Compositions and methods for performing methylation detection assays

Abstract

Provided herein are techniques relating to compositions and methods for detecting tissue cell-specific DNA, such as epithelial cell-specific DNA, in blood or blood products from a subject. The techniques also relate to using tissue cell-specific DNA as an internal control for methylation assays. Provided herein are techniques relating to characterizing the presence or absence and / or amount of different kinds of nucleic acids in a sample, e.g., a blood sample, a stool sample, etc., that can be relevant to the health status of a subject. For example, in some embodiments, the techniques relate to detecting and measuring DNA associated with a particular tissue in a sample type that does not normally contain DNA from that tissue. In preferred embodiments, the techniques relate to detecting and / or measuring epithelial cells and / or epithelial cell-specific DNA in a blood or blood product sample.

Description

[0001] This application is a divisional application of Chinese Invention Patent Application with the application date of December 11, 2015, application number 201580062917.7, and the invention name of "Compositions and methods for performing methylation detection assays".

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 091,069, filed December 12, 2014, which is incorporated herein by reference. TECHNICAL FIELD

[0003] Provided herein are technologies relating to compositions and methods for detecting epithelial cell-specific DNA in blood or blood products from a subject, where the presence and amount of epithelial cell DNA in the blood or blood products is indicative of the presence or magnitude of a medical condition in the subject. The technologies also relate to the use of tissue cell-specific DNA, such as epithelial cell-specific DNA, as an internal control for methylation assays in samples such as stool or tissue samples from a subject. BACKGROUND

[0004] Methylated DNA has been investigated as a class of potential biomarkers in tissues of most tumor types. In many cases, DNA methyltransferases add methyl groups to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. In a biologically compelling mechanism, acquired methylation events in the promoter regions of tumor suppressor genes are thought to silence expression, thus contributing to neoplasia. DNA methylation can be a more chemically and biologically stable diagnostic tool than RNA or protein expression (Laird (2010) "Principles and challenges of genome-wide DNA methylation analysis" Nat Rev Genet 11: 191-203). Furthermore, in other cancers such as sporadic colon cancer, methylation markers provide excellent specificity and are more informative and sensitive than individual DNA mutations (Zou et al. (2007) "Highly methylated genes in colorectal neoplasia: implications for screening" Cancer Epidemiol Biomarkers Prev 16:2686-96).

[0005] Nucleic acids from patient samples (e.g., blood, feces, and tissue samples) analyzed for the presence and / or methylation status of mutations associated with disease or disease risk typically undergo numerous processing steps during analysis. These steps may include, for example, filtration, precipitation, capture, washing, elution, and / or chemical modification. To analyze DNA to determine its methylation status (e.g., to test the percentage of DNA methylation), processing often involves treatment with bisulfite to convert unmethylated dC bases to dU residues, making them more easily distinguishable from methyl-C residues protected from bisulfite conversion.

[0006] Precise quantification of DNA for testing (e.g., determining the percentage of methylation, the presence and amount of DNA carrying mutations, etc.) typically requires normalization to a control nucleic acid, such as an endogenously invariant gene with known characteristics (e.g., known sequence, known copy number per cell). Normalization can control sample-to-sample variations that occur, such as in sample processing, assay efficiency, etc., and allows for precise sample-to-sample data comparisons.

[0007] Cancer-specific marker DNA or circulating cell-free DNA in blood or blood products present within circulating cancer cells or complexes has been used to characterize solid tumors in subjects, such as breast cancer. However, the utility of analyzing specific cancer markers in blood is limited to evaluating specific source tumors or cancer types already characterized by those markers, and the detection of specific markers in a subject's blood may have limited use in detecting other conditions or cancers. Summary of the Invention

[0008] This document provides techniques for characterizing the presence or absence and / or quantity of different types of nucleic acids in samples such as blood samples, fecal samples, etc., which may be related to, for example, the health status of a subject. For instance, in some embodiments, the techniques involve detecting and measuring DNA associated with a specific tissue in a sample type that typically does not contain DNA from that tissue. In a preferred embodiment, the techniques involve detecting and / or measuring epithelial cell and / or epithelial cell-specific DNA in blood or blood product samples.

[0009] In some embodiments, the technology provides a method for monitoring a disease state in a subject, the method comprising the steps of: obtaining a first blood product sample from the subject at a first time point; initiating a treatment regimen, wherein the treatment regimen includes a therapeutic intervention; obtaining a second blood product sample from the subject at a second time point, wherein the second time point is after the initiation of the treatment regimen; and determining the amount of epithelial cell-specific DNA in the first blood product sample and the second blood product sample, wherein the difference in the amount of epithelial cell-specific DNA between the first blood product sample and the second blood product sample indicates a change in the disease state in the subject. The technology is not limited regarding the determination of the first and second blood product samples. For example, in some embodiments, the first blood product sample is determined before the start of the treatment regimen, while in other embodiments, the first blood product sample is determined during or after the treatment regimen (e.g., at the same time as the second blood product sample). In a preferred embodiment, the method includes generating a record, such as a patient record like a hard copy or electronic medical record, wherein the record reports the determination result, such as a reported value (e.g., comparing the amount or change in the amount of epithelial cell-specific DNA in the sample) or a value-based diagnostic result.

[0010] This method is not limited to any particular treatment regimen. In some embodiments, the treatment regimen may include active interventions, such as monitoring the subject's condition. In a preferred embodiment, said treatment regimen includes one or more of surgery, pharmacological therapy, chemotherapy, immunotherapy, nutritional therapy, radiation therapy, thermotherapy, and physical therapy.

[0011] The difference in the amount of epithelial cell-specific DNA between a first blood product sample and a second blood product sample indicates, for example, a relapse, progression, or regression of a disease state in the subject. In some embodiments, no treatment regimen is used after the first sample is collected, and the difference in the amount of epithelial cell-specific DNA between the first and second blood product samples indicates the initial occurrence of a disease state in the subject. In some embodiments, the disease state indicated by the presence of epithelial cell-specific DNA in the blood or blood product sample is cancer, such as metastatic cancer.

[0012] In some preferred embodiments, the epithelial cell-specific DNA includes DNA that is methylated in epithelial cells and unmethylated in blood cells. In such embodiments, a preferred method includes treating DNA from a blood product sample with a bisulfite reagent to produce transformed epithelial cell-specific DNA. In a preferred embodiment, the epithelial cell-specific DNA includes ZDHHC1 DNA, and in a particularly preferred embodiment, the DNA comprises at least a portion of the sequence shown in SEQ ID NO:26.

[0013] The method is not limited to any particular form of blood or blood product sample; in some preferred embodiments, the blood product is plasma.

[0014] The method is not limited to any specific means of measuring the sample. In some preferred embodiments, the assay includes the use of polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or DNA target capture. In a particularly preferred embodiment, the assay includes the use of flanking endonuclease assays.

[0015] In some embodiments, the technique provides a composition for analyzing samples from a subject. For example, in some embodiments, the composition comprises a DNA strand containing the nucleotide sequence of SEQ ID NO:33 and / or a DNA strand containing the nucleotide sequence of SEQ ID NO:27. In some embodiments, the composition further comprises a detection probe oligonucleotide, wherein the detection probe oligonucleotide contains a region complementary to a portion of the DNA strand. In a preferred embodiment, the detection probe oligonucleotide contains a region complementary to a portion of SEQ ID NO:27 and / or a portion of SEQ ID NO:33. In a particularly preferred embodiment, the detection probe oligonucleotide comprises a reporter molecule. The reporter molecule is not limited to any particular detectable portion. In a preferred embodiment, the reporter molecule comprises a fluorophore. In some embodiments, the detection probe comprises flanking sequences.

[0016] In some preferred embodiments, the composition further comprises one or more of FRET box oligonucleotides, flanking endonucleases (e.g., FEN-1 endonuclease), and / or DNA polymerases (e.g., thermostable DNA polymerase). In a preferred embodiment, the DNA polymerase is a bacterial DNA polymerase. In some embodiments, the technique provides, for example, a reaction mixture for detection assays, the reaction mixture comprising any combination of the above compositions.

[0017] In some embodiments, the technique involves performing a methylation assay. In particular, in some embodiments, the technique involves an internal control for the methylation assay.

[0018] In some embodiments, the technology provides a method for characterizing a blood or blood product sample from a subject, the method comprising measuring the sample to detect the presence of tissue-cell-specific DNA, wherein the presence of tissue-cell-specific DNA indicates the presence of tissue-cell or tissue-cell-derived DNA in the blood or blood product sample. Tissue-cell DNA may be present within tissue cells or other complexes (e.g., nucleosomes, episomes, immune complexes, microparticles, etc.) in the blood, or it may be in the form of circulating cell-free DNA (ccfDNA). In some embodiments, the tissue-cell-specific DNA is epithelial cell-specific DNA. In some preferred embodiments, the blood product sample is a plasma sample.

[0019] In some particularly preferred embodiments, the tissue-cell-specific DNA is epithelial cell-specific DNA that is methylated in epithelial cells and unmethylated in blood cells, and the application of the technique preferably includes treating DNA from a sample with a bisulfite reagent to produce transformed tissue-cell-specific DNA. In a particularly preferred embodiment, as described below, the epithelial cell-specific DNA comprises ZDHHC1 DNA.

[0020] Methods for analyzing tissue- and cell-specific DNA are not limited to any particular DNA analysis method. In some embodiments, the assay includes the use of polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, and / or DNA target capture. In preferred embodiments, the assay includes a flanking endonuclease assay. In some preferred embodiments, the assay is a flanking endonuclease assay, such as the QUARTS assay.

[0021] In some embodiments, the technique provides a reference DNA that can be used to determine the total human DNA input in a sample as a means of determining the relative amount of test nucleic acids in the sample (e.g., the methylation percentage of a cancer marker gene). In some preferred embodiments, the technique provides a reference DNA having a methylation profile similar to that of a marker DNA to which it is being compared, such that the reference DNA can be exposed to the same preparation steps as the marker DNA and will behave like the marker DNA.

[0022] In some embodiments, the technology provides control or labeled DNA specific to tissue cells (e.g., epithelial cells). In specific embodiments, the technology provides labeled DNA, for example, that is highly methylated in tissue cells—e.g., normal and cancerous epithelial cells—but not methylated in blood, such as in lymphocytes. These labeled DNAs have numerous applications. For example, in some embodiments, these markers are used as control or reference DNA in quantifying tissue-derived DNA in samples that may also contain blood cells, such as lymphocytes, which will create background in the detection of other control DNAs, such as β-actin. These tissue cell-specific markers can also be used to detect tissue cells in samples where tissue cells or tissue DNA are not normally present (e.g., in blood), where the presence of tissue cells or tissue DNA can indicate the presence of disease (e.g., cancer metastasis).

[0023] For example, in some embodiments, the technology provides a method for performing quantitative nucleic acid detection assays, including determining the amount of at least one marker gene in a sample from a subject; determining the amount of ZDHHC1 DNA in the same sample; and comparing the amount of the at least one marker gene in the sample with the amount of ZDHHC1 DNA to determine the amount of the at least one marker gene in the sample relative to the amount of ZDHHC1 DNA. In some embodiments, an external control, such as a calibration standard, may be used to determine the absolute quantification of the marker gene and / or ZDHHC1 DNA.

[0024] In some embodiments, the technique includes treating DNA from a sample with a bisulfite reagent to produce transformed ZDHHC1 DNA and at least one transformed marker gene, such that determining the amount of marker gene and ZDHHC1 DNA includes determining the amount of transformed marker gene and transformed ZDHHC1 DNA.

[0025] The methods described above for determining nucleic acids are not limited to any particular method. In some embodiments, the assay includes one or more of polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some preferred embodiments, the assay of labeled DNA and the assay of ZDHHC1 DNA are performed in a single reaction. In a particularly preferred embodiment, the assay is a flanking endonuclease assay, such as the QUARTS assay.

[0026] In some embodiments, the amount of transformed marker gene relative to the amount of transformed ZDHHC1 DNA indicates, for example, the methylation status of the marker gene in the test sample, and the methylation status includes an increase or decrease in methylation of the marker gene relative to its normal methylation status. In some preferred embodiments, the percentage of increased methylation indicates a disease state.

[0027] Other embodiments provide methods for detecting tissue cells in blood or blood products, including: detecting the presence of methylated ZDHHC1 in a blood or blood product sample from a subject, wherein the presence of methylated ZDHHC1 indicates the presence of tissue cells, such as epithelial cells, in the blood. In some embodiments, the presence of tissue cells in the sample indicates metastatic cancer in the subject. In some embodiments, the blood product is plasma. In some embodiments, the assay includes using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some embodiments, the assay is a flanking endonuclease assay, such as the QUARTS assay. In some embodiments, the cancer is colorectal cancer.

[0028] Other embodiments provide a method for detecting metastatic cancer in blood or blood products from a subject, comprising: detecting the presence of methylated ZDHHC1 in a blood or blood product sample from the subject, wherein the presence of methylated ZDHHC1 indicates the presence of metastatic cancer in the subject. Still other embodiments provide a kit comprising: a) at least one oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to ZDHHC1; and b) a bisulfite. In some embodiments, the oligonucleotide is selected from one or more of, for example, capture oligonucleotides, a pair of nucleic acid primers, nucleic acid probes, or INVADER oligonucleotides. In some embodiments, the kit further comprises one or more nucleic acids that specifically hybridize to one or more target genes. In some embodiments, the kit further comprises a solid support (e.g., magnetic beads). In some embodiments, the solid support comprises one or more capture reagents (e.g., oligonucleotides complementary to ZDHHC1 and / or other target genes).

[0029] Another embodiment provides a composition comprising a complex of ZDHHC1 nucleic acid and at least one oligonucleotide, wherein at least a portion of the oligonucleotide hybridizes with the ZDHHC1 nucleic acid. In some embodiments, the composition further comprises one or more additional reaction mixtures comprising a complex of a target nucleic acid and one or more oligonucleotides that specifically hybridize with one or more target genes.

[0030] Another embodiment provides a method for screening tumors in samples obtained from a subject, the method comprising: a) determining the amount of at least one methylation marker gene in a sample from the subject, said at least one methylation marker gene being selected from the group consisting of vimentin, septin9, NDRG4, and BMP3 in the sample; determining the amount of methylated ZDHHC1 DNA in the sample, and comparing the amount of said at least one methylation marker gene in the sample with the amount of methylated ZDHHC1 DNA to determine the methylation status of at least one marker gene in the sample. In some embodiments, at least one marker is at least two, three, four, or all markers. In some embodiments, the assay further includes a step of identifying a KRAS mutation score in the sample. In some embodiments, the K-ras mutation score is measured by quantitative allele-specific PCR. In some embodiments, the assay includes detecting the methylation status of ZDHHC1, BMP3, and NDRG4, and identifying a KRAS mutation score in the sample. In some embodiments, the method further includes a step of determining the presence of hemoglobin in the sample. In some embodiments, the patient has inflammatory bowel disease. In some preferred embodiments, the sample is a fecal sample, tissue sample, pancreatic juice sample, pancreatic cyst fluid sample, blood sample, or urine sample. The tumor may include, for example, a pancreatic tumor, colorectal tumor, bile duct tumor, gastric tumor, esophageal tumor, or adenoma.

[0031] Some embodiments provide a kit comprising: a) at least one oligonucleotide, wherein at least a portion of the oligonucleotide specifically hybridizes to ZDHHC1; and b) at least one additional oligonucleotide, wherein at least a portion of the oligonucleotide specifically hybridizes to a marker selected from vimentin, cytoschizoprotein 9, NDRG4, and BMP3. In some embodiments, the kit comprises at least two additional oligonucleotides. In some embodiments, the kit also comprises bisulfite. In some embodiments, the kit further comprises at least one oligonucleotide, wherein at least a portion of the oligonucleotide specifically hybridizes to KRAS. In some embodiments, the kit further comprises reagents for detecting the presence of hemoglobin in fecal samples.

[0032] Some embodiments provide compositions comprising: a) a complex of ZDHHC1 nucleic acid and at least one oligonucleotide, wherein at least a portion of the oligonucleotide hybridizes with ZDHHC1 nucleic acid; and b) a complex of a target nucleic acid selected from the group consisting of vimentin, cytoschizoprotein 9, NDRG4, and BMP and one or more oligonucleotides specifically hybridizing with the target nucleic acid. Attached Figure Description

[0033] These and other features, aspects, and advantages of the present invention will become more readily understood with reference to the following accompanying drawings:

[0034] Figures 1A-1E A graph comparing the presence of the methylated gene ZDHHC1 in β-actin (BTACT) and bisulfite-converted DNA from fecal, blood, cell line, and tissue samples is provided.

[0035] Figures 2A-2C A graph is provided showing the percentage of methylation of the marker gene NDRG4, as determined by comparison with the control gene BTACT or ZDHHC1 as measured in fecal samples (2A), cell lines (2B), and colorectal cancer tissue samples (2C).

[0036] Figures 3A-3C A graph is provided showing the percentage of methylation of the marker gene BMP3, determined by comparison with the control gene BTACT or ZDHHC1 in fecal samples (3A), cell lines (3B), and colorectal cancer tissue samples (3C).

[0037] Figures 4A-4E A table is provided showing the detection levels of ZDHHC1 markers in plasma samples from subjects with specified cancers and from healthy subjects.

[0038] definition

[0039] To aid in understanding the technology of this invention, numerous terms and phrases are defined below. Further definitions are set forth throughout the detailed description.

[0040] As used herein, “a”, “an”, or “the” may mean one or more. For example, a “a” widget may mean one or more widgets.

[0041] As used herein, the terms “subject” and “patient” refer to any animal, such as a dog, cat, bird, livestock, and especially a mammal, preferably a human. In some cases, a subject is also a “user” (and therefore a user is also a subject or patient).

