group, respectively, that are capable of generating a detectable signal by FRET when in close proximity to each other, as described above. Thus, upon ligation (step D), the complementary 5' and 3' tails of the ligation product hybridize to each other, bringing their respective donor and acceptor moieties into close proximity to each other to generate a detectable FRET signal (step E). Figure 62 Figure 62

[0343] Figure 62 An RT-PCR-LDR-qPCR legacy prevention reaction is shown for detecting low levels of a alternatively spliced transcript. Figure 62 Step A shows a wild type transcript containing exon 3a (top) and a low level of an alternatively spliced transcript containing exon 3b (bottom) to be detected. The method includes isolating mRNA and treating it with UDG to effect legacy prevention Figure 62 (step B). cDNA is generated using a 3' transcript specific primer (i.e., to exon 4) and reverse transcriptase in the presence of dUTP. Taq polymerase is activated for a limited cycle of PCR amplification (12-20) to maintain the relative ratios of different amplicons Figure 62 (step B). In this embodiment, primers specific to the alternatively spliced variant (i.e., exon 3b) and that do not hybridize to the wild type variant (i.e., exon 3a) are utilized to generate only an amplification product corresponding to the alternatively spliced variant. The PCR product incorporates dUTP, allowing for legacy prevention Figure 62 (step C). The biotinylated PCR product is immobilized to a solid support and the region of interest is detected using exon junction specific ligation probes, as Figure 62 shown in step D. Exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. A ligase covalently seals the two oligonucleotides together Figure 62 (step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (Fi-Q) that span the ligation junction Figure 63 (step E-F). The alternatively spliced variant is detected during real-time PCR by release of the fluorescent group of the TaqMan TM probes. The sample is treated with UDG to effect legacy prevention, which also destroys the original target amplicon Figure 62 ​​(Step E). When PCR is used in the presence of dUTP, only the true LDR product is amplified. Neither the original PCR primers nor the LDR probe amplify the LDR product, thus providing additional legacy protection.

[0344] Figure 63 and 64 Similar RT-PCR-LDR-qPCR and RT-PCR-qLDR legacy prevention responses for detecting low levels of alternative splicing transcripts are shown, as per [reference to...]. Figure 63 As stated and shown. Figure 64 In this embodiment, the exon junction-specific ligation probe is designed to contain a UniTaq primer sequence (Ai, Ci') and a UniTaq tag sequence (Bi'). Therefore, in this embodiment, the ligation product corresponding to the alternative splicing variant is subsequently amplified, detected, and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci), as described above and as in […]. Figure 64 As shown in step EG. Figure 64 In the implementation scheme, the exon junction-specific ligation probes of the ligation pair contain complementary tail sequences and respectively contain acceptor or donor groups, which are capable of generating a detectable signal through FRET that occurs when they are in close proximity to each other, as described above. Therefore, after ligation ( Figure 65 Step D) involves the complementary 5' and 3' ends of the ligation product hybridizing with each other, thereby bringing their respective donor and acceptor portions (D, F2) into close proximity to generate a detectable FRET signal. Figure 65 Step E).

[0345] Figure 65 An overview of the residual preventative response of RT-PCR-LDR-qPCR for detecting alternative splicing is presented. Figure 65 Step A illustrates a gene with three exons, a variable start site, and a first exon at the DNA level. Figure 65 Step B illustrates examples of normal (1-2-3) and alternative splicing variant (1a-2-3) mRNAs. The method includes isolating mRNA from whole blood cells, exogenous organisms, or CTCs, and generating cDNA using reverse transcriptase and primers complementary to exon 2, such as... Figure 58 As shown in step B, cDNA is amplified by PCR using exon 2 primers and forward primers complementary to exon 1 or exon 1a to generate amplicones for both splice variants. Figure 58Exon junction specific ligation oligonucleotide probes containing either a tag-primer sequence (Ai, Ci'; left panel) or a UniTaq primer and tag sequence (Ai, Bi'-Ci'; right panel) hybridize to their corresponding target sequences in the PCR product and if perfect complementarity exists at the junction, the ligase covalently seals the two oligonucleotides together. As described above, using tag specific primers (Ai, Ci) and TaqMan TM probes (F1-Q or F2-Q; left panel) or UniTaq primers (F1-Bi-Q-Ai, F2-Bi-Q-Ai and Ci, Figure 66 , right panel) amplify and detect the ligation product. Figure 66

[0346] Figure 66 RT-PCR-LDR-qPCR legacy prevention reaction for quantifying transcripts containing a wild type start site and alternative transcription start sites is shown. Figure 66 Step A shows wild type transcripts containing exon 1 (top) and alternative transcripts with exon la as a start site (bottom). The method includes isolating mRNA and treating it with UDG to achieve legacy prevention Figure 66 , step B). cDNA is generated using 3' transcript specific primers and reverse transcriptase in the presence of dUTP. The cDNA is PCR amplified using exon 2 specific primers and forward primers complementary to either exon 1 or exon la to generate amplicons for both splice variants. Limited PCR amplification (12-20 cycles) is performed to maintain the relative ratio of different amplicons Figure 66 , step B). In another embodiment, the region of interest is amplified using 20-40 PCR cycles. The primers contain identical 8-11 base tails to prevent primer dimerization. The PCR product incorporates dU, allowing legacy prevention Figure 66 , step C). As Figure 66 shown in step D, exon junction specific ligation oligonucleotide probes containing tag-primer specific portions (Ai, Ci') suitable for subsequent TaqMan TM PCR amplification hybridize to their corresponding target sequences in a base specific manner. The ligase covalently seals the two oligonucleotides together Figure 66 , step D) and the ligation product is aliquoted into separate wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q and F2-Q) that span the ligation junction Figure 67 , steps E-F). Real-time PCR and detection of different labels is used to quantify the relative amounts of the two splice variants TM ​probes to quantify and distinguish between wild type and alternative transcript start site variants. The sample is treated with UDG to enable carryover prevention, which also destroys the original target amplicon Figure 67 , step E). When PCR is used in the presence of dUTP, only the true LDR product is amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional carryover prevention.

