Detection of colonoma formation by analysis of methylated DNA

Abstract

Provided herein are techniques for neoplasia screening and particularly, but not exclusively, to methods, compositions and related uses for detecting the presence of cancer, in particular colorectal cancer.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of January 26, 2018, the priority date of January 27, 2017, the application number 201880012946.6, and the invention name “Detection of colon neoplasia by analyzing methylated DNA”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 451,327 filed on January 27, 2017 and U.S. Provisional Patent Application No. 62 / 622,107 filed on January 25, 2018, each of which is incorporated by reference in its entirety. Technical Field

[0004] Provided herein are techniques related to detecting neoplasia, and particularly, but not exclusively, to methods, compositions, and related uses for detecting tumors such as colon cancer. Background Art

[0005] Colorectal cancer remains the second most common cancer in men and women combined in the United States (Siegel R et al., CA Cancer J Clin 2013; 63: 11-30). The underlying biological characteristics of progression from pre-lesions to cancer favor screening (Vogelstein B et al., Science 2013; 339: 1546-58). There is evidence support and guidelines support any of several tests and strategies (Levin B et al., Gastroenterology 2008; 134: 1570-95; Rex DK et al., Am J Gastroenterol 2009; 104: 739-50; Karl J et al., Clin Gastroenterol Hepatol 2008; 6: 1122-8). From a societal perspective, screening is considered cost-effective (Karl J et al., Clin Gastroenterol Hepatol 2008;6:1122-8; Heitman SJ et al., PLoS Med 2010;7:e1000370; Parekh M et al., Aliment Pharmacol Ther 2008;27:697-712; Sharaf RN et al., Am J Gastroenterol 2013;108:120-32).

[0006] Colorectal cancer arises from cumulative genetic and epigenetic changes, providing a basis for analyzing tumor-specific changes in stool (Berger BM et al., Pathology 2012; 44: 80-8). Previous large-scale studies of early generation stool-based DNA tests in a screening setting have shown only reasonable sensitivity for colorectal cancer and low sensitivity for advanced adenomas (Ahlquist DA et al., Ann Intern Med 2008; 149: 441-50, W81; Imperiale TF et al., NEngl J Med 2004; 351: 2704-14). Important advances have been made since then, including stabilizing buffers (Boynton KA et al., Clin Chem 2003;49:1058-65; Zou H et al., Cancer Epidemiol Biomarkers Prev 2006;15:1115-9), more discriminatory markers (Ahlquist DA et al., Gastroenterology 2012;142:248-56; Bardan E et al., Israel journal of medical sciences 1997;33:777-80), platforms with higher analytical sensitivity (Ahlquist DA et al., Gastroenterology 2012;142:248-56; Aronchick CA et al., Gastrointestinal endoscopy 2000;52:346-52), use of logistic regression analysis rather than individual marker values ​​to determine results, and automation.

[0007] Although screening has reduced colorectal cancer mortality (Mandel JS et al., N Engl J Med. 1993, 328: 1365-71; Hardcastle JD et al., Lancet. 1996, 348: 1472-7; Kronborg O et al., Scand J Gastroenterol. 2004, 39: 846-51; Winawer SJ et al., J Natl Cancer Inst. 1993, 85: 1311-8; Singh H et al., JAMA. 2006, 295: 2366-73), the observed reduction is modest (Singh H et al., JAMA. 2006; 295, 2366-73; Heresbach D et al., Eur J Gastroenterol Hepatol. 2006, 18: 427-33) and more than half of adults in the United States have not yet been screened (Meissner HI, Cancer Epidemiol Biomarkers 2006). Prev.2006,15:389-94).

[0008] An emerging cancer screening approach involves measuring tumor-specific DNA alterations in body samples from cancer patients, such as stool, serum, and urine (Osborn NK, Ahlquist DA. Gastroenterology 2005;128:192–206; Ahlquist DA et al. Gastroenterology 2000;119:1219–27; Ahlquist DA et al. Gastroenterology 2002;122:Suppl A40; Chen WD et al. J Natl Cancer Inst 2005;97:1124–32; Zou H et al. Cancer Epidemiol Biomarkers Prev 2006;15:1115–9; Zou HZ, Clin Cancer Res 2002;8:188–91; Hoque MO, J Clin Oncol 2005;23:6569–75; Belinsky SA et al. Cancer Res 2006;66:3338–44; Itzkowitz SH et al., Clin Gastroenterol Hepatol 2007;5:111–7' Kann L et al., Clin Chem 2006;52:2299–302). The selection of markers with high precision is important if efficiency and effectiveness are to be achieved in cancer screening applications. Due to the molecular heterogeneity of colorectal neoplasia, a panel of markers is often required for high detection rates.

[0009] Several methylated genes have been detected in stool and serum / plasma samples from colorectal cancer patients (