[0042] As used herein, the terms “sample” and “test specimen” are used interchangeably and in the broadest sense. In a certain sense, “sample” is intended to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, feces, urine, etc. Environmental samples include environmental materials such as surface material, soil, water, mud, sludge, biofilms, water, crystals, and industrial samples. However, these examples should not be construed as limiting the types of samples applicable to this invention.

[0043] As used herein, and in some contexts as such, "remote sample" refers to a sample collected indirectly from a location not of cellular, tissue, or organ origin. For example, a sample is a remote sample when evaluating pancreatic-derived sample material in a fecal sample (e.g., a sample not obtained directly from the pancreas).

[0044] When used to refer to nucleic acid capture, detection, or analysis methods, the term "target" typically refers to a nucleic acid that is characteristic (e.g., a specific nucleotide sequence to be detected or analyzed) in a sample suspected of containing the target nucleic acid. In some embodiments, the target is a nucleic acid with a specific sequence for which the methylation state is to be determined. When used to refer to polymerase chain reaction (PCR), "target" typically refers to a region of nucleic acid bound by primers used in the PCR. Therefore, the aim is to identify the "target" from other nucleic acid sequences that may be present in the sample. A "segment" is defined as a region of nucleic acid within the target sequence. The term "sample template" refers to nucleic acids derived from the sample that are analyzed in the presence of the target.

[0045] As used herein, the term "site" refers to a specific location within a defined region or segment of a nucleic acid, such as a mutation, polymorphism, or C residue of a CpG dinucleotide, like a gene on a chromosome or any other characterized sequence or RNA molecule. A site is not limited to any particular size or length and can refer to a portion of a chromosome, gene, functional genetic element, or a single nucleotide or base pair. As used herein in reference to methylable CpG sites, a site refers to a C residue in a CpG dinucleotide.

[0046] As used herein, a “capture agent” refers to any agent capable of binding to an analyte (e.g., a target). Preferably, a “capture agent” refers to any agent capable of specifically binding to an analyte (e.g., having a higher binding affinity and / or specificity to the analyte than to any other part). Any part such as a cell, organelle, inorganic molecule, organic molecule, or mixture or complex thereof can be used as a capture agent if it has the necessary binding affinity and / or specificity to the analyte. Capture agents can be peptides, proteins (e.g., antibodies or receptors), oligonucleotides, nucleic acids, vitamins, oligosaccharides, carbohydrates, lipids, small molecules, or complexes thereof. Capture agents containing nucleic acids (e.g., oligonucleotides) can capture nucleic acid targets by sequence-specific hybridization (e.g., by forming conventional Watson-Crick base pairs) or by other binding interactions. When a capture oligonucleotide hybridizes with a target nucleic acid, the hybridization may involve a portion of the oligonucleotide or the entire oligonucleotide sequence, and the oligonucleotide may bind to a portion of or the entire target nucleic acid sequence.

[0047] In the context of nucleic acids, the term "amplifying" or "amplification" refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small number of polynucleotides (e.g., a single polynucleotide molecule), where the amplification product or amplicon is usually detectable. Polynucleotide amplification encompasses a variety of chemical and enzymatic processes. In polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., U.S. Patent No. 5,494,810; which is incorporated herein by reference in its entirety), the production of multiple copies of DNA from one or more copies of a target or template DNA molecule is a form of amplification. Other types of amplification include, but are not limited to, allele-specific PCR (see, for example, U.S. Patent No. 5,639,611; which is incorporated herein by reference in its entirety), assembly PCR (see, for example, U.S. Patent No. 5,965,408; which is incorporated herein by reference in its entirety), helicase-dependent amplification (see, for example, U.S. Patent No. 7,662,594; which is incorporated herein by reference in its entirety), hot-start PCR (see, for example, U.S. Patent Nos. 5,773,258 and 5,338,671; each of which is incorporated herein by reference in its entirety), sequence-specific PCR, inverse PCR (see, for example, Triglia, et al. (1988) Nucleic Acids Res., 16:8186; which is incorporated herein by reference in its entirety), and ligation-mediated PCR (see, for example, Guilfoyle, R.).Nucleic Acids Research, 25:1854-1858 (1997); US Patent No. 5,508,169; each of these references is incorporated herein by reference in its entirety), methylation-specific PCR (see, for example, Herman, et al., (1996) PNAS 93(13)9821-9826; which is incorporated herein by reference in its entirety), microprimer PCR, multiplex ligation-dependent probe amplification (see, for example, Schouten, et al., (2002) Nucleic Acids Research 30(12):e57; which is incorporated herein by reference in its entirety), multiplex PCR (see, for example, Chamberlain, et al., (1988) Nucleic Acids Research 16(23)11141-11156; Ballabio, et al., (1990) Human Genetics 84(6)571-573; Hayden, et al., (2008) BMC Genetics 9:80; each of these references is incorporated herein by reference in its entirety), nested PCR, overlap extension PCR (see, for example, Higuchi, et al., (1988) Nucleic Acids Research 16(15)7351-7367; which is incorporated herein by reference in its entirety), real-time PCR (see, for example, Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030; each of these references is incorporated herein by reference in its entirety), reverse transcription PCR (see, for example, Bustin, SA (2000) J. Molecular Endocrinology 25:169-193; which is incorporated herein by reference in its entirety), solid-phase PCR, thermal asymmetric interleaved PCR and Touchdown PCR (see, for example, Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al. (1996) Biotechniques 20(3) 478-485; each of these references is incorporated herein by reference in its entirety). Polynucleotide amplification can also be achieved using digital PCR (see, for example, Kalinina, et al., Nucleic Acids Research. 25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA).96; 9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525; Each of these references is incorporated herein by reference in its entirety.

[0048] The term "polymerase chain reaction" ("PCR") refers to the methods described in KBMullis U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, which describe methods for increasing the concentration of a segment of a target sequence in a mixture of genomic or other DNA or RNA without cloning or purification. This method for amplifying a target sequence involves introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by precise thermal cycling in the presence of DNA polymerase. The two primers are complementary to the corresponding strands of their double-stranded target sequence. To achieve amplification, the mixture is denatured, and then the primers are annealed to their complementary sequences within the target molecule. After annealing, the primers are extended with polymerase to form a new pair of complementary strands. The denaturation, primer annealing, and polymerase extension steps can be repeated many times (i.e., denaturation, annealing, and extension constitute a "cycle"; many "cycles" may exist) to obtain high concentrations of amplified segments of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers to each other, and therefore this length is a controllable parameter. Due to the reproducible aspect of the process, the method is referred to as a “polymerase chain reaction” (“PCR”). Because the desired amplified segment of the target sequence becomes the dominant sequence in the mixture (in terms of concentration), they are claimed to be “PCR-amplified” and are “PCR products” or “amplifiers.” Those skilled in the art will understand that the term “PCR” encompasses many variations of the methods originally described, such as real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single-primer PCR, and arbitrary primed PCR.

[0049] As used herein, the term "nucleic acid assay" refers to any method for determining the nucleotide composition of a target nucleic acid. Nucleic acid assays include, but are not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays (Hologic, Inc.), and are described in, for example, U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999); Hall et al., PNAS, USA, 97:8272 (2000); and US In 2009 / 0253142, each of the aforementioned references is incorporated herein by reference in its entirety for all purposes; enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Patent Nos. 6,110,684, 5,958,692, and 5,851,770, which are incorporated herein by reference in their entirety); polymerase chain reaction (PCR) as described above; branching hybridization methods (e.g., Chiron, U.S. Patent Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, which are incorporated herein by reference in their entirety); rolling circle replication (e.g., U.S. Patent Nos. 6,210,884 and 6,183,960). And 6,235,502 (which is incorporated herein by reference in its entirety); NASBA (e.g., U.S. Patent No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Patent No. 6,150,097, which is incorporated herein by reference in its entirety); electronic sensor technology (Motorola, U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170 and 6,063,573, which are incorporated herein by reference in their entirety); cyclic probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769 and 5,660,988, which are incorporated herein by reference in their entirety); Dade Behring signal amplification methods (e.g., U.S. Patent Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated herein by reference in their entirety); ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88,189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Patent No. 5,288,609, which is incorporated herein by reference in its entirety).

[0050] In some implementations, target nucleic acids are amplified (e.g., by PCR), and the amplified nucleic acids are simultaneously detected using an invasive cleavage assay. Assays configured to be used in combination with amplification assays for detection (e.g., invasive cleavage assays) are described in U.S. Patent Publication US 20090253142A1 (Application Serial No. 12 / 404,240), which is incorporated herein by reference in its entirety for all purposes. Further amplification configurations incorporating invasive cleavage detection, known as the Quarts method, are described in U.S. Patent Nos. 8,361,720; 8,715,937; 8,916,344 and 9,127,318, which are incorporated herein by reference in their entirety for all purposes. As used herein, the term "invasive cleavage structure" refers to a cleavage structure comprising: i) a target nucleic acid; ii) an upstream nucleic acid (e.g., an invasive or "INVADER" oligonucleotide); and iii) a downstream nucleic acid (e.g., a probe), wherein the upstream and downstream nucleic acids are annealed to a contiguous region of the target nucleic acid, and wherein an overlap is formed between the 3' portion of the upstream nucleic acid and the duplex formed between the downstream nucleic acid and the target nucleic acid. The overlap occurs where one or more bases from the upstream and downstream nucleic acids occupy the same position relative to a base of the target nucleic acid, regardless of whether the overlapping base of the upstream nucleic acid is complementary to the target nucleic acid, and regardless of whether those bases are natural or non-natural bases. In some embodiments, such as those disclosed, for example, in U.S. Patent No. 6,090,543, the 3' portion of the upstream nucleic acid overlapping with the downstream duplex is a non-basic chemical portion, such as an aromatic ring structure, which is incorporated herein by reference in its entirety. In some embodiments, one or more nucleic acids may be attached to each other, for example, by covalent bonds such as nucleic acid stem-rings or by non-nucleic acid chemical bonds (e.g., multi-carbon chains). As used herein, the term “flanking endonuclease assay” includes the “INVADER” invasive cleavage assay and the Quarts assay as described above.

[0051] As used herein, the term "complementarity" or "complementarity" for reference to polynucleotides (i.e., nucleotide sequences) refers to polynucleotides in relation to base pairing rules. For example, the sequence "5′-AGT-3′" is complementary to the sequence "3′-TCA-5′". Complementarity can be "partial," where only some nucleic acid bases match according to base pairing rules. Alternatively, there may be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and in detection methods that rely on binding between nucleic acids.

[0052] As used herein, the term "primer" refers to an oligonucleotide (whether naturally occurring or synthetically produced during purification restriction digestion) that can act as the starting point for synthesis when placed under conditions that induce the synthesis of primer extension products complementary to the nucleic acid strand (e.g., in the presence of nucleotides and inducing agents such as biocatalysts, e.g., DNA polymerases, etc.). For maximum amplification efficiency, primers are typically single-stranded, but can alternatively be partially or fully double-stranded. The primer portion that hybridizes to the template nucleic acid is long enough to initiate the synthesis of the extension product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, primer source, and method of use. Primers may include markers, tags, capture portions, etc.

[0053] As used herein, the term "nucleic acid molecule" refers to a molecule containing nucleic acids, including but not limited to DNA or RNA. This term encompasses any sequence containing known DNA and RNA base analogs, including but not limited to: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinecytosine, pseudoisocytosine, 5-(carboxyhydroxy-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl The following are listed: 5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N-isopentenyladenine, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, Q-nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate, uracil-5-oxyacetic acid, pseudouracil, Q-nucleoside, 2-thiocytosine, and 2,6-diaminopurine.

[0054] As used herein, the term “nucleobase” is synonymous with other terms used in the art, including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleoside triphosphate (NTP),” or “dNTP.”

[0055] "Oligonucleotide" refers to a nucleic acid containing at least two monomeric units (e.g., nucleotides), typically more than three monomeric units, and more often than ten monomeric units. The exact size of an oligonucleotide often depends on various factors, including its final function or purpose. For further illustration, oligonucleotides are typically less than 200 residues long (e.g., between 15 and 100 residues); however, as used herein, the term is also intended to cover longer polynucleotide chains. Oligonucleotides are often referred to by their length. For example, a 24-residue oligonucleotide is called a "24-mer." Typically, nucleoside monomers are bound together by phosphodiester bonds or their analogues (including thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phosphoroanilothioate, phosphoranilidate, aminophosphates, etc.), including associated counterions such as H+. + NH4 + Na + (If such counterions are present) Linkage. Furthermore, oligonucleotides are typically single-stranded. Oligonucleotides are optionally prepared by any suitable method, including but not limited to the isolation of existing or natural sequences, DNA replication or amplification, reverse transcription, cloning and restriction digestion of suitable sequences, or direct chemical synthesis by methods such as: the phosphotriester method of Narang et al. (1979) Meth Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth Enzymol. 68:109-151; the diethylphosphamide method of Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862; the triester method of Matteucci et al. (1981) J Am Chem Soc. 103:3185-3191; automated synthesis; or the solid support method of U.S. Patent No. 4,458,066 entitled “Method for the Preparation of Polynucleotides”, granted to Caruthers et al. on July 3, 1984; or other methods known to those skilled in the art. All of these references are included by way of citation.

[0056] The "sequence" of a biopolymer refers to the order and characteristics of its monomeric units (such as nucleotides, amino acids, etc.). The sequence of nucleic acids (e.g., the base sequence) is typically read in the 5′ to 3′ direction.

[0057] As used herein, the term "gene" refers to a nucleic acid (e.g., DNA) sequence containing the coding sequence required to produce a polypeptide, precursor, or RNA (e.g., non-coding RNA such as ribosomal RNA, transfer RNA, spliceosome RNA, microRNA). The polypeptide or non-coding RNA may be encoded by a full-length coding sequence or any portion thereof, provided that the desired activity or functional properties of the full-length or fragmented polypeptide (e.g., enzyme activity, ligand binding, signal transduction, immunogenicity, etc.) are preserved. This term also encompasses the coding region of a structural gene and the sequence adjacent to the coding region at both the 5′ and 3′ ends, at either end for a distance of approximately 1 kb or longer, such that the gene corresponds to the length of full-length mRNA. A sequence located at the 5′ end of the coding region and present on the mRNA is called a 5′ untranslated sequence. A sequence located at or downstream of the coding region and present on the mRNA is called a 3′ untranslated sequence. The term "gene" encompasses both the cDNA and genomic forms of a gene. The genomic form or clone of a gene contains coding regions interrupted by non-coding sequences called "introns," "insertion regions," or "insertion sequences." Introns are gene segments transcribed into nuclear RNA (e.g., hnRNA); introns may contain regulatory elements (e.g., enhancers). Introns are removed or "cut out" from the nucleus or primary transcript; therefore, introns are not present in messenger RNA (mRNA) transcripts. mRNA functions during translation to specify the sequence or order of amino acids in the nascent polypeptide.

[0058] In addition to introns, the genomic form of a gene may also include sequences located at the 5′ and 3′ ends of the RNA transcript. These sequences are called “flanking” sequences or regions (these flanking sequences are located at the 5′ or 3′ untranslated sequences present on the mRNA transcript). The 5′ flanking region may contain regulatory sequences that control or influence gene transcription, such as promoters and enhancers. The 3′ flanking region may contain sequences that guide transcription termination, post-transcriptional cleavage, and polyadenylation.

[0059] When referring to a gene, the term "wildtype" means a gene that has the characteristics of a gene isolated from a naturally occurring source. When referring to a gene product, the term "wildtype" means a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term "naturally occurring," as used with respect to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and is not intentionally modified by humans in a laboratory is considered naturally occurring. Wild-type genes are often the genes or alleles most frequently observed in a population and are therefore arbitrarily designed as the "normal" or "wild-type" form of the gene. In contrast, the terms "modified" or "mutant," when referring to a gene or gene product, respectively, refer to a gene or gene product that exhibits modifications (e.g., altered characteristics) in sequence and / or functional properties compared to a wild-type gene or gene product. It should be noted that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered characteristics compared to a wild-type gene or gene product.

[0060] The term "allele" refers to a variation in a gene; variations include, but are not limited to, variants and mutants, polymorphic loci and single nucleotide polymorphic loci, frameshift and splicing mutations. Alleles may be naturally present in a population or may appear during the lifespan of any particular individual in the population.

[0061] Therefore, when referring to nucleotide sequences, the terms "variant" and "mutant" refer to a nucleic acid sequence that differs from another normally associated nucleotide sequence by one or more nucleotides. A "change" is the difference between two different nucleotide sequences; typically, one sequence is a reference sequence.

[0062] As used herein, the term "solid support" includes all materials on which a target (e.g., DNA) can be immobilized. Natural or synthetic materials, whether or not chemically modified, can be used as solid supports, particularly polymers such as polyvinyl chloride, polyethylene, polystyrene, polyacrylates, or polyamides, or copolymers based on vinyl aromatic monomers, esters of unsaturated carboxylic acids, vinylidene chloride, dienes, or compounds having nitrile functional groups (acrylonitrile); polymers of vinyl chloride and propylene, polymers of vinyl chloride and vinyl acetate; copolymers based on styrene or substituted derivatives of styrene; synthetic fibers, such as nylon; inorganic materials, such as silica, glass, ceramics, or quartz; latex, magnetic particles; and metal derivatives. Other examples include, but are not limited to, microtiter plates, sheets, cones, tubes, holes, beads (e.g., magnetic beads), granules, etc., or flat supports such as silica or silicon wafers.

[0063] As used herein, the terms “magnetic particle” and “magnetic bead” are used interchangeably and refer to particles or beads that respond to a magnetic field. Typically, magnetic particles comprise materials that do not have a magnetic field but form magnetic dipoles when exposed to one, such as materials that can be magnetized in the presence of a magnetic field but are not magnetic in the absence of such a field. The term “magnetic” as used in this context includes materials that are paramagnetic or superparamagnetic. As used herein, the term “magnetic” also encompasses temporary magnetic materials, such as ferromagnetic or ferrimagnetic materials with low Curie temperatures, provided that the temporary magnetic material is paramagnetic within the temperature range for separating biological materials using silica magnetic particles containing said material according to the method of the invention. The term “miscible” as used in references to particles or beads refers to particles that are, for example, free in a column (i.e., not fixed) but can be added to a sample and distributed in the sample solution by mixing actions (e.g., vortexing, stirring, shaking, repeated pipetting, etc.).