[0347] Figure 66 Another RT-PCR-LDR-qPCR carryover prevention reaction for quantifying wild type and alternatively spliced mRNA transcripts is shown. Figure 67 Step A shows wild type transcripts containing exon 1 (top) and alternative transcripts with exon la as a start site (bottom). Steps A-D of the method are essentially the same as described for Figure 68 the exception that the exon junction specific ligation probes are designed to contain UniTaq primer sequences (Ai, Ci') and UniTaq tag sequences (Bi'). Thus, in this embodiment, the ligation products corresponding to the wild type and variant transcripts are subsequently amplified, detected and quantified using real-time PCR with UniTaq-specific primers (Fl-Bi-Q-Ai, Ci) as described above and shown in Figure 68 steps E-F.

[0348] Figure 68 An RT-PCR-qLDR carryover prevention reaction for quantifying wild type and alternatively spliced mRNA transcripts is shown. Figure 68 Step A shows wild type transcripts containing exon 1 (top) and alternative transcripts with exon la as a start site (bottom). The method includes isolating mRNA and treating with UDG to enable carryover prevention Figure 68 , step B). cDNA is generated using 3' transcript specific primers and reverse transcriptase in the presence of dUTP. The cDNA is PCR amplified using exon 2 primers and forward primers complementary to exon 1 or exon la to generate amplicons for both splice variants. The primers contain identical 8-11 base tails to prevent primer dimer and a universal primer specific portion to enable subsequent universal PCR amplification using biotin labeled primers to append 5' biotin to the amplicon containing the region of interest. The PCR products incorporate dU, allowing carryover prevention Figure 68 , step C). The biotinylated PCR products are immobilized to a solid support and the region of interest is detected using exon junction specific ligation probes as Figure 68As shown in step D. In this embodiment, the exon junction-specific ligation probes of the ligation pair contain complementary tail sequences and, respectively, acceptor or donor groups, which are capable of generating a detectable signal via FRET that occurs when they are in close proximity to each other, as described above. Therefore, after ligation ( Figure 69 Step D) involves the complementary 5' and 3' ends of the ligation product hybridizing with each other, thereby bringing their respective donor and acceptor portions into close proximity to generate a detectable FRET signal. Figure 69 Step E).

[0349] Figure 69 This demonstrates a RT-PCR-LDR-qPCR legacy prevention response for detecting low levels of variable initiation site transcripts. Figure 69 Step A shows a wild-type transcript containing exon 1 (top) and a variable transcript with exon 1a as the start site (bottom). The method includes isolating mRNA and treating it with UDG to achieve legacy prevention. Figure 69 Step B). cDNA is generated using 3' transcript-specific primers and reverse transcriptase in the presence of dUTP. Taq polymerase is activated for finite-cycle PCR amplification (12-20 cycles) to maintain the relative ratio of different amplicons. Figure 69 (Step B). In this implementation, primers specific to the variable transcript (i.e., exon 1a) and not hybridizing with the wild-type variant (i.e., exon 1) are used to generate only the amplification product corresponding to the variable transcript. The PCR product incorporates dUTP, thereby allowing for residual prevention ( Figure 69 Step C). Figure 69 As shown in step D, the exon junction-specific oligonucleotide probe containing primer-specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridizes to its corresponding target sequence in a base-specific manner. The ligase covalently seals the two oligonucleotides together. Figure 69 Step D), and the ligation products are aliquoted into individual wells to use tag primers (Ai, Ci) and TaqMan across the ligation junctions. TM Detection using probe (F1-Q) Figure 70 (Step EF). During real-time PCR, TaqMan is released... TM The probe's fluorescent group is used to detect variable transcripts. Samples are treated with UDG for legacy prevention, which also destroys the original target amplicon (…). Figure 69 (Step E). When PCR is used in the presence of dUTP, only the true LDR product is amplified. Neither the original PCR primers nor the LDR probe amplify the LDR product, thus providing additional legacy protection.

[0350] Figure 70 and71 Similar RT-PCR-LDR-qPCR and RT-PCR-qLDR legacy prevention reactions for detecting low levels of alternative start site transcripts are shown as described and shown in ​ (Steps A-C) with the use of primers specific for amplification of only alternatively transcribed. In ​ embodiments, the exon junction specific ligation probes are designed to contain the UniTaq primer sequence (Ai, Ci') and the UniTaq tag sequence (Bi'). Thus, in this embodiment, the ligation products corresponding to the alternative start site transcripts are subsequently amplified, detected and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above and as shown in Figure 70 Steps E-G. In Figure 71 embodiments, the exon junction specific ligation probes of the ligation pair contain complementary tail sequences and contain either an acceptor group or a donor group, respectively, that are capable of generating a detectable signal by FRET occurring upon close proximity to each other, as described above. Thus, after ligation (Step D), the complementary 5' and 3' tail ends of the ligation products hybridize to each other, bringing their respective donor and acceptor moieties (D, F2) into close proximity to each other to generate a detectable FRET signal (Step E). Figure 71 Figure 71