[0064] The term "probe" refers to an oligonucleotide (e.g., a nucleotide sequence) capable of hybridizing with another target oligonucleotide, whether naturally present during purification restriction digestion or produced synthetically, recombinantly, or by PCR amplification. Probes can be single-stranded or double-stranded. Probes can be used for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). It is contemplated that in some embodiments, any probe used in this invention may be labeled with any "reporter molecule" so that it can be detected in any detection system, including but not limited to enzymes (e.g., ELISA and enzyme-based histochemical assays), fluorescence, radioactivity, and luminescence systems. This invention is not intended to be limited to any particular detection system or label.

[0065] As used herein, "methylation" refers to cytosine methylation at position C5 or N4, adenine methylation at position N6, or other types of nucleic acid methylation. DNA amplified in vitro is typically unmethylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template was either unmethylated or methylated.

[0066] Therefore, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to a nucleotide base containing a methyl moiety that is not present in a typically known nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine does contain a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide, and 5-methylcytosine is. In another example, thymine contains a methyl moiety at position 5 of its pyrimidine ring; however, for the purposes of this document, thymine is considered not to be a methylated nucleotide when present in DNA, as thymine is a typical nucleotide base of DNA.

[0067] As used in this article, "methylated nucleic acid molecule" refers to a nucleic acid molecule containing one or more methylated nucleotides.

[0068] As used herein, the “methylation state,” “methylation profile,” and “methylation state” of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule containing methylated cytosine is considered methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated.

[0069] The methylation status of a specific nucleic acid sequence (e.g., a gene marker or DNA region as described herein) can indicate the methylation status of each base in the sequence, or the methylation status of a subset of bases (e.g., one or more cytosines) within the sequence, or information about the methylation density of regions within the sequence, while providing or not providing precise information about the location where methylation occurs within the sequence.

[0070] The methylation state of a nucleotide site in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a specific site. For example, when the nucleotide at the 7th nucleotide position in a nucleic acid molecule is 5-methylcytosine, the methylation state of the cytosine at the 7th nucleotide position is methylated. Similarly, when the nucleotide at the 7th nucleotide position in a nucleic acid molecule is cytosine (and not 5-methylcytosine), the methylation state of the cytosine at the 7th nucleotide position is unmethylated.

[0071] Methylation status may optionally be represented or indicated by a "methylation value" (e.g., representing methylation frequency, fraction, ratio, percentage, etc.). Methylation values ​​can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification spectra after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Therefore, a value (e.g., methylation value) represents the methylation status and can thus be used as a quantitative indicator of methylation status across multiple copies of a site. This is particularly useful when it is desirable to compare the methylation status of a sequence in a sample with a threshold or reference value.

[0072] As used herein, “methylation frequency” or “methylation percentage (%)” refers to the number of molecules or sites that are methylated relative to the number of unmethylated molecules or sites.

[0073] Therefore, methylation status describes the methylation state of nucleic acids (e.g., genomic sequences). Furthermore, methylation status refers to the characteristics of a nucleic acid segment at a specific genomic site associated with methylation. These characteristics include, but are not limited to, the location of methylated C residues, the frequency or percentage of methylated C residues within any specific region of the entire nucleic acid, and allelic methylation differences due to, for example, differences in allelic origin. The terms “methylation status,” “methylation profile,” and “methylation state” also refer to the relative concentration, absolute concentration, or pattern of methylated or unmethylated C residues within any specific region of the entire nucleic acid in a biological sample. For example, if cytosine (C) residues within a nucleic acid sequence are methylated, it may be described as “hypermethylated” or having “increased methylation,” while if a sequence of cytosine (C) residues within a DNA sequence is not methylated, it may be described as “hypomethylated” or having “decreased methylation.” Similarly, if cytosine (C) residues within a nucleic acid sequence are methylated compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.), then the sequence is considered hypermethylated or has increased methylation compared to the other nucleic acid sequence. Alternatively, if cytosine (C) residues within a DNA sequence are methylated compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.), then the sequence is considered hypomethylated or has decreased methylation compared to the other nucleic acid sequence. Furthermore, the term "methylation pattern" as used herein refers to the aggregate site of methylated and unmethylated nucleotides on a nucleic acid region. When the number of methylated and unmethylated nucleotides is the same or similar throughout the region, but the positions of the methylated and unmethylated nucleotides differ, two nucleic acids may have the same or similar methylation frequency or methylation percentage, but different methylation patterns. Sequences are said to be "differentially methylated" or have "different methylation" or "different methylation states" when they differ in their degree of methylation (e.g., one sequence has increased or decreased methylation relative to another sequence), frequency, or pattern. The term "differential methylation" refers to the difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern in a cancer-negative sample. It can also refer to the difference in level or pattern between patients with cancer recurrence after surgery and those without. Specific levels or patterns of differential methylation and DNA methylation are prognostic and predictive biomarkers, such as once the correct cutoff or predictive characteristic is defined.

[0074] Methylation state frequency can be used to describe a population of individuals or a sample from a single individual. For example, a nucleotide site with a methylation state frequency of 50% is methylated in 50% of cases and unmethylated in 50% of cases. This frequency can be used, for example, to describe the degree to which nucleotide sites or nucleic acid regions are methylated in a population of individuals or an assembly of nucleic acids. Therefore, when the methylation in a first population or assembly of nucleic acid molecules differs from that in a second population or assembly of nucleic acid molecules, the methylation state frequency of the first population or assembly will differ from that of the second population or assembly. This frequency can also be used, for example, to describe the degree to which nucleotide sites or nucleic acid regions are methylated in a single individual. For example, this frequency can be used to describe the degree to which a group of cells from a tissue sample are methylated or unmethylated at nucleotide sites or nucleic acid regions.

[0075] The term "highly methylated" refers to nucleic acids methylated at a specific site (e.g., a CpG dinucleotide or a set of dinucleotides or a CpG-rich region) at a rate greater than that observed at a comparable site in the same DNA in another tissue or sample type in a measurable manner. "Highly methylated" can also refer to the average methylation rate within multiple C residues in a single specific C residue or region, as a fraction of the copy number of said site in the sample being measured. In some embodiments, without limiting the term to any particular methylation level, hypermethylated sites may be >10% methylated, preferably >20% to 40%, more preferably >50% to 75%, and still more preferably between 75% and 100%.

[0076] As used in this article, "nucleotide site" refers to the location of a nucleotide in a nucleic acid molecule. The nucleotide site of a methylated nucleotide refers to the location of a methylated nucleotide in a nucleic acid molecule.

[0077] Typically, methylation of human DNA occurs on dinucleotide sequences containing adjacent guanine and cytosine (also known as CpG dinucleotide sequences), in which cytosine is located at the 5′ position of guanine. Most cytosine within a CpG dinucleotide is methylated in the human genome; however, some remain unmethylated in specific CpG dinucleotide-rich genomic regions (called CpG islands) (see, for example, Antequera et al. (1990) Cell 62:503–514).

[0078] As used herein, a “CpG island” refers to a G:C-rich region of genomic DNA that represents an increase in the number of CpG dinucleotides relative to the total genomic DNA. The length of a CpG island may be at least 100, 200, or more base pairs, wherein the G:C content of the region is at least 50%, and the observed CpG frequency is a ratio of 0.6 to the expected frequency; in some cases, the length of a CpG island may be at least 500 base pairs (where the G:C content of the region is at least 55%), and the observed CpG frequency is a ratio of 0.65 to the expected frequency. The observed CpG frequency relative to the expected frequency can be calculated using the method provided in Gardiner-Garden et al. (1987) J. Mol. Biol. 196:261–281. For example, the observed CpG frequency relative to the expected frequency can be calculated using the formula R = (A × B) / (C × D), where R is the ratio of the observed CpG frequency to the expected frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is typically determined within CpG islands (e.g., at promoter regions). It will be understood that other sequences in the human genome tend to be DNA methylated, such as CpA and CpT (see Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237–5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204:340–351; Grafstrom (1985) Nucleic Acids Res. 13:2827–2842; Nyce (1986) Nucleic Acids Res. 14:4353–4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145:888–894).

[0079] As used herein, the term "tissue cell" refers to any tissue cell in the body (e.g., in a human or animal body), including, for example, epithelial cells, muscle cells, nerve cells, and bone cells. Tissue cells do not include blood cells. As used herein, blood typically contains plasma, red blood cells, white blood cells (including leukocytes and lymphocytes), and platelets. White blood cells include neutrophils, monocytes, eosinophils, and basophils, and lymphocytes include T cells, B cells, and natural killer cells.

[0080] "Tissue-cell specific control DNA" and "tissue-cell specific DNA" refer to DNA that can detect the presence of tissue or cell-free DNA derived from tissue, and is minimally detectable or undetectable in blood or normal blood components (e.g., plasma, leukocytes, etc., as listed above). As used herein, DNA that is methylated only in tissue and not similarly methylated in blood (or vice versa) can be tissue-cell specific DNA with respect to its methylation state, even if the primary sequence of the DNA is identical in both cell types. "Epithelial specific control DNA" refers to tissue-specific control DNA used to detect DNA found in epithelial cells.

[0081] As used herein, a reagent or methylation-specific reagent that modifies the nucleotides of a nucleic acid molecule according to its methylation state refers to a compound or composition or other agent that can alter the nucleotide sequence of a nucleic acid molecule in a manner that reflects its methylation state. Methods of treating nucleic acid molecules with such a reagent may include contacting the nucleic acid molecule with the reagent, coupling it (if necessary) with additional steps to achieve the desired nucleotide sequence change. This change in the nucleotide sequence of a nucleic acid molecule can produce a nucleic acid molecule in which each methylated nucleotide is modified into a different nucleotide. This change in the nucleotide sequence of a nucleic acid molecule can produce a nucleic acid molecule in which each unmethylated nucleotide is modified into a different nucleotide. This change in the nucleotide sequence of a nucleic acid molecule can produce a nucleic acid molecule in which each of the selected unmethylated nucleotides (e.g., each unmethylated cytosine) is modified into a different nucleotide. Using such a reagent to alter the nucleotide sequence of a nucleic acid molecule can produce a nucleic acid molecule in which each nucleotide (e.g., each methylated cytosine) of a methylated nucleotide is modified into a different nucleotide. As used herein, the use of a reagent to modify a selected nucleotide refers to a reagent that modifies one of the four commonly present nucleotides in a nucleic acid molecule (C, G, T, and A for DNA; and C, G, U, and A for RNA) such that the reagent modifies one nucleotide without modifying the other three. In one exemplary embodiment, such a reagent modifies an unmethylated selected nucleotide to produce a different nucleotide. In another exemplary embodiment, such a reagent can deaminate an unmethylated cytosine nucleotide. An exemplary reagent is a bisulfite.

[0082] As used herein, the term "bisulfite reagent" refers to a reagent, in some embodiments, comprising bisulfite, disulfite, hydrogen sulfite, or combinations thereof to distinguish, for example, methylated cytidine and unmethylated cytidine in a CpG dinucleotide sequence.

[0083] The term "methylation assay" refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0084] The term “MS AP-PCR” (methylation-sensitive arbitrary primer polymerase chain reaction) refers to an art-recognized technique that allows the use of CG-rich primers to perform a global scan of the genome to focus on regions most likely to contain CpG dinucleotides, and this art-recognized technique is described by Gonzalgo et al. (1997) Cancer Research 57:594–599.

[0085] The term "MethyLight" TM "Refers to the fluorescence-based real-time PCR technique recognized in the field, as described by Eads et al. (1999) Cancer Res. 59:2302–2306.

[0086] The term "Heavy Methyl" TM "Methylation-specific blocking probes (also referred to as blocking agents in this paper) that cover the CpG sites between or by amplification primers enable the determination of methylation-specific selective amplification of nucleic acid samples.

[0087] The term "Heavy Methyl" TM MethyLight TM "Determination refers to Heavy Methyl TM MethyLight TM The determination was made by MethyLight. TM The measured changes, among which MethyLight TM Determine the methylation-specific blocking probe combination at the CpG position between the amplification primers and the covering primers.

[0088] The term “Ms-SNuPE” (methylation-sensitive single nucleotide primer extension) refers to the art-recognized assay described by Gonzalgo and Jones (1997) Nucleic Acids Res. 25:2529–2531.

[0089] The term “MSP” (methylation-specific PCR) refers to the methylation assay recognized in the art as described by Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821–9826 and by U.S. Patent No. 5,786,146.

[0090] The term “COBRA” (Combined Bisulfite Limitation Analysis) refers to the art-recognized methylation assay described by Xiong and Laird (1997) Nucleic Acids Res. 25:2532–2534.

[0091] The term “MCA” (methylated CpG island amplification) refers to the methylation assay described by Toyota et al. (1999) Cancer Res. 59:2307–12 and WO 00 / 26401A1.

[0092] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of nucleic acid purification systems and reaction assays, said delivery system includes systems that allow the storage, transport, or delivery of reagents and devices (e.g., inhibitor adsorbents, particles, denaturants, oligonucleotides, rotary filters, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for performing the procedure, etc.) from one location to another. For example, a kit includes one or more canisters (e.g., boxes) containing the associated reaction reagents and / or support materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers, each containing a sub-part of the total kit components. The containers may be delivered jointly or separately to the intended recipient. For example, the first container may contain materials and buffers for sample collection, while the second container contains capture oligonucleotides and denaturants. The term “fragmented kit” is intended to cover, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system comprising two or more separate containers, each containing a sub-part of the total kit components, is included in the term "fragmented kit." In contrast, a "combination kit" refers to a delivery system containing all components of the reaction assay in a single container (e.g., a single box containing each desired component). The term "kit" includes both fractionated kits and combination kits.

[0093] As used herein, the term "system" refers to a collection of items intended for use for a particular purpose. In some embodiments, said items include instruction manuals provided as information, for example, on an item, on paper, or on a recordable medium (e.g., a disk, CD, flash drive, etc.). In some embodiments, the instruction manual directs the user to an online location, such as a website.

[0094] As used herein, the term "information" refers to any collection of facts or data. In reference to information stored or processed using computer systems (including, but not limited to, the Internet), the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information relating to a subject" refers to facts or data concerning a subject (e.g., a human, plant, or animal). The term "genomic information" refers to information concerning a genome, including but not limited to nucleic acid sequences, genes, methylation percentages, allele frequencies, RNA expression levels, protein expression, genotype-related phenotypes, etc. "Allele frequency information" refers to facts or data concerning allele frequencies, including but not limited to allele characteristics, statistical correlations between the presence of alleles and characteristics of a subject (e.g., a human subject), the presence or absence of alleles in an individual or population, the percentage probability of an allele being present in an individual with one or more specific characteristics, etc. Detailed Implementation

[0095] This document provides techniques for performing assays to detect and quantify DNA (e.g., methylated DNA). In particular, the techniques relate to internal controls for the methylation assay.

[0096] Embodiments of this disclosure provide a marker called “ZDHHC1” for use as a methylation marker and internal control. Experiments conducted during the development of embodiments of this disclosure confirmed that little or no methylated ZDHHC1 was found in normal blood samples (e.g., obtained from disease-free individuals). In contrast to commonly used internal control DNA (e.g., β-actin), ZDHHC1 produces a very low background signal, for example, from blood present in tissue or fecal samples. During the development of the inventive technique, it was found that replacing the ACTB internal control with ZDHHC1 in exemplary methylation assays (e.g., flanking endonuclease assays, such as the QUARTS assay) increased the sensitivity and specificity of the assay.

[0097] Further experiments have confirmed that ZDHHC1 can be used as a marker for detecting epithelial cells in the blood (e.g., as a marker for metastatic cancer). Exemplary implementations are described herein.

[0098] Although the content disclosed herein refers to certain illustrative implementations, it should be understood that these implementations are presented by way of example rather than limitation.

[0099] I. Tissue-cell specific markers

[0100] In assays for detecting and quantifying methylated CpG-rich DNA that has undergone bisulfite conversion, it is typical to also detect a control gene present in the same sample. This control gene validates the DNA input in the assay, regardless of its source (e.g., cancer, normal, feces, tissue). Such a control gene is used, for example, to normalize DNA copy number data obtained across assays from different samples to accurately indicate higher or lower levels of disease-related markers in each sample.

[0101] For a normalized gene to function optimally for methylation assays, it should meet several criteria. An ideal normalized gene should, for example: 1) be present equally in normal and diseased tissues; 2) have approximately the same GC content as the test gene / marker being measured (e.g., DNA markers where hypermethylation is an indicator of disease status); 3) react in the same manner as the test gene / marker for pre-PCR sample processing, such as bisulfite conversion; and 4) have a PCR amplification efficiency similar to that of the test gene / marker being measured.

[0102] The β-actin gene (commonly used as a normalized gene for detecting methylated marker DNA) does not have the same GC content and CpG methylation as methylated markers (e.g., vimentin, cytoschizoprotein 9, NDRG4, BMP3) associated with diseases such as cancer and adenomas, and therefore does not behave like the marker DNA in pre-PCR bisulfite conversion or PCR amplification. In the development of this technique, it has been found that using a normalized gene that meets the above criteria instead of ACTB improves assay sensitivity and specificity. Further in the development of this technique, it has been found that using a marker gene that is highly methylated in normal and diseased tissues but not methylated in blood provides a marker specific to tissue cells (e.g., epithelial cells) and with low presence in blood. The use of the control DNA reduces any background of blood present from samples (e.g., fecal or tissue samples) and can also be used to detect the abnormal presence of said tissue cells in the blood that may occur, for example, during tumor metastasis.