[0351] Figure 72 An overview of a RT-PCR-LDR-qPCR legacy prevention reaction for detecting exon deletions is shown. Figure 72 Step A shows a diagram of a gene with 5 exons and 4 introns at the DNA level. Figure 72 Step B shows an example of a wild type transcript containing exons 1-5 (top) and an alternative transcript in which exon 4 is deleted (bottom, i.e. exons 1-3 and 5). The method includes isolating mRNA from whole blood cells, exosomes or CTCs and generating cDNA using a reverse transcriptase and a primer complementary to exon 5, as shown in Figure 72 Step B. The cDNA is PCR amplified using the exon 5 primer in conjunction with forward primers complementary to exon 3 and exon 4 to generate amplicons of the wild type and the deletion variant. As Figure 72 ​​Exon junction specific ligation oligonucleotide probes containing either a tag-primer sequence (Ai, Ci'; left panel) or UniTaq primer and tag sequence (Ai, Bi'-Ci'; right panel) hybridize to their corresponding target sequences in the PCR product and if perfect complementarity exists at the junction, the ligase covalently seals the two oligonucleotides together. As described above, the tag specific primers (Ai, Ci) and TaqMan TM probes (F1-Q or F2-Q; left panel) or UniTaq primer (F1-Bi-Q-Ai, F2-Bi-Q-Ai and Ci, Figure 72 panel) are used to amplify and detect the ligation product. Figure 72 panel) are used to amplify and detect the ligation product.

[0352] Figure 73 RT-PCR-LDR-qPCR legacy prevention reaction for quantifying wild type transcript and transcript with exon deletion is shown. Figure 73 Step A shows wild type transcript containing exons 1-5 (top) and alternative transcript with only exons 1-3 and 5, missing exon 4 (bottom). The method includes isolating mRNA and treating it with UDG to achieve legacy prevention Figure 73 , Step B). cDNA is generated using 3' transcript specific primers (e.g. exon 5 specific primer) and reverse transcriptase in the presence of dUTP. The cDNA is PCR amplified using exon 5 primer in conjunction with forward primers complementary to exon 3 and exon 4 to generate amplicons for wild type and deletion variant. Limited PCR amplification (12-20 cycles) is performed to maintain the relative ratio of different amplicons Figure 73 , Step B). In another embodiment, the target region is amplified using 20-40 PCR cycles. The primers contain identical 8-11 base tails to prevent primer dimerization. The PCR product incorporates dU, allowing legacy prevention Figure 73 , Step C). As Figure 73 Exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification are hybridized to their corresponding target sequences in a base specific manner, as shown in Step D. The ligase covalently seals the two oligonucleotides together Figure 73 , Step D) and the ligation product is aliquoted into separate wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q and F2-Q) spanning the ligation junction (left panel) or UniTaq primer (F1-Bi-Q-Ai, F2-Bi-Q-Ai and Ci, Figure 73 , Steps E-F). Real-time PCR and TaqMan TMprobes to quantitate and distinguish between wild type and deletion variants. The sample is treated with UDG to enable carryover prevention, which also destroys the original target amplicon Figure 73 When PCR is used in the presence of dUTP, only the true LDR product is amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional carryover prevention.

[0353] Figure 74 Another RT-PCR-LDR-qPCR carryover prevention reaction for quantitating wild type transcripts and transcripts with exon deletions is shown. Figure 74 Step A shows wild type transcripts containing exons 1-5 (top) and alternative transcripts with only exons 1-3 and 5, missing exon 4 (bottom). Steps A-D of the method are essentially the same as described for Figure 73 the wild type and variant transcripts using UniTaq-specific primers (Fl-Bi-Q-Ai, Ci) as described above and shown in Steps E-G. Figure 74

[0354] Figure 75 An RT-PCR-qLDR carryover prevention reaction for quantitating wild type transcripts and transcripts with exon deletions is shown. Figure 75 Step A shows wild type transcripts containing exons 1-5 (top) and alternative transcripts with only exons 1-3 and 5, missing exon 4 (bottom). The method includes isolating mRNA and treating it with UDG to enable carryover prevention (Step B). A 3' transcript-specific primer (e.g., exon 5 specific primer) and reverse transcriptase are used in the presence of dUTP to produce cDNA. The cDNA is PCR amplified using an exon 5 primer in conjunction with a forward primer complementary to exon 3 and exon 4 to produce amplicons for wild type and deletion variants. The primers contain identical 8-11 base tails to prevent primer dimer and a universal primer-specific portion to enable subsequent universal PCR amplification using biotin-labeled primers to append 5' biotin to the amplicon containing the region of interest. The PCR product incorporates dU, allowing carryover prevention (Step C). Figure 75 Figure 75 Figure 75 ​​​As shown in step D. In this embodiment, the exon junction-specific ligation probes of the ligation pair contain complementary tail sequences and, respectively, acceptor or donor groups, which are capable of generating a detectable signal via FRET that occurs when they are in close proximity to each other, as described above. Therefore, after ligation ( Figure 75 Step D) involves the complementary 5' and 3' ends of the ligation product hybridizing with each other, thereby bringing their respective donor and acceptor portions into close proximity to generate a detectable FRET signal. Figure 75 Step E).

[0355] Figure 76 This demonstrates the RT-PCR-LDR-qPCR legacy prevention response used to detect low levels of transcripts with exon deletions. Figure 76 Step A shows a wild-type transcript containing exons 1-5 (top) and a variable transcript containing only exons 1-3 and 5, lacking exon 4 (bottom). The method includes isolating the mRNA and treating it with UDG to achieve legacy prevention. Figure 76 (Step B). cDNA is generated using 3' transcript-specific primers (e.g., exon 5-specific primers) and reverse transcriptase in the presence of dUTP. The cDNA is amplified by PCR using an exon 5 primer combined with a forward primer complementary to exon 3 and a blocking oligonucleotide. The blocking oligonucleotide hybridizes to exon 4 in the wild-type transcript, thus preventing wild-type transcript amplification. Therefore, an amplification product corresponding only to the deleted transcript is produced. The PCR product incorporates dUTP, thus allowing for residual prevention (…). Figure 76 Step C). Figure 76 As shown in step D, the exon junction-specific oligonucleotide probe containing the tag primer-specific portion (Ai, Ci') suitable for subsequent PCR amplification hybridizes to its corresponding target sequence in a base-specific manner. The ligase covalently seals the two oligonucleotides together. Figure 76 Step D), and the ligation products are aliquoted into individual wells to use tag primers (Ai, Ci) and TaqMan across the ligation junctions. TM Detection using probe (F1-Q) Figure 76 (Step EF). During real-time PCR, TaqMan is released... TM The fluorescent group of the probe is used to detect the missing transcript. Samples are treated with UDG for legacy prevention, which also destroys the original target amplicon (…). Figure 76 (Step E). When PCR is used in the presence of dUTP, only the true LDR product is amplified. Neither the original PCR primers nor the LDR probe amplify the LDR product, thus providing additional legacy protection.

[0356] Figure 77 and 78Similar RT-PCR-LDR-qPCR and RT-PCR-qLDR legacy prophylactic reactions for detecting low levels of missing transcripts are shown, as described above and illustrated in Figure 76 the embodiments. In Figure 77 the embodiments, the exon junction specific ligation probes are designed to contain the UniTaq primer sequence (Ai, Ci') and the UniTaq tag sequence (Bi'). Thus, in this embodiment, the ligation products corresponding to the missing transcripts are subsequently amplified, detected and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci), as described above and as illustrated in Figure 77 steps E-G. In Figure 78 the embodiments, the exon junction specific ligation probes of the ligation pair contain complementary tail sequences and contain either an acceptor group or a donor group, respectively, which are capable of generating a detectable signal by FRET occurring upon close proximity to each other, as described above. Thus, after ligation (step D), the complementary 5' and 3' tail ends of the ligation product hybridize to each other, bringing their respective donor and acceptor moieties (D, F2) into close proximity to each other to generate a detectable FRET signal (step E). Figure 78 Figure 78

[0357] Figure 79 An overview of the RT-PCR-LDR-qPCR legacy prophylactic reaction for detecting alternative splicing of intron insertion is shown. Figure 79 Step A shows a schematic of a gene with 5 exons and 4 introns at the DNA level. Figure 79 Step B shows an example of a wild type transcript containing exons 1-5 (top) and an alternatively spliced transcript containing exons 1-5 and intron il insertion (bottom). The method comprises isolating mRNA from whole blood cells, exosomes or CTCs and generating cDNA using a reverse transcriptase and a primer complementary to exon 2, as shown in Figure 79 Step B. The cDNA is PCR amplified using an exon 2 specific primer in conjunction with a forward primer directed to exon 1 to generate amplicons of the wild type and the intron insertion variant. As shown in Figure 79 Step C, the exon junction specific ligation oligonucleotide probes containing a tag-primer sequence (Ai, Ci'; left panel) or a UniTaq primer and tag sequence (Ai, Bi'-Ci'; right panel) hybridize to their corresponding target sequences in the PCR product and, if perfect complementarity exists at the junction, a ligase covalently seals the two oligonucleotides together. As described above, tag-specific primers (Ai, Ci) and TaqMan TM probes (F1-Q or F2-Q; Figure 79 ​​Step D (left figure) or UniTaq primers (F1-Bi-Q-Ai, F2-Bi-Q-Ai and Ci, Figure 79 Step D (right figure) Amplify and detect the ligation product.

[0358] Figure 80 This demonstrates the RT-PCR-LDR-qPCR legacy prevention response used to quantify wild-type transcripts and alternatively spliced ​​transcripts containing intron insertions. Figure 80 Step B shows examples of wild-type transcripts containing exons 1-5 (top) and alternatively spliced ​​transcripts containing exons 1-5 and intron i1 insertions (bottom). The method includes isolating mRNA and treating it with UDG to achieve residual prevention (…). Figure 80 (Step B). cDNA is generated using 3' transcript-specific primers (e.g., exon 2-specific primers) and reverse transcriptase in the presence of dUTP. The cDNA is then amplified by PCR using exon 2-specific primers combined with a forward primer targeting exon 1 to generate amplicones for wild-type and intron insert variants. Limited PCR amplification (12–20 cycles) is performed to maintain the relative ratio of different amplicon types. Figure 80 (Step B). In another embodiment, 20-40 cycles are used to amplify the target region. Primers contain identical 8-11 base tails to prevent primer dimerization. dU is incorporated into the PCR product, thus allowing for residual prevention (…). Figure 80 Step C). Figure 80 As shown in step D, the exon junction-specific oligonucleotide probe containing the tag primer-specific portion (Ai, Ci') suitable for subsequent PCR amplification hybridizes to its corresponding target sequence in a base-specific manner. The ligase covalently seals the two oligonucleotides together. Figure 80 Step D), and the ligation products are aliquoted into individual wells to use tag primers (Ai, Ci) and TaqMan across the ligation junctions. TM Detection is performed using probes (F1-Q and F2-Q). Figure 80 Steps EF). Using real-time PCR and TaqMan to detect different markers. TM Probes are used to quantify and differentiate between wild-type and insert variants. Samples are treated with UDG for legacy prevention, which also destroys the original target amplicon (…). Figure 80 (Step E). When PCR is used in the presence of dUTP, only the true LDR product is amplified. Neither the original PCR primers nor the LDR probe amplify the LDR product, thus providing additional legacy protection.