[0103] The experiments described herein identified genes (e.g., ZDHHC1) that are highly methylated in normal and cancerous tissues. These genes are not highly methylated in blood, and their degree of methylation in blood does not vary with disease state, except that they are associated with metastatic cancer as described in Example 6. This allows for better and more accurate methylation calculations that reflect only the tissue and are independent of blood concentrations in the sample. The genes described herein were used to normalize marker levels between patients and samples.

[0104] ZDHHC1, ZFAND3, ZMYM4, ODZ2, and TRIO were identified as candidate methylation markers. Selecting normalized genes with low methylation in the erythrocyte sedimentation rate (ESR) layer allowed for more sensitive detection of methylation of the target marker (e.g., a lower denominator for the normalized signal, and therefore a larger and more distinguishable methylation percentage for the target marker).

[0105] The normalized gene described in this article is highly methylated in tissues (cancer and normal) and not highly methylated in blood, and offers several advantages over existing markers:

[0106] The 1-GC content and CpG methylation and bisulfite reactivity are more similar to those of the DNA markers studied.

[0107] 2- They show PCR amplification efficiencies that are more similar to the PCR amplification efficiencies of the labeled DNA being measured.

[0108] The low methylation state in the 3-erythrocyte sedimentation rate (ESR) brown layer allows for the detection of a higher percentage of methylation of the target marker in or in the presence of blood.

[0109] II. Methylation Detection and Determination

[0110] The markers described herein (e.g., particularly ZDHHC1) can be used as normalization reagents and indicators of disease states in various methylation assays.

[0111] The most common method for analyzing the presence of 5-methylcytosine in nucleic acids is based on the bisulfite method for detecting 5-methylcytosine in DNA, described by Frommer et al. (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827–31, which is explicitly incorporated herein by reference in its entirety for all purposes) or variations thereof. The bisulfite method mapping 5-methylcytosine is based on the observed reaction of cytosine, rather than 5-methylcytosine, with bisulfite ions (also known as bisulfites). This reaction typically proceeds according to the following steps: First, cytosine reacts with bisulfite to form sulfonated cytosine. Next, the sulfonation intermediate undergoes spontaneous deamination to produce sulfonated uracil. Finally, sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because the uracil base pairs with adenine (and thus behaves like thymine), while the 5-methylcytosine base pairs with guanine (and thus behaves like cytosine). This allows methylated cytosine to be distinguished from unmethylated cytosine by, for example, bisulfite genome sequencing (Grigg G and Clark S, Bioessays (1994) 16:431–36; Grigg G, DNA Seq. (1996) 6:189–98), methylation-specific PCR (MSP) as disclosed in, for example, U.S. Patent No. 5,786,146, or assays using comparative sequence-specific probe cutting (e.g., Quarts flanking endonuclease assays) (see, for example, Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56:A199; U.S. Patent Nos. 8,361,720 and U.S. Patent Application Serial Nos. 12 / 946,745; 12 / 946,752 and 61 / 705,603).

[0112] Some conventional techniques involve methods including blocking the DNA to be analyzed in an agarose matrix to prevent DNA diffusion and renaturation (bisulfite reacts only with single-stranded DNA), and replacing precipitation and purification steps with rapid dialysis (Olek A, et al. (1996) "A modified and improved method for bisulfite based cytosine methylation analysis" Nucleic Acids Res. 24:5064-6). Therefore, the methylation status of individual cells can be analyzed, thus demonstrating the utility and sensitivity of the methods. A review of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0113] Bisulfite techniques typically involve amplifying short, specific fragments of a known nucleic acid following bisulfite treatment, followed by determination of the product by sequencing (Olek and Walter (1997) Nat. Genet. 17:275–6) or primer extension (Gonzalgo and Jones (1997) Nucleic Acids Res. 25:2529–31; WO 95 / 00669; US Patent No. 6,251,594) to analyze the position of individual cytosines. Some methods utilize enzymatic digestion (Xiong and Laird (1997) Nucleic Acids Res. 25:2532–4). Detection by hybridization has also been described in the art (Olek et al., WO 99 / 28498). In addition, the use of bisulfite technology for the detection of methylation of individual genes has been described (Grigg and Clark (1994) Bioessays 16:431–6; Zeschnigk et al. (1997) Hum Mol Genet. 6:387–95; Feil et al. (1994) Nucleic Acids Res. 22:695; Martin et al. (1995) Gene 157:261–4; WO 9746705; WO9515373).

[0114] Various methylation assays can be used in conjunction with bisulfite treatment according to the techniques of the present invention. These assays allow for the determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Among other techniques, the assays also involve sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blotting analysis, and the use of methylation-sensitive restriction enzymes.

[0115] For example, methylation patterns and 5-methylcytosine distribution have been simplified for genome sequencing by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827–1831). Additionally, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA can be used to assess methylation status, as illustrated by Sadri and Hornsby (1997) Nucleic Acids Res. 24:5058–5059 or by a method known as COBRA (Combined Bisulfite Restriction Assay) (Xiong and Laird (1997) Nucleic Acids Res. 25:2532–2534).

[0116] COBRA TM The analysis is a quantitative methylation assay used to determine the level of DNA methylation at specific loci in a small amount of genomic DNA (Xiong and Laird, Nucleic Acids Res. 25:2532-2534, 1997). In short, restriction enzyme digestion was used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. First, methylation-dependent sequence differences were introduced into genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). Then, PCR amplification of the bisulfite-converted DNA was performed using primers specific to the target CpG islands, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specifically labeled hybridization probes. The methylation level in the original DNA sample was expressed as a linear quantification across a broad spectrum of DNA methylation levels by the relative amounts of digested and undigested PCR products. Furthermore, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0117] For COBRA TM Typical reagents for analysis (e.g., those available in typical COBRA-based reagents) TM The kits found may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, gene regions, labeled regions, bisulfite-treated DNA sequences, CpG islands, etc.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; kinase labeling kits for oligonucleotide probes; and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0118] Such as "MethyLight" TM (Real-time PCR based on fluorescence) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE TM The assays for (methylation-sensitive single nucleotide primer extension) reactions (Gonzalgo and Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR (“MSP”; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; US Patent No. 5,786,146) and methylation CpG island amplification (“MCA”; Toyota et al., Cancer Res. 59:2307-12, 1999) were used alone or in combination with one or more of these methods.

[0119] HeavyMethyl TM "Assays and techniques are quantitative methods for assessing methylation differences in DNA based on methylation-specific amplification using bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") covering CpG sites between or by amplification primers enable methylation-specific selective amplification of nucleic acid samples."

[0120] The term "Heavy Methyl" TM MethyLight TM "Determination refers to Heavy Methyl TM MethyLight TM The determination was made by MethyLight. TM The measured changes, among which MethyLight TM Determine the methylation-specific blocking probe combination at the CpG position between the heavy methylated amplification primers and the primers covering the amplification. TM The assay can also be used in combination with methylation-specific amplification primers.

[0121] For HeavyMethyl TM Typical reagents for analysis (e.g., those available in typical MethyLight-based systems) TM The kit may include, but is not limited to: PCR primers for specific sites (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, or bisulfite-treated DNA sequences or CpG islands); blocking oligonucleotides; optimized PCR buffers and deoxynucleotides; and Taq polymerase.

[0122] Methylation-specific PCR (MSP) allows for the assessment of the methylation status of virtually any set of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes (Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; US Patent No. 5,786,146). In short, DNA is modified with sodium bisulfite, which converts unmethylated cytosine into uracil, and the products are subsequently amplified using primers specific to both methylated and unmethylated DNA. MSP requires only a small amount of DNA, is sensitive to 0.1% methylation alleles at a given CpG island site, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents used for MSP analysis (e.g., those found in typical MSP-based kits) may include, but are not limited to: methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, gene regions, labeled regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; and specific probes.

[0123] MethyLight TM The assay utilizes fluorescence-based real-time PCR (e.g., MethyLight offers high-throughput quantitative methylation assays that do not require further steps after PCR (Eads et al., Cancer Res. 59:2302-2306, 1999). In short, MethyLight... TM The method begins with a mixed genomic DNA sample, which is converted into a mixed methylation-dependent sequence differential aggregate in a sodium bisulfite reaction according to a standard procedure (the bisulfite method converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a “biased” reaction, for example, using PCR primers that overlap with known CpG dinucleotides. Sequence identification occurs at both the amplification and fluorescence detection levels.

[0124] MethyLight TM The assay is used as a quantitative test for methylation patterns in nucleic acid samples, such as genomic DNA, where sequence identification occurs at the probe hybridization level. In the quantitative version, the PCR reaction provides methylation-specific amplification in the presence of a fluorescent probe that overlaps with a specific putative methylation site. Unbiased control for the amount of input DNA is provided by a reaction in which neither the primers nor the probe cover any CpG dinucleotides. Alternatively, this can be achieved by using a control oligonucleotide (e.g., HeavyMethyl) that does not cover known methylation sites. TMQualitative testing of genomic methylation can be achieved using fluorescence-based versions and MSP technology, or by using oligonucleotide probe biased PCR aggregates covering potential methylation sites.

[0125] MethyLight TM The method is compatible with any suitable probe (e.g. probe, Probes, etc., are used together. For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and used... Probes (e.g., using MSP primers and / or Heavy Methyl blocking oligonucleotides and) The probe was used to perform one of two PCR reactions. The probe is dual-labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific to regions with relatively high GC content, allowing it to denature at approximately 10°C higher during PCR cycling compared to either the forward or reverse primer. This allows The probe remains fully hybridized during the PCR annealing / extension step. As Taq polymerase enzymatically synthesizes new strands during PCR, these strands will eventually reach the annealing stage. Probe. Taq polymerase 5′ to 3′ endonuclease activity will be obtained through digestion The probe is used to replace it to release the fluorescent reporter molecule, so that the signal that is not quenched can be quantitatively detected using a real-time fluorescence detection system.

[0126] For MethyLight TM Typical reagents for analysis (e.g., those available in typical MethyLight-based systems) TM The kits found may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.). or Probes; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0127] QM TM (Quantitative methylation) assays are an alternative quantitative test for methylation patterns in genomic DNA samples, where sequence identification occurs at the probe hybridization level. In this quantitative version, PCR reactions provide unbiased amplification in the presence of fluorescent probes that overlap with specific putative methylation sites. Unbiased control for the amount of input DNA is provided by reactions in which neither the primers nor the probes cover any CpG dinucleotides. Alternatively, this can be achieved by using control oligonucleotides (Heavy Methyl) that do not cover known methylation sites. TMQualitative testing of genomic methylation can be achieved using fluorescence-based versions and MSP technology, or by using oligonucleotide probe biased PCR aggregates covering potential methylation sites.

[0128] During the amplification process, QM TM The method can be used with any suitable probe, such as probe, Probes are used together. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and... Probe. The probe is dual-labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific to regions with relatively high GC content, allowing it to denature at approximately 10°C higher during PCR cycling compared to either the forward or reverse primer. This allows The probe remains fully hybridized during the PCR annealing / extension step. As Taq polymerase enzymatically synthesizes new strands during PCR, these strands will eventually reach the annealing stage. Probe. Taq polymerase 5′ to 3′ endonuclease activity will be obtained through digestion The probe is used to replace it, releasing the fluorescent reporter molecule so that its currently unquenched signal can be quantitatively detected using a real-time fluorescence detection system. (For QM) TM Typical reagents for analysis (e.g., those available in typical QM-based systems) TM The kits found may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.). or Probes; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0129] Ms-SNuPE TM This technique is a quantitative method for assessing differential methylation at specific CpG sites based on bisulfite treatment of DNA, followed by single nucleotide primer extension (Gonzalgo and Jones, Nucleic Acids Res. 25:2529-2531, 1997). In short, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil, while leaving 5-methylcytosine unchanged. The desired target sequence is then amplified using PCR primers specific to the bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the target CpG site. Small amounts of DNA (e.g., microdissected pathological sections) can be analyzed, and it avoids the use of restriction enzymes to determine the methylation status at CpG sites.

[0130] For Ms-SNuPE TMTypical reagents for analysis (e.g., those available in typical Ms-SNuPE-based solutions) TM The kits found may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, gene regions, labeled regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kits; positive control primers; and Ms-SNuPE for specific loci. TM Primers; reaction buffer (for Ms-SNuPE reaction); and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.

[0131] Reduction sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosine into uracil, followed by restriction enzyme digestion (e.g., by enzymes that recognize sites including CG sequences, such as MspI), and then complete sequencing of the fragments after coupling to adaptor ligands. The selection of restriction enzymes enriches fragments within CpG-dense regions, reducing the number of redundant sequences that might map to multiple gene locations during analysis. Therefore, RRBS reduces the complexity of nucleic acid samples by selecting a subset of restriction fragments for sequencing (e.g., by size selection using preparative gel electrophoresis). In contrast to whole-genome bisulfite sequencing, each fragment produced by restriction enzyme digestion contains DNA methylation information for at least one CpG dinucleotide. Therefore, RRBS enriches samples with promoters, CpG islands, and other genomic features that have high-frequency restriction enzyme cleavage sites in these regions, and thus provides a method for assessing the methylation status of one or more genomic loci.

[0132] A typical protocol for RRBS includes the following steps: digestion of nucleic acid samples with restriction enzymes such as MspI, completion of overhangs and A-tails, ligation of adaptors, bisulfite conversion, and PCR. See, for example, et al. (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" NatMethods 7:133–6; Meissner et al. (2005) "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis" Nucleic Acids Res. 33:5868–77.

[0133] In some implementations, quantitative allele-specific real-time target and signal amplification (QuARTS) assays are used to assess methylation status. Three reactions occur sequentially in each QuartS assay, including amplification (reaction 1) and target probe cleavage (reaction 2) in a primary reaction; and FRET cleavage and fluorescence signal generation in a secondary reaction (reaction 3). When the target nucleic acid is amplified with specific primers, a specific detection probe with flanking sequences loosely binds to the amplicon. The presence of a specific invasive oligonucleotide at the target binding site causes a 5′ nuclease, such as FEN-1 endonuclease, to release the flanking sequence by cleaving between the detection probe and the flanking sequence. The flanking sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flanking sequence acts as an invasive oligonucleotide on the FRET cassette and enables cleavage between the FRET cassette fluorophore and a quencher, which generates a fluorescence signal. The cleavage reaction can cleave multiple probes at each target site and thus release multiple fluorophores from each flanking site, providing exponential signal amplification. QuartS can detect multiple targets in a single reaction well by using different dyes with the FRET cassette. See, for example, Zou et al. (2010), “Sensitive quantification of methylated markers with a novel methylation specific technology” (Clin Chem 56:A199).

[0134] The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or a combination thereof, as disclosed herein for distinguishing between methylated and unmethylated CpG dinucleotide sequences. The methods of treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO 2013 / 116375, each of which is incorporated herein by reference in its entirety). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent such as, but not limited to, n-alkylenglycol or diethylene glycol dimethyl ether (DME) or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is used at a concentration between 1% and 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger such as, but not limited to, benzodihydropyran derivatives (e.g., 6-hydroxy-2,5,7,8,-tetramethylbenzodihydropyran-2-carboxylic acid) or trihydroxybenzoic acid and its derivatives (e.g., gallic acid) (see: PCT / EP2004 / 011715, which is incorporated herein by reference in its entirety). In some preferred embodiments, the bisulfite reaction comprises treatment with ammonium bisulfite, for example, as described in WO 2013 / 116375.

[0135] In some embodiments, the bisulfite-treated DNA is purified prior to quantification. This can be done by any means known in the art, such as, but not limited to, via Microcon TM Column (by Millipore) TM Ultrafiltration is performed (in accordance with the manufacturer's instructions). Purification is carried out according to a modified manufacturer's protocol (see, for example, PCT / EP2004 / 011715, which is incorporated herein by reference in its entirety). In some embodiments, bisulfite-treated DNA is bound to a solid support (e.g., magnetic beads), and desulfonation and washing occur as the DNA binds to the support. Examples of such embodiments are provided, for example, in WO 2013 / 116375. In some preferred embodiments, the support-bound DNA is immediately ready for methylation assay after desulfonation and washing on the support. In some embodiments, the desulfonated DNA is eluted from the support prior to assay.

[0136] In some embodiments, fragments of the treated DNA are amplified using multiple sets of primer oligonucleotides according to the invention (e.g., see Table 2) and an amplification enzyme. Amplification of several DNA segments can be performed simultaneously in the same reaction vessel. Typically, polymerase chain reaction (PCR) is used for amplification.

[0137] In another embodiment of the method, the methylation state of a CpG position within or near the marker is detected using methylation-specific primer oligonucleotides. This technique (MSP) is described in U.S. Patent No. 6,265,171 to Herman. The use of primers specific to the methylation state of bisulfite-treated DNA allows for differentiation between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide. Thus, the sequence of the primers contains at least one CpG dinucleotide. MSP primers specific to unmethylated DNA contain a "T" at the C position in the CpG. The fragment obtained by amplification may carry a marker that can be detected directly or indirectly. In some embodiments, the marker is a fluorescent marker, a radionuclide, or a resolvable molecular fragment with a typical mass that can be detected in a mass spectrometer. When the marker is a mass marker, some embodiments provide a marker amplicon with a single positive or negative net charge, thereby allowing for better delectability in a mass spectrometer. Detection can be performed and visualized, for example, by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or by using electron spray mass spectrometry (ESI).

[0138] Methods for isolating DNA suitable for these assays are known in the art. In particular, some embodiments include nucleic acid isolation as described in U.S. Patent Application Serial No. 13 / 470,251 (“Nucleic Acid Isolation,” published as US 2012 / 0288868), which is incorporated herein by reference in its entirety.