[0359] Figure 81 This demonstrates another RT-PCR-LDR-qPCR legacy preventative response for quantifying wild-type transcripts and alternatively spliced ​​transcripts containing intron insertions. Figure 81Step B shows an example of wild type transcript containing exons 1-5 (top) and alternative spliced transcript containing exon 1-5 and intron il insertion (bottom). Steps A-D of the method are essentially the same as for Figure 80 the same except that the exon junction specific ligation probes are designed to contain UniTaq primer sequences (Ai, Ci') and UniTaq tag sequences (Bi'). Thus, in this embodiment, the ligation products corresponding to wild type and variant transcripts are subsequently amplified, detected and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above and in Figure 81 shown in steps E-G.

[0360] Figure 82 RT-PCR-qLDR legacy prevention reaction for quantifying wild type transcript and alternative spliced transcript containing intron insertion is shown. Figure 82 Step B shows an example of wild type transcript containing exons 1-5 (top) and alternative spliced transcript containing exon 1-5 and intron il insertion (bottom). The method includes isolating mRNA and treating with UDG to achieve legacy prevention Figure 82 using a 3' transcript specific primer (e.g. exon 2 specific primer) and reverse transcriptase. The cDNA is PCR amplified using an exon 2 specific primer in conjunction with a forward primer to exon 1 to generate amplicons of wild type and intron insertion variant. The primers contain identical 8-11 base tails to prevent primer dimer and a universal primer specific portion to enable subsequent universal PCR amplification with biotin labeled primers to append 5' biotin to the amplification product containing the region of interest. The PCR product incorporates dU, thus allowing legacy prevention Figure 82 , step C). The biotinylated PCR product is immobilized to a solid support and the region of interest is detected using exon junction specific ligation probes as Figure 82 shown in step D. In this embodiment, the exon junction specific ligation probes of the ligation pair contain complementary tail sequences and either contain an acceptor group or a donor group, respectively, that are capable of generating a detectable signal by FRET occurring upon close proximity to each other as described above. Thus, upon ligation Figure 82 , step D), the complementary 5' and 3' tails of the ligation product hybridize to each other, bringing their respective donor and acceptor moieties in close proximity to each other to generate a detectable FRET signal Figure 82 , step E).

[0361] Figure 83RT-PCR-LDR-qPCR legacy prevention reaction for detecting low level transcripts containing intron insertion is shown. Figure 83 Step B shows an example of a wild type transcript containing exons 1-5 (top) and an alternative spliced transcript containing exons 1-5 and an intron il insertion (bottom). The method includes isolating mRNA and treating with UDG to achieve legacy prevention Figure 83 , Step B). cDNA is generated using a 3' transcript specific primer (e.g., exon 2 specific primer) and reverse transcriptase in the presence of dUTP. The cDNA is PCR amplified using an exon 2 primer in conjunction with an intron specific forward primer. The intron specific primer does not amplify the wild type transcript, thereby amplifying only transcripts containing intron il. The PCR product incorporates dU, allowing for legacy prevention Figure 83 , Step C). As Figure 83 shown in Step D, exon junction specific ligation oligonucleotide probes containing primer specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridize in a base specific manner to their corresponding target sequences. A ligase covalently seals the two oligonucleotides together Figure 83 , Step D), and the ligation products are aliquoted into individual wells for detection using tag primers (Ai, Ci) and TaqMan TM probes (F1-Q) that span the ligation junction Figure 83 , Steps E-F). Transcripts containing intron insertion are detected by release of the fluorescent group of the TaqMan TM probes during real-time PCR. The sample is treated with UDG to achieve legacy prevention, which also destroys the original target amplicon Figure 83 , Step E). When PCR is used in the presence of dUTP, only the true LDR product is amplified. Neither the original PCR primers nor the LDR probes amplify the LDR product, providing additional legacy prevention.

[0362] Figure 84 and 85 show similar RT-PCR-LDR-qPCR and RT-PCR-qLDR legacy prevention reactions for detecting low levels of intron insertion transcripts as described and shown in relation to Figure 83 . In the embodiment of Figure 84 , the exon junction specific ligation probes are designed to contain UniTaq primer sequences (Ai, Ci') and UniTaq tag sequences (Bi'). Thus, in this embodiment, the ligation products corresponding to the intron insertion transcripts are subsequently amplified, detected and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci) as described above and as shown in Figure 84 Steps E-G.Figure 85 In the implementation scheme, the exon junction-specific ligation probes of the ligation pair contain complementary tail sequences and respectively contain acceptor or donor groups, which are capable of generating a detectable signal through FRET that occurs when they are in close proximity to each other, as described above. Therefore, after ligation ( Figure 85 Step D) involves the complementary 5' and 3' ends of the ligation product hybridizing with each other, thereby bringing their respective donor and acceptor portions (D, F1) into close proximity to generate a detectable FRET signal. Figure 85 Step E).

[0363] The method of this invention is suitable for quantifying or counting the amount of one or more target nucleotide sequences in a sample. For example, the method of this invention can be used to count the relative copy number of one or more target nucleic acid molecules in a sample, such as... Figures 86-91 As shown in the image.

[0364] Figure 86 This illustrates a PCR-LDR legacy prevention reaction for counting DNA copy numbers. The method includes isolating DNA from CTCs, tumor-specific exogens, or another biological sample, and treating it with UDG to achieve legacy prevention. Figure 86 Step B). The target chromosomal region is amplified using finite-cycle PCR (12-20 cycles) to maintain the relative ratio of different amplicones. Figure 86 (Step B). In another embodiment, 20-40 cycles are used to amplify the target chromosomal region. For accurate counting, the sample is dispersed into 12, 24, 48, or 96 wells prior to PCR amplification. Primers contain identical 8-11 base tails to prevent primer dimerization. dU is incorporated into the PCR product, thus allowing for residual prevention (…). Figure 86 Step C). Figure 86 As shown in step D, the locus-specific oligonucleotide probe containing primer-specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridizes to its corresponding target sequence in a base-specific manner. The ligase covalently seals the two oligonucleotides together. Figure 86 Step D), and the ligation products are aliquoted into individual wells to use tag primers (Ai, Ci) and TaqMan across the ligation junctions. TM Detection using probe (F1-Q) Figure 86 Steps EF). DNA copy number was determined based on the Poisson distribution of the signal in different wells or chambers. Samples were treated with UDG for legacy prevention, which also destroyed the original target amplicon ( Figure 86 (Step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probe amplify the LDR product, thus providing additional legacy protection.

[0365] Figure 87 Another PCR-LDR-qPCR Legacy Prevention reaction for counting DNA copy number is shown. The method includes essentially the same steps (i.e. steps A-D) as the method shown in Figure 86 ; however, in this embodiment, the locus-specific ligation probes are designed to contain the UniTaq primer sequence (Ai, Ci') and the UniTaq tag sequence (Bi'). Thus, in this embodiment, the ligation products are subsequently amplified, detected and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci), as described above and shown in Figure 87 Steps E-G. Copy number is determined based on the Poisson distribution of signals in different wells or chambers.

[0366] Figure 88 A PCR-qLDR Legacy Prevention reaction for counting DNA copy number is also shown. The method includes isolating DNA from CTCs, tumor-specific exosomes or other biological samples and treating it with UDG to achieve Legacy Prevention Figure 88 , step B). The target chromosomal region is amplified using PCR with limited cycles (12-20 cycles) to maintain the relative ratios of different amplicons Figure 88 , step B). In another embodiment, the target chromosomal region is amplified using 20-40 cycles. For accurate counting, the sample is aliquoted into 12, 24, 48 or 96 wells prior to PCR amplification. The primers contain identical 8-11 base tails to prevent primer dimerization and a universal primer-specific portion to enable subsequent universal PCR amplification using biotin-labeled primers to append 5' biotin to the amplification products containing the target region. The PCR products are dU-incorporated, thus allowing Legacy Prevention Figure 88 , step C). The biotinylated PCR products are immobilized to a solid support and the target region is detected using locus-specific ligation probes as shown in Figure 88 Step D. In this embodiment, the exon junction-specific ligation probes of the ligation pair contain complementary tail sequences and either contain an acceptor group or a donor group, respectively, that are capable of generating a detectable signal by FRET that occurs upon close proximity to each other, as described above. Thus, upon ligation Figure 88 , step D), the complementary 5' and 3' tails of the ligation products hybridize to each other, thus bringing their respective donor and acceptor moieties in close proximity to each other to generate a detectable FRET signal Figure 88 , step E). Copy number is determined based on the Poisson distribution of signals in different wells or chambers.

[0367] Figure 89This illustrates an RT-PCR-LDR-qPCR residual prevention reaction for counting RNA copy numbers. The method includes isolating RNA from whole blood cells, exogenous organisms, CTCs, or other biological samples, and treating it with UDG to achieve residual prevention. Figure 89 Step B). cDNA is generated using 3' transcript-specific primers and reverse transcriptase in the presence of dUTP. Taq polymerase is activated for finite-cycle PCR amplification (12-20 cycles) to maintain the relative ratio of different amplicons. Figure 89 (Step B). For accurate counting, disperse the samples into 12, 24, 48, or 96 wells before PCR amplification. Primers contain identical 8-11 base tails to prevent primer dimerization. PCR products incorporate dU, thus allowing for residual prevention (…). Figure 89 Step C). Figure 89 As shown in step D, the locus-specific ligation oligonucleotide probe containing the tag primer-specific portions (Ai, Ci') suitable for subsequent PCR amplification hybridizes to its corresponding target sequence in a base-specific manner. The ligase covalently seals the two oligonucleotides together. Figure 89 Step D), and the ligation products are aliquoted into individual wells to use tag primers (Ai, Ci) and TaqMan across the ligation junctions. TM Detection using probe (F1-Q) Figure 89 Step EF). RNA copy number was quantified using real-time PCR based on the Poisson distribution of signals in different wells or chambers. Samples were treated with UDG for legacy prevention, which also destroyed the original target amplicon ( Figure 89 (Step E). When PCR is used in the presence of dUTP, only the true LDR product will be amplified. Neither the original PCR primers nor the LDR probe amplify the LDR product, thus providing additional legacy protection.