[0139] In some embodiments, the markers described herein are used for QUARTS assays on fecal samples. In some embodiments, methods are provided for producing DNA samples, particularly methods for producing DNA samples comprising small volumes (e.g., less than 100, less than 60 μL) of highly purified, low-abundance nucleic acids, and substantially and / or virtually free of substances that inhibit assays used to test the DNA sample (e.g., PCR, INVADER, QUARTS assays, etc.). These DNA samples can be used in diagnostic assays that qualitatively detect the presence of genes, gene variants (e.g., alleles), or gene modifications (e.g., methylation) present in samples taken from patients, or quantitatively measure their activity, expression, or amount. For example, some cancers are associated with the presence of specific mutated alleles or specific methylation states, therefore, the detection and / or quantification of said mutated alleles or methylation states has predictive value in the diagnosis and treatment of cancer.

[0140] Many valuable genetic markers are present in extremely low amounts in samples, and the occurrence of such markers is rare. Therefore, even sensitive detection methods such as PCR require large amounts of DNA to provide sufficient low-abundance targets to meet or replace the detection threshold of the assay. Furthermore, even the presence of low amounts of inhibitory substances impairs the accuracy and precision of these assays involving the detection of such low-abundance targets. Therefore, this paper provides methods for providing the necessary management of volume and concentration to produce such DNA samples.

[0141] Some biological samples, such as fecal samples, contain a wide variety of different compounds that inhibit PCR. Therefore, DNA extraction procedures include methods for removing and / or inactivating PCR inhibitors. Accordingly, in some embodiments, as described in Example 1, methods, systems, and kits for processing and preparing samples, and specifically, but not exclusively, for removing assay inhibitors from samples containing nucleic acids.

[0142] In some embodiments, the samples include blood, serum, plasma, gastric secretions, pancreatic juice, gastrointestinal biopsy samples, microdissected cells from gastrointestinal biopsies, gastrointestinal cells exfoliated into the gastrointestinal lumen, and / or gastrointestinal cells recovered from feces. In some embodiments, the subject is a human. These samples may be derived from the upper gastrointestinal tract, lower gastrointestinal tract, or may include cells, tissues, and / or secretions from both the upper and lower gastrointestinal tracts. Samples may include cells, secretions, or tissues from the liver, bile ducts, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, the samples include cytotoxic fluid, ascites, urine, excretions, pancreatic juice, fluids obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a fecal sample.

[0143] The samples can be obtained by any number of means known in the art (as will be apparent to a person skilled in the art). For example, urine and fecal samples can be readily obtained, while blood, ascites, serum, or pancreatic juice samples can be obtained, for example, via a parenteral route using needles and syringes. Cell-free or substantially cell-free samples can be obtained by subjecting the samples to various techniques known to those skilled in the art, including but not limited to centrifugation and filtration. While non-invasive techniques are generally preferred for obtaining samples, samples such as tissue homogenates, tissue sections, and biopsy specimens are still preferred. The techniques are not limited to methods for preparing samples and providing nucleic acids for testing. For example, in some embodiments, DNA can be isolated from fecal samples, blood samples, or plasma samples using direct gene capture, as detailed in or by related methods such as U.S. Patent Nos. 8,808,990 and 9,169,511 and WO 2012 / 155072.

[0144] The analysis of markers can be performed alone or simultaneously with other markers within a test sample. For example, several markers can be combined into a single test for efficient processing of multiple samples and potentially providing greater diagnostic and / or prognostic accuracy. Furthermore, those skilled in the art will recognize that the values ​​of the markers can be tested on multiple samples from the same subject (e.g., at consecutive time points). The testing of sequential samples allows for the identification of changes in marker methylation status over time. Changes in methylation status, and the absence of changes in methylation status, can provide useful information about the disease state, including but not limited to determining the approximate time of event onset, the presence and amount of treatable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and determining subject outcomes (including the risk of future events).

[0145] Biomarker analysis can be performed in various physical forms. For example, microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single-sample formats can be developed to facilitate point-of-care treatment and diagnosis, for example, in mobile transport or emergency room settings.

[0146] Implementations of the technology described herein are intended to be provided in the form of a kit. The kit includes implementations of the compositions, devices, instruments, etc., described herein, as well as instructions for use. The instructions describe appropriate methods for preparing analytes from samples (e.g., for collecting samples and preparing nucleic acids from samples). Individual components of the kit are packaged in appropriate containers and packaging (e.g., vials, boxes, blister packs, ampoules, jars, bottles, tubes, etc.), and the components are packaged together in appropriate containers (e.g., one or more boxes) for convenient storage, transport, and / or use by the user. It should be understood that liquid components (e.g., buffers) may be provided in lyophilized form for user reconfiguration. The kit may include controls or references for evaluating, validating, and / or ensuring kit performance. For example, a kit for determining the amount of nucleic acid present in a sample may include controls containing known concentrations of the same or another nucleic acid for comparison, and in some embodiments includes detection reagents (e.g., primers) specific to the control nucleic acid. The kit is suitable for use in a clinical setting and in some embodiments is suitable for use in the user's home. In some embodiments, the kit components provide functionality for a system for preparing nucleic acid solutions from samples. In some implementations, certain components of the system are provided by the user.

[0147] III. Other Applications

[0148] In some implementations, diagnostic tests identify the presence of a disease or symptom in an individual. In some implementations, the disease is cancer (e.g., cancer of the gastrointestinal system).

[0149] This disclosure is not limited to specific markers. In some embodiments, markers associated with aberrant methylation of gastrointestinal tumors are utilized (e.g., one or more of vimentin, cytoschizoprotein 9, NDRG4; see also U.S. Provisional Patent Application No. 62 / 091,053, filed December 12, 2014, which is incorporated herein by reference in its entirety for all purposes). In some embodiments, the assay also includes the detection of mutated KRAS genes (see, for example, Example 1). In some embodiments, the assay also includes the detection of hemoglobin in fecal samples (see, for example, Example 1).

[0150] In some embodiments, the technology relates to methods for treating patients (e.g., patients with gastrointestinal cancer, patients with early-stage gastrointestinal cancer, or patients who may develop gastrointestinal cancer), the methods comprising determining the methylation status of one or more markers as provided herein, and administering treatment to the patient based on the results of the methylation status determination. Treatment may include administering a drug compound, a vaccine, performing surgery, imaging the patient, or performing another test. Preferably, the use is a method for clinical screening, prognostic assessment, monitoring treatment outcomes, identifying patients most likely to respond to a particular therapeutic treatment, imaging a patient or subject, and methods for drug screening and development.

[0151] In some embodiments of the technology, methods for diagnosing gastrointestinal cancer in a subject are provided. As used herein, the terms "diagnosing" and "diagnosis" refer to methods by which a technician can estimate and even determine whether a subject has a given disease or symptom or is likely to develop a given disease or symptom in the future. Technicians often make diagnoses based on one or more diagnostic indicators, such as biomarkers (e.g., those described herein), whose methylation status indicates the presence, severity, or absence of a symptom.

[0152] Clinical cancer prognosis, along with diagnosis, involves determining the aggressiveness of the cancer and the likelihood of tumor recurrence in order to plan the most effective treatment. If a more precise prognosis is possible, or even if the potential risk of developing cancer can be assessed, appropriate treatment can be selected for the patient, and in some cases, less drastic therapies can be chosen. Assessment of cancer biomarkers (e.g., determining methylation status) can be used to isolate subjects with a good prognosis and / or a low risk of developing cancer, who will not require therapy or will require limited therapy compared to those who may benefit from more intensive treatment and are more likely to develop cancer or suffer cancer recurrence.

[0153] Therefore, as used herein, “performing a diagnosis” or “diagnosing” also includes determining the risk of developing cancer or determining prognosis, which can provide predictions of clinical outcomes (with or without medical treatment), selection of appropriate treatment (or whether treatment will be effective), or monitoring of current treatment and potential changes in treatment, based on measurements of the diagnostic biomarkers disclosed herein (e.g., those described herein). Furthermore, in some embodiments of the currently disclosed subject matter, multiple measurements of biomarkers over time can be performed to facilitate diagnosis and / or prognosis. Temporary changes in biomarkers can be used to predict clinical outcomes, monitor the progression of gastrointestinal cancer, and / or monitor the efficacy of appropriate therapies against cancer. In such embodiments, for example, it may be desirable to see changes in the methylation status of one or more biomarkers disclosed herein (and potentially one or more additional biomarkers, if monitored) in biological samples over time during an effective treatment process.

[0154] The currently disclosed subject matter also provides, in some embodiments, methods for determining whether to initiate or continue preventive or therapeutic treatment of cancer in a subject. In some embodiments, the method includes having a series of biological samples provided by the subject over a period of time; analyzing the series of biological samples to determine the methylation status of at least one biomarker disclosed herein in each biological sample; and comparing any measurable changes in the methylation status of one or more biomarkers in each biological sample. Any changes in the biomarker methylation status over the time period can be used to predict the risk of developing cancer, predict clinical outcomes, determine whether to initiate or continue preventive or therapeutic treatment of cancer, and determine whether the current therapy is effective in treating cancer. For example, a first time point can be selected before initiating treatment, and a second time point can be selected some time after initiating treatment. Methylation status can be measured in each sample taken from different time points, and qualitative and / or quantitative differences can be recorded. Changes in the methylation status of biomarker levels from different samples can be associated with gastrointestinal cancer risk, prognosis, determination of treatment efficacy, and / or cancer progression in the subject.

[0155] In a preferred embodiment, the methods and compositions of the present invention are used to treat or diagnose diseases in their early stages, such as before the onset of disease symptoms. In some embodiments, the methods and compositions of the present invention are used to treat or diagnose diseases in the clinical phase.

[0156] As described above, in some embodiments, multiple measurements of one or more diagnostic or prognostic biomarkers can be performed, and temporary changes in the markers can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be measured at an initial time and again at a second time. In such embodiments, an increase in the marker from the initial time to the second time can be a diagnosis of a specific type or severity of cancer or a given prognosis. Similarly, a decrease in the marker from the initial time to the second time can indicate a specific type or severity of cancer or a given prognosis. Furthermore, the degree of change in one or more markers can be associated with the severity of cancer and future adverse events. Those skilled in the art will understand that while in some embodiments, comparative measurements of the same biomarker can be performed at multiple time points, it is also possible to measure a given biomarker at one time point and a second biomarker at a second time point, and the comparison of these markers can provide diagnostic information.

[0157] As used herein, the phrase “prognosis determination” refers to a method by which a technician can predict the course or outcome of a symptom in a subject. The term “prognosis” does not refer to the ability to predict the course or outcome of a symptom with 100% accuracy, or even that a given course or outcome is more or less likely to occur based on the methylation status of a biomarker. Rather, a technician will understand that the term “prognosis” refers to an increased likelihood of a certain course or outcome occurring; that is, the course or outcome is more likely to occur in a subject exhibiting a given symptom compared to those individuals who do not exhibit the symptom. For example, in individuals who do not exhibit the symptom (e.g., have a normal methylation status for one or more target genes), the chance of a given outcome (e.g., having gastrointestinal cancer) may be very low.

[0158] In some implementations, statistical analysis correlates prognostic indicators with a predisposition to adverse outcomes. For example, in some implementations, a methylation state differing from that in normal control samples obtained from patients without cancer, as determined by a level of statistical significance, may indicate that a subject is more likely to have cancer than a subject with a level of methylation more similar to that in the control samples. Furthermore, changes in methylation state from baseline (e.g., “normal”) levels may reflect patient prognosis, and the degree of change in methylation state may be associated with the severity of adverse events. Statistical significance is often determined by comparing two or more populations and establishing confidence intervals and / or p-values. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is incorporated herein by reference in its entirety. The exemplary confidence intervals for this topic are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, and 99.99%, while the exemplary p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0159] In other embodiments, a threshold change in the methylation status of the prognostic or diagnostic biomarkers disclosed herein can be established, and the degree of change in the methylation status of the biomarkers in a biological sample can be simply compared to the threshold change in methylation status. Preferred threshold changes in the methylation status of the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In other embodiments, a "nomograph" can be established, in which the methylation status of a predictive or diagnostic indicator (biomarker or combination of biomarkers) is directly associated with the relevant treatment for a given outcome. Those skilled in the art will recognize that the uncertainty of this measurement is the same as the uncertainty of the label concentration because individual sample measurements are referenced, rather than population averages.

[0160] In some embodiments, control samples and biological samples are analyzed simultaneously, allowing results from the biological samples to be compared with those from the control samples. Additionally, a standard curve is anticipated to be available for comparison with the determination results of the biological samples. If fluorescent labeling is used, such a standard curve presents the methylation status of the biomarker as a function of the measurement unit (e.g., fluorescence signal intensity). Using samples taken from multiple donors, a standard curve can be provided for the control methylation status of one or more biomarkers in normal tissue, and a standard curve for the "at-risk" level of one or more biomarkers in tissue taken from donors with metaplasia or gastrointestinal cancer. In some embodiments of the method, a subject is identified as having metaplasia upon identifying abnormal methylation status of one or more of the biomarkers described herein in a biological sample obtained from the subject. In other embodiments of the method, the detection of abnormal methylation status of one or more of the biomarkers in a biological sample obtained from the subject results in the subject being identified as having cancer.

[0161] In some implementations, a subject is diagnosed with gastrointestinal cancer if a measurable difference in the methylation status of at least one biomarker in the sample is present compared to a control methylation status. Conversely, when no change in methylation status is identified in the biological sample, the subject can be identified as not having gastrointestinal cancer, not at risk of cancer, or having a low risk of cancer. In this respect, subjects with cancer or at risk of cancer can be distinguished from subjects with low to substantially no cancer or at risk of cancer. Those subjects at risk of developing gastrointestinal cancer can be placed in more intensive and / or periodic screening programs (including endoscopic monitoring). On the other hand, those subjects with low to substantially no risk can avoid endoscopic examination until a future screening time, for example, screening according to the technology of the present invention, indicates that the risk of gastrointestinal cancer has already appeared in those subjects.

[0162] As described above, according to embodiments of the method of the present invention, detecting changes in the methylation state of one or more biomarkers can be a qualitative or quantitative determination. Therefore, steps indicating a subject's diagnosis of gastrointestinal cancer or at risk of developing gastrointestinal cancer include certain threshold measurements, such as a difference in the methylation state of one or more biomarkers in a biological sample compared to a predetermined control methylation change. In some embodiments of the method, the control methylation state is any detectable biomarker methylation state. In other embodiments of the method, the predetermined methylation state is the methylation state of the control sample, tested simultaneously with the biological sample. In other embodiments of the method, the predetermined methylation state is identified based on and / or by a standard curve. In other embodiments of the method, the predetermined methylation state is specifically a state or a range of states. Therefore, the predetermined methylation state can be selected, in part, based on the implementation of the method practiced and the desired specificity, within permissible limits that will be apparent to those skilled in the art.

[0163] Furthermore, regarding diagnostic methods, preferred subjects are vertebrate subjects. Preferred vertebrates are warm-blooded; preferred warm-blooded vertebrates are mammals. Most preferably, preferred mammals are humans. As used herein, the term "subject" includes both human and animal subjects. Therefore, this invention provides for veterinary therapeutic use. Thus, the present invention provides for the diagnosis of mammals such as humans and those important due to their endangerment, such as the Siberian tiger; those of economic importance, such as animals raised on farms for human consumption; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to: carnivores such as cats and dogs; pigs, including domestic pigs, castrated pigs, and wild boars; ruminants and / or ungulates such as cattle, bulls, sheep, giraffes, deer, goats, bison, and camels; pinnipeds and horses. Therefore, the diagnosis and treatment of livestock are also provided, including but not limited to domesticated pigs, ruminants, ungulates, horses (including racehorses), etc. The disclosed subject matter also includes systems for diagnosing gastrointestinal cancer in subjects. These systems may be provided, for example, as commercial kits used to screen for or diagnose gastrointestinal cancer in subjects from whom biological samples have been collected. Exemplary systems provided according to the invention include assessing the methylation status of the markers described herein.

[0164] In recent years, it has become apparent that circulating epithelial cells, representing metastatic tumor cells, can be detected in the blood of many cancer patients. The molecular profile of rare cells is important in biological and clinical research. The application scope includes characterization of circulating epithelial cells (CEpC) in peripheral blood of cancer patients for disease prognosis and personalized treatment (see, for example, Cristofanilli M, et al. (2004) N Engl J Med 351:781–791; Hayes DF, et al. (2006) Clin Cancer Res 12:4218–4224; Budd GT, et al. (2006) Clin Cancer Res 12:6403–6409; Moreno JG, et al. (2005) Urology 65:713–718; Pantel et al. (2008) Nat Rev 8:329–340; and Cohen SJ, et al. (2008) J Clin Oncol 26:3213–3221).

[0165] Experiments conducted during the development of embodiments of this disclosure revealed the unexpected result that the presence of methylated ZDHHC1 in blood or plasma was associated with the presence of epithelial cells in the blood of patients with metastatic cancer. Therefore, embodiments of this disclosure provide compositions and methods for detecting the presence of metastatic cancer in a subject by identifying the presence of methylated ZDHHC1 in plasma or whole blood. The presence of methylated ZDHHC1 can be identified using any suitable method (e.g., those described herein).

[0166] Experimental Examples

[0167] Example 1

[0168] DNA isolation methods and QUARTS assay

[0169] The following provides exemplary methods for DNA isolation prior to analysis, such as those applicable to embodiments of the described technology, and exemplary QUARTS assays. This embodiment describes the application of the Quarts technology to DNA from feces and various tissue samples; however, the technology can be readily applied to other nucleic acid samples, such as those shown in other embodiments.

[0170] Target DNA was collected from fecal samples.

[0171] Collect all feces in a plastic container. For example, add a preservative buffer such as 150 mM EDTA, 500 mM Tris-Cl and 10 mM NaCl (pH 9.0) to the feces at a rate of approximately 4 ml per gram of feces. The buffered feces can be used directly or stored at -80°C.