[0368] Figure 90 This illustrates another RT-PCR-LDR-qPCR legacy prevention reaction for counting RNA copy numbers. For accurate counting, the sample is dispersed into 12, 24, 48, or 96 wells prior to PCR amplification. The method includes... Figure 89 The method shown is essentially the same as the steps (i.e., step AD); however, in this embodiment, the locus-specific ligation probe is designed to contain UniTaq primer sequences (Ai, Ci') and a UniTaq tag sequence (Bi'). Therefore, in this embodiment, the ligation product is subsequently amplified, detected, and quantified using real-time PCR with UniTaq-specific primers (F1-Bi-Q-Ai, Ci), as described above and in... Figure 90 As shown in step EG, the copy number is determined based on the Poisson distribution of the signal in different holes or chambers.

[0369] Figure 91 RT-PCR-qLDR legacy prevention reactions are shown for counting RNA copy number. The method includes isolating RNA from whole blood cells, exosomes, CTCs, or another biological sample, and treatment with UDG to effect legacy prevention Figure 91 , step B). For accurate counting, the sample is d...

Claims

1. A device for use in combination with a microtiter plate to simultaneously add liquid to two or more wells in a row and / or column of the microtiter plate, the microtiter plate having opposing top and bottom surfaces, wherein the top surface has openings to the wells and the bottom surface defines closed ends of the wells, the device comprising: a first layer defined by a first border and a second border, wherein metering chambers extend between the first and second borders of the first layer and are in fluid communication with each other through metering chamber channels, the first layer configured to fit in proximity to the microtiter plate in an operational position, wherein the first border of the first layer is closest to the top surface of the microtiter plate and each of the metering chambers is in fluid communication with a single well in a row and / or column of the microtiter plate, the first layer further comprising a fill chamber in fluid communication with one or more of the metering chambers, and a second layer defined by a first border and a second border, wherein a fill port extends between the first and second borders of the second layer, and, the second layer comprising an air passageway extending between the first and second borders of the second layer, wherein the air passageway is aligned with the metering chambers, the second layer configured to fit in proximity to the first layer in an operational position, wherein the first border of the second layer is closest to the second border of the first layer and the fill port is aligned with the fill chamber, wherein, when the first layer, the second layer, and the microtiter plate are positioned relative to each other in their operational positions, liquid entering the device through the fill port will pass through the fill chamber, the metering chambers, and into two or more wells in a row and / or column of the microtiter plate.

2. The device of claim 1, further comprising: an intermediate layer defined by a first border and a second border, wherein intermediate layer passageways extend between the first and second borders of the intermediate layer, the intermediate layer configured to fit between the microtiter plate and the first layer in an operational position, wherein the first border of the intermediate layer is adjacent to the top surface of the microtiter plate and the second border of the intermediate layer is adjacent to the first border of the first layer, and one of the intermediate layer passageways is aligned with a single well in a row and / or column of the microtiter plate, wherein, when the first layer, the second layer, the intermediate layer, and the microtiter plate are positioned relative to each other in their operational positions, liquid entering the device through the fill port will pass through the fill chamber, the metering chambers, the intermediate layer passageways, and into the wells of the microtiter plate.

3. The device of claim 2, wherein the intermediate layer passageways comprise hydrophobic walls.

4. The device of claim 2, wherein the intermediate layer passageways are burst valves.

5. The device of claim 2, wherein the first layer further comprises an overflow chamber in fluid communication with the fill chamber.

6. The device of claim 5, wherein the intermediate layer further comprises an overflow passageway connecting the overflow chamber to the fill chamber.

7. The device of claim 5, wherein the second layer has an overflow air passageway extending between the first and second borders of the second layer, wherein the overflow air passageway is aligned with the overflow chamber.

8. The device of claim 2, further comprising: a third layer having a first border and a second border, wherein a fill port connector extends between the first and second borders of the third layer, the third layer configured to fit between the first and second layers in an operational position, wherein the first border of the third layer is adjacent to the second border of the first layer and the fill port connector is aligned with the fill port, wherein, when the first layer, the second layer, the third layer, the intermediate layer, and the microtiter plate are positioned relative to one another in their operational positions, liquid entering the device through the fill port will pass through the fill port connector, the fill chamber, the metering chamber, the intermediate layer passageway, and into the well of the microtiter plate.

9. The device of claim 1, wherein the device is configured to allow filling of the metering chamber by capillary action.

10. The device of claim 1, wherein the device is configured to allow filling of the metering chamber by mechanical force.

11. The device of claim 1, wherein the first layer is provided with a pair of spaced apart fill chambers on opposite ends of the metering chamber and the second layer is provided with a pair of spaced apart fill ports, each of the fill ports in fluid communication with one of the pair of spaced apart fill chambers, whereby one of the pair of spaced apart fill ports provides liquid to one of the pair of spaced apart fill chambers and one half of the metering chamber, and the other of the pair of spaced apart fill ports provides liquid to the other of the pair of spaced apart fill chambers and the other half of the metering chamber.