[0172] An exemplary procedure for isolating target nucleic acids from fecal samples:

[0173] 1. For example, a fecal sample is homogenized with a buffer solution to form a fecal homogenate. The homogenate is then processed by centrifugation or filtration to separate residual solids from the liquid, producing a "fecal supernatant".

[0174] 2. The fecal supernatant is treated to remove assay inhibitors (e.g., with polyvinylpyrrolidone as described in U.S. Patent No. 8,993,341, which is incorporated herein by reference in its entirety) to produce a “clarified supernatant”.

[0175] 3. Mix 10 ml of the clarified supernatant (representing approximately 4 grams of fecal equivalent) with guanidine thiocyanate (GTC) to a final concentration of 2.4 M;

[0176] 4. The mixture is then heated in a 90°C water bath for 10 minutes to denature the DNA (and proteins) present in the feces.

[0177] 5. Add paramagnetic particles containing covalently attached (coupled) oligonucleotides complementary to the target sequence (“target-specific capture probe”) to the sample. Then incubate the sample (e.g., at an ambient temperature of approximately 22°C–25°C) for one hour to allow the target DNA to hybridize with the capture probe on the magnetic particles.

[0178] 6. The mixture of clarified supernatant, GTC, and particles is exposed to a magnetic field to separate the particles (now containing target DNA hybridized with the capture probe) from the fecal supernatant / GTC mixture, which is then transferred to a new tube. See, for example, U.S. Patent Application Serial No. 13 / 089,116, which is incorporated herein by reference.

[0179] The denaturation / hybridization / separation cycle (steps 4-6) can be repeated, for example, at least four or more times, to continuously extract different target DNAs from the same fecal supernatant sample.

[0180] FFPE tissue DNA

[0181] DNA was isolated from formalin-fixed paraffin-embedded (FFPE) tissue using the QIAamp DNA FFPE Tissue Kit (Qiagen Sciences, Germantown, MD).

[0182] DNA isolation from cells and plasma

[0183] For cell lines, for example, " The RSC ccfDNA Plasma Kit (Promega Corp., Madison, WI) isolates genomic DNA from cell conditioned medium. Following the kit protocol, use 1 mL of cell conditioned medium (CCM) instead of plasma and process according to the kit procedure.

[0184] An exemplary procedure for isolating DNA from a 4 mL plasma sample is as follows:

[0185] Add 300 μL of proteinase K (20 mg / mL) to a 4 mL plasma sample and mix.

[0186] • Add 3 μL of 1 μg / μL fish DNA to the plasma proteinase K mixture.

[0187] Add 2 mL of plasma lysis buffer to the plasma.

[0188] The plasma lysis buffer is:

[0189] -4.3M guanidine thiocyanate

[0190] -10% IGEPEAL CA-630 (branched octylphenoxy poly(vinyloxy)ethanol)

[0191] (5.3 g of IGEPA CA-630 combined with 45 mL of 4.8 M guanidine thiocyanate)

[0192] • Incubate the mixture at 55°C for 1 hour while shaking at 500 rpm.

[0193] Add 3 mL of plasma lysis buffer and mix.

[0194] • Add 200 μL of magnetic silica-bonded beads [16 μg beads / μL] and mix again.

[0195] Add 2 mL of 100% isopropanol and mix.

[0196] • Incubate at 30°C for 30 minutes while shaking at 500 rpm.

[0197] Place the tube on the magnet and collect the beads. Drain and discard the supernatant.

[0198] • Add 750 μL of GuHCl-EtOH to the container containing the bound beads and mix.

[0199] The GuHCl-EtOH washing buffer is:

[0200] -3M GuHCl

[0201] -57% EtOH.

[0202] Oscillate at 400 rpm for 1 minute.

[0203] Transfer the sample to a deep-well plate or a 2 mL microcentrifuge tube.

[0204] Place the tube on the magnet and allow the beads to collect for 10 minutes. Drain and discard the supernatant.

[0205] Add 1000 μL of washing buffer (10 mM Tris HCl, 80% EtOH) to the beads and incubate at 30°C for 3 minutes with shaking.

[0206] Place the tube on the magnet and collect the beads. Drain and discard the supernatant.

[0207] Add 500 μL of washing buffer to the beads and incubate at 30°C for 3 minutes with shaking.

[0208] Place the tube on the magnet and collect the beads. Drain and discard the supernatant.

[0209] Add 250 μL of washing buffer and incubate at 30°C for 3 minutes with shaking.

[0210] Place the tube on the magnet and collect the beads. Drain and discard any remaining buffer solution.

[0211] Add 250 μL of washing buffer and incubate at 30°C for 3 minutes with shaking.

[0212] Place the tube on the magnet and collect the beads. Drain and discard any remaining buffer solution.

[0213] Dry the beads at 70°C for 15 minutes under oscillation.

[0214] • Add 125 μL of elution buffer (10 mM Tris HCl, pH 8.0, 0.1 mM EDTA) to the beads and incubate at 65 °C for 25 minutes with shaking.

[0215] Place the tube on the magnet and let the beads collect for 10 minutes.

[0216] • Extract the supernatant containing DNA and transfer it to a new container or tube.

[0217] QuARTS assay

[0218] The QuARTS technology combines polymerase-based target DNA amplification with invasive cleavage-based signal amplification. This technology is described, for example, in U.S. Patent 8,361,720; U.S. Patent No. 8,715,937; U.S. Patent No. 8,916,344; and U.S. Patent Application Serial No. 14 / 036,649, each of which is incorporated herein by reference. The fluorescence signal generated by the QuARTS reaction is monitored in a manner similar to real-time PCR, allowing for the quantification of the amount of target nucleic acid in a sample.

[0219] An exemplary Quarts reaction typically includes approximately 400-600 nmol / L (e.g., 500 nmol / L) of each primer and detection probe, approximately 100 nmol / L of invasive oligonucleotides, approximately 600-700 nmol / L of each FRET box (FAM, e.g., commercially available from Hologic, Inc.; HEX, e.g., commercially available from BioSearch Technologies, IDT; and Quasar670, e.g., commercially available from BioSearch Technologies), and 6.675 ng / μL of FEN-1 endonuclease (e.g., ...). 2.0, Hologic, Inc.), 1 unit of Taq DNA polymerase in a 30 μl reaction volume (e.g., Hologic, Inc.). DNA polymerase (Promega Corp., Madison, WI), 10 mmol / L 3-(n-morpholino)propanesulfonic acid (MOPS), 7.5 mmol / L MgCl2, and 250 μmol / L of each dNTP. Exemplary Quarts cycling conditions consist of the following: an initial incubation of 3 minutes at 95°C, followed by 10 cycles of: 20 seconds at 95°C, 30 seconds at 67°C, and 30 seconds at 70°C. After 10 cycles, typically an additional 37 cycles are performed: 20 seconds at 95°C, 1 minute at 53°C, 30 seconds at 70°C, and 30 seconds at 40°C. In some applications, quantitative cycling (C... q The analysis provides a measure of the initial number (e.g., copy number) of the target DNA strands in the sample.

[0220] For fecal DNA testing, as described above, capture probes are typically used to capture target nucleic acid fragments from the clarified supernatant. Examples of capture probes are shown below, and they typically contain a 5′–6 C amino-modified bond (Integrated DNA Technology, Coralville, IA):

[0221] For NDRG4:

[0222] / 5AmMC6 / TCCCTCGCGCGTGGCTTCCGCCTTCTGCGCGGCTGGGGTGCCCGGTGG-3′(SEQ IDNO:1)

[0223] For BMP3:

[0224] / 5AmMC6 / GCGGGACACTCCGAAGGCGCAAGGAG-3′(SEQ ID NO:2)

[0225] For KRAS:

[0226] / 5AmMC6 / GGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGC-3′ (SEQ ID NO: 3) and

[0227] / 5AmMC6 / CTCTATTGTTGGATCATATTCGTCCACAAAATGATTCTGAATTAGC-3′(SEQ ID NO:4)

[0228] Captured DNA for methylation assays was treated with bisulfite using, for example, the EZ-96 DNA Methylation Kit (Zymo Research, Irvine CA) or with ammonium bisulfite as described in WO 2013 / 116375, which is incorporated herein by reference. Typically, transformed samples were eluted with 20 ng / µL tRNA (Sigma) in 50 µL of 10 mM Tris, 0.1 mM EDTA, pH 8.0; 10 µL of bisulfite-treated DNA was assayed in 30 µL reaction volumes on a 96-well PCR plate using the Quarts method. The PCR plate was cycled in a LightCycler 480 (Roche) PCR plate.

[0229] QuARTS assays may involve individual labels or multiple combinations of labels, and typically additionally include oligonucleotides for detecting reference nucleic acids such as β-actin, or labels discussed in the embodiments of the present invention below.

[0230] In this implementation, the primers and probes (Integrated DNA Technology, Coralville, IA) are as follows for each of the following targets:

[0231] For NDRG4:

[0232] Primer 5′-CGG TTT TCG TTC GTT TTT TCG-3′, (SEQ ID NO: 5)

[0233] Primer 5′-GTA ACT TCC GCC TTC TAC GC-3′, (SEQ ID NO:6)

[0234] Probe 5′-CGC CGA GGG TTC GTT TAT CG / 3′C6 / (SEQ ID NO:7)

[0235] For BMP3:

[0236] Primer 5′-GTT TAA TTT TCG GTT TCG TCG TC-3′ (SEQ ID NO: 8)

[0237] Primer 5′-CTC CCG ACG TCG CTA CG-3′ (SEQ ID NO:9)

[0238] Probe 5′-CGC CGA GGC GGT TTT TTG CG / 3′C6 / (SEQ ID NO:10)

[0239] For β-actin treated with bisulfite:

[0240] Primer 5′-TTT GTT TTT TTG ATT AGG TGT TTA AGA-3′ (SEQ ID NO: 52)

[0241] Primer 5′-CAC CAA CCT CAT AAC CTT ATC-3′ (SEQ ID NO:59)

[0242] Probe 5′-CCA CGG ACG ATA GTG TTG TGG / 3′C6 / (SEQ ID NO:60)

[0243] For example, each assay in the assay plate includes a bisulfite-treated DNA sample, a standard curve sample, a positive control, and a negative control. The standard curve can be prepared using, for example, target strands cut from engineered plasmids at 300 to 1000 strands. Bisulfite-treated CpGenome universally methylated DNA (Millipore, Billerica, MA) and human genomic DNA (Merck, Germany) are used as positive and negative controls. The C-methylated DNA of the target gene is then analyzed. p The DNA strand number was determined by comparison with a standard curve of the relevant assay. The methylation percentage of each marker was determined by dividing the number of methylated gene strands by the number of control DNA (e.g., β-actin or candidate control markers provided herein) strands and multiplying by 100.

[0244] KRAS mutation

[0245] Seven mutations at codons 12 / 13 of the KRAS gene were evaluated using the Quarts assay. Each mutation assay was designed as a singlet assay. The KRAS mutation-specific forward primers and probes were as follows:

[0246] For the G12S mutation:

[0247] Primer 5′-CTT GTG GTA GTT GGA GCA A-3′ (SEQ ID NO:11)

[0248] Probe 5′-GCG CGT CCA GTG GCG TAG GC / 3′C6 / (SEQ ID NO:12);

[0249] For G12C mutation

[0250] Primer 5′-AAA CTT GTG GTA GTT GGA CCT T-3′ (SEQ ID NO:13)

[0251] Probe 5′-GCG CGT CCT GTG GCG TAG GC / 3′C6 / (SEQ ID NO:14);

[0252] For G12R mutation

[0253] Primer 5′-TAT AAA CTT GTG GTA GTT GGA CCT C-3′ (SEQ ID NO:15)

[0254] Probe 5′-GCG CGT CCC GTG GCG TAG GC / 3′C6 / (SEQ ID NO:16);

[0255] For G12D mutation

[0256] Primer 5′-ACT TGT GGT AGT TGG AGC TCA-3′ (SEQ ID NO:17)

[0257] Probe 5′-GCG CGT CCA TGG CGT AGG CA / 3′C6 / (SEQ ID NO:18);

[0258] For G12V mutation

[0259] Primer 5′-ACT TGT GGT AGT TGG AGC TCT-3′ (SEQ ID NO:19)

[0260] Probe 5′-GCG CGT CCT TGG CGT AGG CA / 3′C6 / (SEQ ID NO:20);

[0261] For G12A mutation

[0262] Primer 5′-AAC TTG TGG TAG TTG GAG ATG C-3′ (SEQ ID NO:21)

[0263] Probe 5′-GCG CGT CCC TGG CGT AGG CA / 3′C6 / (SEQ ID NO:22);

[0264] For G13D mutation

[0265] Primer 5′-GGT AGT TGG AGC TGG TCA-3′ (SEQ ID NO:23)

[0266] Probe 5′-GCG CGT CCA CGT AGG CAA GA / 3′C6 / (SEQ ID NO:24)

[0267] For all KRAS mutants, the reverse primer used is:

[0268] 5′-CTA TTG TTG GAT CAT ATT CGT C-3′ (SEQ ID NO: 25)

[0269] The Quarts cycling conditions and reagent concentrations for KRAS are the same as those in the methylation assay. Each plate contains standards prepared from engineered plasmids, positive and negative controls, and a water blank, and is run on a LightCycler 480 (Roche) or ABI 7500 (Thermo Scientific). The target gene's C... p Or C T The DNA strand number was determined by comparing the result with a standard curve from this assay. The concentration of each mutation marker in 50 μL of KRAS was calculated based on a 500-fold dilution factor and an amplification efficiency of 1.95. This value was divided by the β-actin concentration or ZDHH in the methylation assay, and then multiplied by 100 to determine the mutation percentage.

[0270] In the assays discussed below, “BTACT” refers to the characterization of β-actin in methylation assays, and “ACT” or “ACTB” refers to the characterization of β-actin in mutation assays.

[0271] Example 2

[0272] Identification and testing of candidate control genes

[0273] As described above, in some embodiments, a control gene for the technology is selected based on its methylation status. In the first step, a control gene is selected from normal and cancer epithelial tissue cells. both Highly methylated genes are used as candidate control genes. As a second step, selected candidate genes are screened to identify genes in which the methylated form of the gene is minimally present in blood and blood fractions. In a preferred embodiment, candidate genes may be further analyzed to select genes with similar GC and CpG methylation levels to the marker genes to be analyzed, such that their bisulfite reactivity and PCR amplification behavior are similar to those of the marker genes to be analyzed.

[0274] ZDHHC1, ZFAND3, ZMYM4, ODZ2, and TRIO were identified as methylation genes that may be suitable for use as controls.

[0275] These candidate markers have the following loci (refer to GRCh37 / hg19 assembly):

[0276] ZDHHC1 footprint: Chr 16, 67428559-67428628

[0277] ZMYM4's footprint: Chr 1, 35877002-35877078

[0278] ZFAND3 Footprint: Chr 6, 37841985-37842061

[0279] ODZ2 footprint: Chr 5, 167285650-167285775

[0280] TRIO Footprints: Chr 5, 14461291-14461417

[0281] The ZDHHC1, ZFAND3, and ZMYM4 genes were selected for further analysis and were determined using the Quarts technique to compare gene methylation in normal and cancer samples and to assess the presence of markers in blood (e.g., serum). The oligonucleotides used in the assays are illustrated below. The term "wild-type" refers to gene sequences that are not affected by bisulfite conversion and are unaffected by methylation status.

[0282] ZDHHC1 - Zinc finger, containing DHHC type 1

[0283] Unprocessed target sequence:

[0284] 5'-GGGGCCGGGGCCGACAGCCCACGCTGGCGCGGCAGGCGCGTGCGCCCGCCGTTTTCGTGAGCCCGAGCAG-3' (SEQ ID NO: 26)

[0285] Target sequences for bisulfite treatment:

[0286] 5'-GGGGUCGGGGUCGAUAGUUUACGUTGGCGCGGUAGGCGCGTGCGUUCGUCGTTTCGTGAGUUCGAGUAG-3'

[0287] (SEQ ID NO:33)

[0288] Bisulfite-treated replication target sequence:

[0289] 5'-GGGGTCGGGGTCGATAGTTTACGTTGGCGCGGTAGGCGCGTGCGTTCGTCGTTTTCGTGAGTTCGAGTAG-3' (SEQ ID NO: 27)

[0290]

[0291] QuARTS assay for oligonucleotides (all shown from 5' to 3'):

[0292]

[0293] ZFAND3 - Zinc finger, AN1 type domain 3

[0294] Unprocessed target sequence:

[0295]

[0296] Target sequences for bisulfite treatment:

[0297]

[0298]

[0299] QuARTS assay for oligonucleotides (all shown from 5' to 3'):

[0300]

[0301] ZMYM4 - Zinc Index, MYM Type 4

[0302] Unprocessed target sequence:

[0303]

[0304] Target sequences for bisulfite treatment:

[0305]

[0306] QuARTS assay for oligonucleotides (all shown from 5' to 3'):

[0307]

[0308]

[0309] Quasar 670A3 FRET box:

[0310]

[0311] [FP = forward primer; RP = reverse primer; 3′C6 = 3′ hexane; 5amm6 = 5′ amino; CP = capture probe;] 670 dye; BHQ2 = black hole quencher 2]

[0312] Using the above oligonucleotide combination, methylation analysis of the cancer markers NDRG4 and BMP3 was performed on various sample types (blood, plasma, and two human colorectal cancer cell lines HT29 and HT116) using either β-actin (BTACT) (for normalization) or one of three candidate control genes (ZDHHC1, ZMYM, and ZFAND). Determinations were performed in duplicate as described in Example 1. Table 1 shows the average values ​​of replicates:

[0313] Table 1

[0314]

[0315] These data show that all three candidate markers, like BTACT, exhibited strong positive signals in the cancer cell lines HT29 and HT116. However, both ZFAND3 and ZMYM4v2, like BTACT, showed significant signals in blood samples, potentially producing undesirable background in samples containing a certain amount of blood (e.g., tissue or fecal samples).

[0316] This example demonstrates that ZDHHC1 has a low background signal in blood and plasma and is readily detectable in epithelial cell lines. ZDHHC1 was selected for further analysis.

[0317] Example 3

[0318] Comparison of β-actin and ZDHHC1 as normalized cancer marker assays

[0319] ZDHHC1 markers were tested in parallel with BTACT to compare these DNAs as controls and determine the methylation percentage of the NDRG4 and BMP3 marker genes. DNA isolated from formalin-fixed paraffin-embedded tissue samples was characterized, and the assay signals were normalized to β-actin or ZDHHC1. The results are shown in Table 3 below.

[0320] These data show that the methylation percentage of NDRG4 and BMP3 markers relative to ZDHHC1 is comparable to the methylation percentage of the same markers relative to β-actin, indicating that ZDHHC1 can be used instead of β-actin for normalization.

[0321] Table 3

[0322]

[0323]

[0324] As shown in Table 3, a comparison of the methylation % values ​​measured using ZDHHC1 and those measured using BTACT shows that these controls performed similarly on these tissue samples, and that ZDHHC1 can be used instead of BTACT to measure the methylation of cancer marker genes.

[0325] Example 4

[0326] ZDHHC1 and β-actin DNA in normal and cancerous tissue samples

[0327] This example describes a comparison of the number of ZDHHC1 and β-actin chains in extended sampling of different cancerous and normal tissue samples. DNA from normal and abnormal tissue types (including bile duct, colon, esophagus, head, lung, pancreas, small intestine, and stomach) was tested.

[0328] DNA isolated from formalin-fixed paraffin-embedded tissue samples was characterized using the median signal measurements of β-actin (ACTB) and ZDHHC1 as shown in Table 4 below.

[0329] Table 4

[0330]

[0331]

[0332] These data confirmed the presence of methylated ZDHHC1 controls in all tissue types tested, as well as in both normal and abnormal tissue types (e.g., adenoma, carcinoma, metaplasia). The results showed that ZDHHC1 methylation levels were equal between cancerous and normal tissues.

[0333] Example 5

[0334] The effect of ZDHHC1 on normalized cancer marker assays in complex samples

[0335] Further experiments were conducted using ZDHHC1 as the normalization marker in the assay to detect cancer in more complex samples (e.g., feces, blood, etc.) as well as in normal tissue samples and colorectal cancer tissue samples. Table 5A shows the strands detected for NDRG4 methylation markers and two control DNAs, and shows the methylation percentage of NDRG4 as determined using each control DNA. Data for the BMP3 marker detected in the same assay reaction are shown in Table 5B.

[0336] Table 5A

[0337]

[0338]

[0339] Table 5B

[0340]

[0341] These data show that the presence of methylated ZDHHC1 control DNA was substantially homogeneous in stool samples from normal and colorectal cancer-positive subjects, and confirmed that the marker was substantially absent in blood samples. These data also confirm that the presence of ZDHHC1 is substantially equivalent to that in β-actin DNA samples without blood (e.g., cell lines).

[0342] Example 6

[0343] ZDHHC1 in plasma samples from subjects with metastatic cancer

[0344] This example describes the detection of ZDHHC1 in plasma samples from patients with metastatic cancer.

[0345] Plasma samples from normal, advanced adenomas (AA), and adenocarcinomas (ACA), and from patients were used for the QUARTS assay. Plasma samples from colon cancer subjects and normal subjects were processed using the Qiagen Cyclic Nucleic Acid Kit. The initial plasma volume ranged from 0.75 to 2.0 ml. DNA was converted to bisulfite and tested with a mixture of ZDHHC1 and BTACT oligonucleotides. Results showed higher levels of ZDHHC1 chains in stage IV cancer samples with liver metastases.

[0346] With one exception, none of the samples contained ZDHHC1 marker chains. One sample showing abundant ZDHHC1 marker chains in plasma was a stage IV metastasis. These data support the use of ZDHHC1 as a general marker for metastasis in the detection of epithelial cells in blood / plasma. The results are summarized in Table 6:

[0347] Table 6

[0348]

[0349] * Includes a sample characterized by a stage.

[0350] In a separate study of two AA samples, two of the eight ACA samples were classified as stage IV with observed metastasis, and stage IV was detected in one of the 34 normal samples, showing a significantly elevated ZDHHC1 / BTACT ratio (41%), while one sample appeared to have a normal ZDHHC1 / BTACT ratio (4.3%). No elevated ZDHHC1 / BTACT ratios were observed in the other samples.

[0351] Example 7

[0352] ZDHHC1 in plasma samples from subjects with cancer

[0353] As attached Figures 4A-4E As detailed, plasma levels of ZDHHC1 were measured in an additional cohort of patient samples, comprising 57 samples from cancer patients and 52 normal samples. DNA was extracted from 4 ml of plasma and subjected to bisulfite conversion. ZDHHC1 DNA was pre-amplified for 10 cycles and then detected using Quarts flanking assays as described in Example 1, employing the primers and probes described in Example 2. Results are shown in… Figures 4A-4E The table below shows the average data for each sample type:

[0354]

[0355] Example 8

[0356] Detecting ZDHHC1 to monitor disease status and / or progression

[0357] The presence of epithelial cells or epithelial cell DNA in patient samples (e.g., blood product samples, such as plasma samples) can be analyzed as a means of monitoring disease status (e.g., onset, progression, response to therapy, postoperative status, remission, relapse, etc.). In some implementations, samples are obtained from the patient at multiple time points, and the amount of ZDHHC1 DNA present (whether free, circulating in cells, or in complexes) is measured at each time point. The amount of ZDHHC1 DNA in samples obtained at different time points is compared to assess changes in disease status.

[0358] At the first point in time, blood samples were obtained from the patient, and plasma samples were prepared.

[0359] At the second time point, a second blood sample was obtained from the patient, and a second plasma sample was prepared.

[0360] Test the ZDHHC1 DNA in the whole blood sample or further process the sample to produce plasma fractions.

[0361] For example, the presence and amount of ZDHHC1 DNA in each plasma sample can be tested using the methods described in Examples 1 and 6 above. In some embodiments, it is anticipated that the first sample will not be tested immediately (e.g., blood or plasma, or DNA isolated therefrom, will be stored for later testing), and the first and second samples will be tested simultaneously. In other embodiments, the first plasma sample is tested before the second blood sample is collected, and the results are stored for later comparison.

[0362] The technology is not limited to a specific event or action occurring between a first time point and a second time point. For example, the first time point could be a time when there is no suspicion of disease; for instance, the first measurement might establish a baseline with the expectation of monitoring for future disease development in the subject. Alternatively, the first time point can be obtained at a point where symptoms or disease may be present in the subject, but the disease state is preferred as an action rather than a proactive therapeutic intervention, such as monitoring changes in the disease state, such as metastasis. In other cases, proactive therapies, such as surgery, drug therapy, etc., can be administered to the subject between the two time points, and the efficacy of the therapy can be monitored using measurements of epithelial cell DNA in the blood.

[0363] This invention provides:

[0364] 1. A method for characterizing blood or blood products, comprising:

[0365] a) Blood or blood product samples provided by the subject;

[0366] b) Analyze the sample to detect the presence of tissue-cell-specific DNA;

[0367] The presence of the tissue cell-specific control DNA indicates the presence of tissue cells in the blood or blood product sample.

[0368] 2. The method of claim 1, wherein the tissue-cell-specific DNA is epithelial cell-specific DNA.

[0369] 3. The method of claim 2, wherein the epithelial cell-specific DNA comprises DNA that is methylated in epithelial cells and not methylated in blood cells.

[0370] 4. The method of claim 1, further comprising treating DNA from said sample with a bisulfite reagent to produce transformed tissue-cell-specific DNA.

[0371] 5. The method of claim 3, wherein the epithelial cell-specific DNA comprises ZDHHC1 DNA.

[0372] 6. The method of claim 1, wherein the blood product is plasma.

[0373] 7. The method of claim 1, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or DNA target capture.

[0374] 8. The method as described in item 1, wherein the assay is a flanking endonuclease assay.

[0375] 9. A method for monitoring a disease state in a subject, the method comprising the following steps:

[0376] a) Obtain a first blood product sample from the subject at the first time point;

[0377] b) Initiate a treatment plan, wherein the treatment plan includes therapeutic intervention;

[0378] c) Obtain a second blood product sample from the subject at a second time point, wherein the second time point is after the initiation of the treatment regimen; and

[0379] d) Determine the amount of epithelial cell-specific DNA in the first blood product sample and the second blood product sample.

[0380] The difference in the amount of epithelial cell-specific DNA between the first blood product sample and the second blood product sample indicates a change in the disease state in the subject.

[0381] 10. The method of claim 9, wherein the treatment regimen includes one or more of surgery, pharmacological therapy, chemotherapy, immunotherapy, nutritional therapy, radiotherapy, thermotherapy, and physical therapy.

[0382] 11. The method of claim 9 or 10, wherein the difference in the amount of epithelial cell-specific DNA between the first blood product sample and the second blood product sample indicates recurrence, progression, or regression of the disease state in the subject.

[0383] 12. A method for monitoring the disease state of a subject, the method comprising the following steps:

[0384] a) Obtain a first blood product sample from the subject at the first time point;

[0385] b) Obtain a second blood product sample from the subject at the second time point; and

[0386] c) Determine the amount of epithelial cell-specific DNA in the first blood product sample and the second blood product sample.

[0387] The difference in the amount of epithelial cell-specific DNA between the first blood product sample and the second blood product sample indicates the occurrence of a disease state in the subject.

[0388] 13. The method as described in item 9 or item 12, wherein the disease state is cancer.

[0389] 14. The method as described in item 13, wherein the cancer is a metastatic cancer.

[0390] 15. The method of claim 9 or claim 12, wherein the epithelial cell-specific DNA comprises DNA that is methylated in epithelial cells and not methylated in blood cells.

[0391] 16. The method of claim 15, comprising treating DNA from the blood product sample with a bisulfite reagent to produce transformed epithelial cell-specific DNA.

[0392] 17. The method of claim 9 or claim 12, wherein the epithelial cell-specific DNA comprises ZDHHC1 DNA.

[0393] 18. The method of claim 9 or claim 12, wherein the blood product is plasma.

[0394] 19. The method of claim 9 or claim 12, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or DNA target capture.

[0395] 20. The method as described in item 9 or item 12, wherein the assay includes a flanking endonuclease assay.

[0396] 21. A method for quantitative nucleic acid detection, comprising:

[0397] a) Determine the amount of at least one marker gene in a sample from a subject;

[0398] b) Determine the amount of tissue-cell-specific DNA in the sample; and

[0399] c) Compare the amount of the at least one marker gene in the sample with the amount of the tissue-cell-specific DNA to determine the amount of the at least one marker gene in the sample relative to the amount of the tissue-cell-specific DNA.

[0400] 22. The method of claim 21, wherein the tissue-cell-specific DNA is epithelial cell-specific DNA.

[0401] 23. The method of claim 21, wherein the tissue-cell-specific DNA comprises DNA that is methylated in tissue cells and not methylated in blood cells.

[0402] 24. The method of claim 23, wherein the tissue-cell specific DNA comprises ZDHHC1 DNA.

[0403] 25. The method of claim 21, comprising treating DNA from the sample with a bisulfite reagent to produce transformed tissue-cell-specific DNA and at least one transformed marker gene, wherein determining the amount of the at least one marker gene and the tissue-cell-specific DNA comprises determining the amount of the transformed marker gene and the transformed tissue-cell-specific DNA.

[0404] 26. The method of claim 21, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation and / or target capture.

[0405] 27. The method of claim 21, wherein the determination of the labeled DNA and the determination of the tissue-cell-specific DNA are performed in a single reaction.

[0406] 28. The method of claim 21, wherein the assay is a flanking endonuclease assay.

[0407] 29. The method of claim 25, wherein the amount of the transformed marker gene relative to the amount of transformed tissue-cell-specific DNA indicates the methylation state of the marker gene, wherein the methylation state includes an increase or decrease in methylation of the marker gene relative to the normal methylation state of the marker gene.

[0408] 30. The method of claim 29, wherein the increase or decrease in methylation of the marker gene relative to the normal methylation state of the marker gene indicates a disease.

[0409] 31. The method as described in item 29, wherein the disease is a gastrointestinal tumor.

[0410] 32. The method of claim 31, wherein the tumor is located in the upper gastrointestinal region of the patient.

[0411] 33. The method of claim 31, wherein the tumor is located in the lower gastrointestinal region of the patient.

[0412] 34. The method of claim 31, wherein the tumor includes pancreatic tumor, colorectal tumor, bile duct tumor, gastric tumor, esophageal tumor, or adenoma.

[0413] 35. The method as described in item 31, wherein the tumor is precancerous.

[0414] 36. The method of claim 31, wherein the sample is a fecal sample, tissue sample, pancreatic juice sample, pancreatic cyst fluid sample, blood sample, or urine sample.

[0415] 37. The method of claim 31, wherein the sample is derived from a subject suffering from inflammatory bowel disease.

[0416] 38. A method for detecting cancer in a blood or blood product sample from a subject, comprising:

[0417] a) Blood or blood product samples provided by the subject;

[0418] b) Analyze the sample to detect the presence of tissue-cell-specific DNA;

[0419] The presence of tissue-cell-specific DNA in the blood or blood product indicates the presence of cancer in the subject.

[0420] 39. The method of claim 38, wherein the presence of the tissue-cell-specific DNA in the blood or blood product indicates the presence of metastatic cancer in the subject.

[0421] 40. The method of claim 38, wherein the tissue-cell-specific DNA is epithelial cell-specific DNA.

[0422] 41. The method of claim 38, wherein the tissue-cell-specific DNA comprises DNA that is methylated in tissue cells and not methylated in blood cells.

[0423] 42. The method of claim 41, wherein the tissue-cell specific DNA comprises ZDHHC1 DNA.

[0424] 43. The method of claim 38, wherein the blood product is plasma.

[0425] 44. The method of claim 38, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or target capture.

[0426] 45. The method of claim 38, wherein the assay is a flanking endonuclease assay.

[0427] 46. ​​The method of claim 38, wherein the metastatic cancer is colorectal cancer.

[0428] 47. A reagent kit comprising:

[0429] a) at least one oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to bisulfite-converted ZDHHC1 DNA; and

[0430] b) Bisulfite reagent.

[0431] 48. The kit as described in item 47, wherein the oligonucleotide is selected from one or more of the following: capture oligonucleotides, a pair of nucleic acid primers, nucleic acid probes, and invasive oligonucleotides.

[0432] 49. The kit as described in item 47, wherein the kit further comprises one or more nucleic acids that specifically hybridize with one or more target or marker genes.

[0433] 50. The kit as described in item 47, wherein the kit further comprises a solid support.

[0434] 51. The kit as described in item 50, wherein the solid support is a magnetic bead.

[0435] 52. The kit of claim 50, wherein the solid support comprises one or more capture reagents.

[0436] 53. The kit as described in item 52, wherein the capture reagent is an oligonucleotide complementary to ZDHHC1 DNA or one or more of the target genes.

[0437] 54. A composition comprising:

[0438] A complex of ZDHHC1 nucleic acid and at least one oligonucleotide, wherein at least a portion of the oligonucleotide hybridizes with the ZDHHC1 nucleic acid.

[0439] 55. The composition of claim 54, wherein the ZDHHC1 nucleic acid is a bisulfite-converted ZDHHC1 nucleic acid.

[0440] 56. The composition of claim 54, wherein the oligonucleotide is selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a nucleic acid probe, and an invasive oligonucleotide.

[0441] 57. The composition of claim 54, wherein the composition further comprises one or more reaction mixtures comprising a complex of a target nucleic acid and one or more oligonucleotides specifically hybridizing with the target nucleic acid.

[0442] 58. A composition comprising a DNA strand containing the nucleotide sequence of SEQ ID NO:33.

[0443] 59. A composition comprising a DNA strand containing the nucleotide sequence of SEQ ID NO:27.

[0444] 60. The composition of claim 58 or 59, further comprising a detection probe oligonucleotide, wherein the detection probe oligonucleotide comprises a region complementary to a portion of the DNA strand.

[0445] 61. The composition of claim 60, wherein the detection probe oligonucleotide comprises a region complementary to a portion of SEQ ID NO:27.

[0446] 62. The composition of claim 61, wherein the detection probe oligonucleotide comprises a reporter molecule.

[0447] 63. The composition of claim 62, wherein the reporter molecule comprises a fluorophore.

[0448] 64. The composition of claim 60, wherein the detection probe comprises a flanking sequence.

[0449] 65. The composition as described in item 60, further comprising a FRET cartridge.

[0450] 66. The composition as described in item 64, further comprising FEN-1 endonuclease.

[0451] 67. The composition of claim 60, further comprising a thermostable DNA polymerase.

[0452] 68. The composition of claim 67, wherein the thermostable DNA polymerase is a bacterial DNA polymerase.

[0453] 69. A reaction mixture comprising the composition as described in item 58 or 59.

[0454] 70. The method of any one of claims 1-3, comprising treating DNA from said sample with a bisulfite reagent to produce transformed tissue-cell-specific DNA.

[0455] 71. The method of any one of items 1-3 and 70, wherein the epithelial cell-specific DNA comprises ZDHHC1 DNA.

[0456] 72. The method of any one of items 1-3 and 71, wherein the blood product is plasma.

[0457] 73. The method of any one of items 1-3 and 72, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or DNA target capture.

[0458] 74. The method of any one of items 1-3 and 73, wherein the assay is a flanking endonuclease assay.

[0459] 75. The method of any one of items 9-12, wherein the disease state is cancer or metastatic cancer.