12. The device of claim 1, wherein the first layer is provided with a pair of spaced apart fill chambers on opposite ends of the metering chamber and the second layer is provided with a pair of spaced apart fill ports, each of the fill ports in fluid communication with one of the pair of spaced apart fill chambers, whereby one of the pair of spaced apart fill ports provides liquid to one of the pair of spaced apart fill chambers and all of the metering chamber, and the other of the pair of spaced apart fill ports provides liquid to the other of the pair of spaced apart fill chambers and all of the metering chamber.

13. The device of claim 1, wherein the device is configured to fill two or more rows and columns of the microtiter plate with liquid.

14. The device of claim 1, wherein the first layer has a first region and a second region, the first region having the metering chambers in two or more rows, the second region having the metering chambers in two or more columns, wherein the first region and the second region are displaced from one another.

15. The device of any one of claims 1-14, wherein the layers are integrated, thereby imparting a monolithic structure to the device.

16. A method of adding liquid to two or more wells in a row and / or column of a microtiter plate, the microtiter plate having opposing top and bottom surfaces, wherein the top surface has openings to the wells and the bottom surface defines closed ends of the wells, the method comprising: providing a device comprising: a first layer having a first boundary and a second boundary, wherein metering chambers extend between the first and second boundaries of the first layer and are in fluid communication with one another through metering chamber channels, the first layer configured to fit in an operative position proximate to the microtiter plate with the first boundary of the first layer closest to the top surface of the microtiter plate and one of the metering chambers in fluid communication with a single well in a row and / or column of the microtiter plate, the first layer further comprising a fill chamber in fluid communication with one or more of the metering chambers, and a second layer having a first boundary and a second boundary, wherein a fill port extends between the first and second boundaries of the second layer, and, the second layer comprising an air passageway extending between the first and second boundaries of the second layer, wherein the air passageway is aligned with the metering chambers, the second layer configured to fit in an operative position proximate to the first layer with the first boundary of the second layer closest to the second boundary of the first layer and the fill port aligned with the fill chamber, wherein, when the first layer, the second layer, and the microtiter plate are positioned relative to one another in their operative positions, liquid entering the device through the fill port will pass through the fill chamber, the metering chambers, and into two or more wells in a row and / or column of the microtiter plate; filling the device with liquid; and discharging the liquid in the device into two or more wells in a row and / or column of the microtiter plate.

17. The method of claim 16, wherein the device further comprises: an intermediate layer having a first border and a second border, wherein intermediate layer pathways extend between the first border and the second border of the intermediate layer, the intermediate layer configured to fit between the microtiter plate and the first layer in an operational position, wherein the first border of the intermediate layer is adjacent to the top surface of the microtiter plate and the second border of the intermediate layer is adjacent to the first border of the first layer, and one of the intermediate layer pathways is aligned with a single well in a row and / or column of the microtiter plate, wherein, when the first layer, the second layer, the intermediate layer, and the microtiter plate are positioned relative to one another in their operational positions, liquid entering the device through the fill port will pass through the fill chamber, the metering chamber, the intermediate layer pathway, and into the well of the microtiter plate.

18. The method of claim 17, wherein the intermediate layer pathway comprises a hydrophobic wall.

19. The method of claim 17, wherein the intermediate layer pathway is a burst valve.

20. The method of claim 17, wherein the device further comprises: a third layer having a first border and a second border, wherein a fill port connector extends between the first border and the second border of the third layer, the third layer configured to fit between the first layer and the second layer in an operational position, wherein the first border of the third layer is adjacent to the second border of the second layer and the fill port connector is aligned with the fill port, wherein, when the first layer, the second layer, the third layer, the intermediate layer, and the microtiter plate are positioned relative to one another in their operational positions, liquid entering the device through the fill port will pass through the fill port connector, the fill chamber, the metering chamber, the intermediate layer pathway, and into the well of the microtiter plate.

21. The method of claim 16, wherein the filling is performed by capillary action.

22. The method of claim 16, wherein the draining is performed by mechanical force.

23. The method of claim 16, wherein the first layer is provided with a pair of spaced apart fill chambers on opposite sides of the metering chamber and the second layer is provided with a pair of spaced apart fill ports, each of the fill ports in fluid communication with one of the pair of spaced apart fill chambers, whereby, during the filling, one of the pair of spaced apart fill ports provides liquid to one of the pair of spaced apart fill chambers and one half of the metering chamber, while the other of the pair of spaced apart fill ports provides liquid to the other of the pair of spaced apart fill chambers and the other half of the metering chamber.

24. The method of claim 16, wherein said first layer is provided with a pair of spaced apart fill chambers on opposite sides of said metering chambers and said second layer is provided with a pair of spaced apart fill ports, each of said fill ports being in fluid communication with one of said fill chambers of said pair of spaced apart fill chambers, whereby during said filling one of said fill ports of said pair of spaced apart fill ports provides liquid to one of said fill chambers of said pair of spaced apart fill chambers and to all of said metering chambers, while the other of said fill ports of said pair of spaced apart fill ports provides liquid to the other of said fill chambers of said pair of spaced apart fill chambers and to all of said metering chambers.

25. The method of claim 16, wherein said device is configured to fill two or more rows and columns of said microtiter plate with liquid during said filling.

26. The method of claim 16, wherein said first layer has a first region and a second region, said first region having said metering chambers in two or more rows and said second region having said metering chambers in two or more columns, wherein said first region and said second region are displaced from one another.

27. The method of any one of claims 16-26, wherein said layers are integrated, thereby imparting a monolithic structure to said device.

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