[0460] 76. The method of any one of items 9-12 and 75, wherein the epithelial cell-specific DNA comprises DNA that is methylated in epithelial cells and not methylated in blood cells.

[0461] 77. The method of claim 76, comprising treating DNA from the blood product sample with a bisulfite reagent to produce transformed epithelial cell-specific DNA.

[0462] 78. The method of any one of items 9-12 and 75-77, wherein the epithelial cell-specific DNA comprises ZDHHC1 DNA.

[0463] 79. The method of any one of items 9-12 and 75-78, wherein the blood product is plasma.

[0464] 80. The method of any one of items 9-12 and 75-79, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or DNA target capture.

[0465] 81. The method of any one of items 9-12 and 75-80, wherein the assay comprises a flanking endonuclease assay.

[0466] 82. The method of claim 21 or 22, wherein the tissue-cell-specific DNA comprises DNA that is methylated in tissue cells and not methylated in blood cells, wherein the tissue-cell-specific DNA preferably comprises ZDHHC1 DNA.

[0467] 83. The method of any one of items 21-22 and 82, comprising treating DNA from the sample with a bisulfite reagent to produce transformed tissue-cell-specific DNA and at least one transformed marker gene, wherein determining the amount of the at least one marker gene and the tissue-cell-specific DNA comprises determining the amount of the transformed marker gene and the transformed tissue-cell-specific DNA.

[0468] 84. The method of any one of items 21-22 and 83, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation and / or target capture.

[0469] 85. The method of any one of items 21-22 and 83-84, wherein the determination of the labeled DNA and the determination of the tissue-cell-specific DNA are performed in a single reaction.

[0470] 86. The method of any one of items 21-22 and 83-85, wherein the assay is a flanking endonuclease assay.

[0471] 87. The method of any one of claims 83-86, wherein the amount of the transformed marker gene relative to the amount of transformed tissue-cell-specific DNA indicates the methylation state of the marker gene, wherein the methylation state includes an increase or decrease in methylation of the marker gene relative to the normal methylation state of the marker gene.

[0472] 88. The method of claim 87, wherein the increase or decrease in methylation of the marker gene relative to the normal methylation state of the marker gene indicates a disease.

[0473] 89. The method as described in item 88, wherein the disease is a gastrointestinal tumor.

[0474] 90. The method of claim 89, wherein the tumor is located in the upper gastrointestinal region of the patient.

[0475] 91. The method of claim 89, wherein the tumor is located in the lower gastrointestinal region of the patient.

[0476] 92. The method of any one of claims 89-91, wherein the tumor comprises a pancreatic tumor, a colorectal tumor, a bile duct tumor, a gastric tumor, an esophageal tumor, or an adenoma.

[0477] 93. The method of any one of items 89-92, wherein the tumor is precancerous.

[0478] 94. The method of any one of claims 89-93, wherein the sample is a fecal sample, tissue sample, pancreatic juice sample, pancreatic cyst fluid sample, blood sample, or urine sample.

[0479] 95. The method of any one of claims 89-94, wherein the sample is derived from a subject suffering from inflammatory bowel disease.

[0480] 96. The method of claim 38 or 39, wherein the tissue-cell-specific DNA is epithelial cell-specific DNA.

[0481] 97. The method of any one of items 38-39 and 96, wherein the tissue-cell-specific DNA comprises DNA that is methylated in tissue cells and not methylated in blood cells, wherein the tissue-cell-specific DNA preferably comprises ZDHHC1 DNA.

[0482] 98. The method of any one of items 38-39 and 97, wherein the blood product is plasma.

[0483] 99. The method of any one of items 38-39 and 98, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or target capture.

[0484] 100. The method of any one of items 38-39 and 99, wherein the assay is a flanking endonuclease assay.

[0485] 101. The method of any one of items 38-39 and 100, wherein the metastatic cancer is colorectal cancer.

[0486] 102. The kit as described in item 47 or 48, wherein the kit further comprises one or more nucleic acids that specifically hybridize with one or more target or marker genes.

[0487] 103. The kit as described in any one of items 47-48 and 102, wherein the kit further comprises a solid support.

[0488] 104. The kit as described in item 103, wherein the solid support is a magnetic bead.

[0489] 105. The kit as described in item 103 or 104, wherein the solid support comprises one or more capture reagents.

[0490] 106. The kit as described in item 105, wherein the capture reagent is an oligonucleotide complementary to ZDHHC1 DNA or one or more of the target genes.

[0491] 107. The composition of claim 54 or 55, wherein the oligonucleotide is selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a nucleic acid probe, and an invasive oligonucleotide.

[0492] 108. The composition of any one of items 54-55 and 107, wherein the composition further comprises one or more reaction mixtures, the one or more reaction mixtures comprising a complex of a target nucleic acid and one or more oligonucleotides specifically hybridizing with the target nucleic acid.

[0493] 109. The composition of claim 60, wherein the detection probe oligonucleotide comprises a region complementary to a portion of SEQ ID NO 27.

[0494] 110. The composition of claim 60 or 109, wherein the detection probe oligonucleotide comprises a reporter molecule.

[0495] 111. The composition of claim 110, wherein the reporter molecule comprises a fluorophore.

[0496] 112. The composition of any one of items 60 or 109-111, wherein the detection probe comprises a flanking sequence.

[0497] 113. The composition of any one of items 60 or 109-112, further comprising a FRET cartridge.

[0498] 114. The composition as described in item 112 or 113, further comprising FEN-1 endonuclease.

[0499] 115. The composition of any one of items 60 or 109-114, further comprising a thermostable DNA polymerase.

[0500] 116. The composition of claim 115, wherein the thermostable DNA polymerase is a bacterial DNA polymerase.

[0501] 117. A reaction mixture comprising the composition as described in any one of items 58-60 and 109-116.

[0502] All publications and patents mentioned in the foregoing specification are incorporated herein by reference in their entirety for all purposes. Various modifications and variations to the compositions, methods, and uses described in this technology will be apparent to those skilled in the art without departing from the scope and spirit of the described technology. Although this technology has been described in conjunction with specific exemplary embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the modes of carrying out the invention that will be apparent to those skilled in pharmacology, biochemistry, medical science, or related fields are intended to fall within the scope of the following claims. sequence list <110> Precision Science Company <120> Compositions and methods for methylation detection and determination <130> EXCT-33905 / WO-1 / ORD <150> US 62 / 091,069 <151> 2014-12-12 <160> 62 <170> PatentIn version 3.5 <210> 1 <211> 48 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 1 tccctcgcgc gtggcttccg ccttctgcgc ggctggggtg cccggtgg 48 <210> 2 <211> 26 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 2 gcgggacact ccgaaggcgc aaggag 26 <210> 3 <211> 44 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 3 ggcctgctga aaatgactga atataaactt gtggtagttg gagc 44 <210> 4 <211> 46 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 4 ctctattgtt ggatcatatt cgtccacaaa atgattctga attagc 46 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 5 cggttttcgt tcgttttttc g 21 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 6 gtaacttccg ccttctacgc 20 <210> 7 <211> 18 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 7 cgccgagggt tcgtttat 18 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 8 gtttaatttt cggtttcgtc gtc 23 <210> 9 <211> 17 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 9 ctcccgacgt cgctacg 17 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 10 cgccgaggcg gttttttgcg 20 <210> 11 <211> 18 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 11 cttgtggtag ttggagca 18 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 12 gcgcgtccag tggcgtaggc 20 <210> 13 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 13 aaacttgtgg tagttggacc tt 22 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 14 gcgcgtcctg tggcgtaggc 20 <210> 15 <211> 25 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 15 tataaacttg tggtagttgg acctc 25 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 16 gcgcgtcccg tggcgtaggc 20 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 17 acttgtggta gttggagctc a 21 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 18 gcgcgtccat ggcgtaggca 20 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 19 acttgtggta gttggagctc t 21 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 20 gcgcgtcctt ggcgtaggca 20 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> twenty one aacttgtggt agttggagat gc 22 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> twenty two gcgcgtccct ggcgtaggca 20 <210> twenty three <211> 18 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> twenty three ggtagttgga gctggtca 18 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> twenty four gcgcgtccac gtaggcaaga 20 <210> 25 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 25 ctattgttgg atcatattcg tc 22 <210> 26 <211> 70 <212> DNA <213> Homo sapiens <400> 26 ggggccgggg ccgacagccc acgctggcgc ggcaggcgcg tgcgcccgcc gttttcgtga 60 gcccgagcag 70 <210> 27 <211> 70 <212> DNA <213> Homo sapiens <400> 27 ggggtcgggg tcgatagttt acgttggcgc ggtaggcgcg tgcgttcgtc gttttcgtga 60 gttcgagtag 70 <210> 28 <211> 12 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 28 gttggcgcgg ta 12 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 29 gtcggggtcg atagtttacg 20 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 30 cgaactcacg aaaacgacga 20 <210> 31 <211> 12 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 31 gacgaacgca cg 12 <210> 32 <211> 19 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 32 actcgaactc acgaaaacg 19 <210> 33 <211> 70 <212> DNA <213> Homo sapiens <400> 33 gggucgggg ucgauaguuu acgutggcgc gguaggcgcg tgcguucguc gttttcgtga 60 guucgaguag 70 <210> 34 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 34 gacgcggagg ttggcgcggt a 21 <210> 35 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 35 gacgcggagg acgaacgcac g 21 <210> 36 <211> 28 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 36 ctcgggctca cgaaaacggc gggcgcac 28 <210> 37 <211> 77 <212> DNA <213> Homo sapiens <400> 37 tctctgtgta ctaatttccc tttttggccg gacgtggtgg ctcacgcctg taatcccagc 60 actttgggag gccaaag 77 <210> 38 <211> 77 <212> DNA <213> Homo sapiens <400> 38 tttttgtgta ttaatttttt tttttggtcg gacgtggtgg tttacgtttg taattttagt 60 attttgggag gttaaag 77 <210> 39 <211> 12 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 39 acgtggtggt tt 12 <210> 40 <211> 28 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 40 tgtgtattaa tttttttttt tggtcgga 28 <210> 41 <211> 28 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 41 cctcccaaaa tactaaaatt acaaacgt 28 <210> 42 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 42 gacgcggaga cgtggtggtt t 21 <210> 43 <211> 35 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 43 gtgctgggat tacaggcgtg agccaccacg tccgg 35 <210> 44 <211> 77 <212> DNA <213> Homo sapiens <400> 44 ccatctatag aaaaatggat tagggccggg cacagtggct cacgcctgta atcccagcac 60 tttgggaggc cgaggca 77 <210> 45 <211> 77 <212> DNA <213> Homo sapiens <400> 45 ttatttatag aaaaatggat tagggtcggg tatagtggtt tacgtttgta attttagtat 60 tttgggaggt cgaggta 77 <210> 46 <211> 12 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 46 aacgtaaacc ac 12 <210> 47 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 47 gaaaaatgga ttagggtcgg gt 22 <210> 48 <211> 28 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 48 tcgacctccc aaaatactaa aattacaa 28 <210> 49 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 49 gacgcggaga acgtaaacca c 21 <210> 50 <211> 36 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 50 cggcctccca aagtgctggg attacaggcg tgagcc 36 <210> 51 <211> 29 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 51 agccggtttt ccggctgaga ctccgcgtc 29 <210> 52 <211> 27 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 52 tttgtttttt tgattaggtg tttaaga 27 <210> 53 <211> 0 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 53 000 <210> 54 <211> 0 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 54 000 <210> 55 <211> 0 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 55 000 <210> 56 <211> 0 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 56 000 <210> 57 <211> 0 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 57 000 <210> 58 <211> 0 <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 58 000 <210> 59 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> DNA primers / probes <400> 59 caccaacctc ataaccttat c 21 <210> 60 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> DNA Primer / Probe <400> 60 ccacggacga tagtgttgtg g 21 <210> 61 <211> 166 <212> DNA <213> Homo sapiens <400> 61 ctctgcaggt tctatttgct ttttcccaga tgagctcttt ttctggtgtt tgtctctctg 60 actaggtgtc taagacagtg ttgtgggtgt aggtactaac actggctcgt gtgacaaggc 120 catgaggctg gtgtaaagcg gccttggagt gtgtattaag taggtg 166 <210> 62 <211> 166 <212> DNA <213> Homo sapiens <400> 62 ttttgtaggt tttatttgtt tttttttaga tgagtttttt ttttggtgtt tgtttttttg 60 attaggtgtt taagatagtg ttgtgggtgt aggtattaat attggtttgt gtgataaggt 120 tatgaggttg gtgtaaagtg gttttggagt gtgtattaag taggtg 166

Claims

1. A method for characterizing blood or blood products, comprising: a) Blood or blood product samples provided by the subject; b) Analyze the sample to detect the presence of tissue-cell-specific DNA; The presence of tissue-cell-specific DNA indicates the presence of tissue cells in the blood or blood product sample. The tissue-cell specific DNA contained in the method comprises methylated ZDHHC1 DNA. i) Treat DNA from the blood or blood product sample with a methylation-specific reagent to produce modified methylated ZDHHC1 DNA; and ii) Amplify the region of the modified methylated ZDHHC1 DNA using primer pairs complementary to the region of the modified methylated ZDHHC1 DNA; The amplification of the modified methylated ZDHHC1 DNA region indicates the presence of tissue-cell-specific DNA in the blood or blood product sample.

2. The method of claim 1, wherein the primer pair is complementary to the modified methylated ZDHHC1 DNA in the region corresponding to SEQ ID NO:26 and its complementary sequence.

3. The method of claim 1, wherein the methylation-specific reagent modifies the methylated cytosine nucleotide to produce different nucleotides.

4. The method of claim 1, wherein the methylation-specific reagent modifies unmethylated cytosine nucleotides to produce different nucleotides.

5. The method of claim 2, wherein the methylation-specific reagent is a bisulfite reagent, and wherein the modified methylated ZDHHC1 DNA in the ZDHHC1 DNA region corresponding to SEQ ID NO:26 and its complementary sequence includes the region of SEQ ID NO:33 or the region of SEQ ID NO:

27.

6. The method of claim 5, wherein the primer pair is selected from primers having sequences selected from SEQ ID NO: 29, 30 and 32.

7. The method of claim 1, wherein the blood product is plasma.

8. The method of claim 1, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, or DNA target capture.

9. The method of claim 1, wherein the assay comprises a valve endonuclease assay.

10. A method for quantitative nucleic acid detection, comprising: a) Determine the amount of at least one marker gene in a sample from a subject, wherein the sample is selected from fecal and tissue samples; b) Determine the amount of methylated ZDHHC1 DNA in the sample; The determination of the sample includes: i) Treat the DNA from the sample with a methylation-specific reagent to produce at least one modified marker gene and modified methylated ZDHHC1 DNA; ii) Amplifying the region of the modified marker gene; and iii) Amplify the region of the modified methylated ZDHHC1 DNA using primer pairs complementary to the region of the modified methylated ZDHHC1 DNA; as well as c) Compare the amount of the at least one marker gene in the sample with the amount of the methylated ZDHHC1 DNA to determine the amount of the at least one marker gene in the sample relative to the amount of the methylated ZDHHC1 DNA.

11. The method of claim 10, wherein the primer pair is complementary to the modified ZDHHC1 DNA in the region corresponding to SEQ ID NO:26 and its complementary sequence.

12. The method of claim 10, wherein the methylation-specific reagent modifies the methylated cytosine nucleotide to produce different nucleotides.

13. The method of claim 10, wherein the methylation-specific reagent modifies unmethylated cytosine nucleotides to produce different nucleotides.

14. The method of claim 11, wherein the methylation-specific reagent is a bisulfite reagent, and wherein the modified methylated ZDHHC1 DNA within the ZDHHC1 DNA region corresponding to SEQ ID NO:26 and its complementary sequence comprises the region of SEQ ID NO:33 or the region of SEQ ID NO:

27.

15. The method of claim 10, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation and / or target capture.

16. The method of claim 10, wherein the determination of the amount of the marker gene and the amount of the methylated ZDHHC1 DNA in the sample is performed in a single reaction.

17. The method of claim 10, wherein the assay comprises a valve endonuclease assay.

18. The method of claim 10, wherein the amount of the modified marker gene relative to the amount of modified methylated ZDHHC1 DNA indicates the methylation state of the marker gene, wherein the methylation state includes an increase or decrease in methylation of the marker gene relative to the normal methylation state of the marker gene.

19. Use of the reagent in the preparation of a kit for a method of quantitative nucleic acid detection, said method comprising: a) Determine the amount of at least one marker gene in a sample from a subject, wherein the sample is selected from fecal and tissue samples; b) Determine the amount of methylated ZDHHC1 DNA in the sample; The determination of the sample includes: i) Treat the DNA from the sample with a methylation-specific reagent to produce at least one modified marker gene and modified methylated ZDHHC1 DNA; ii) Amplify the region of the modified marker gene; and iii) Amplify the region of the modified methylated ZDHHC1 DNA using primer pairs complementary to the region of the modified methylated ZDHHC1 DNA; and c) comparing the amount of the at least one marker gene in the sample with the amount of the methylated ZDHHC1 DNA to determine the amount of the at least one marker gene in the sample relative to the amount of the methylated ZDHHC1 DNA; wherein the reagent comprises a primer pair complementary to the modified methylated ZDHHC1 DNA in the region corresponding to SEQ ID NO: 26 and its complementary sequence.

20. The use as described in claim 19, wherein the assay comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation and / or target capture.

21. The use as described in claim 19, wherein the determination of the marker gene and the determination of the ZDHHC1 DNA are performed in a single reaction.

22. The use as claimed in claim 19, wherein the assay comprises a valve endonuclease assay.

23. The use as claimed in claim 19, wherein the amount of the modified marker gene relative to the amount of modified methylated ZDHHC1 DNA indicates the methylation state of the marker gene, wherein the methylation state includes an increase or decrease in methylation of the marker gene relative to the normal methylation state of the marker gene.

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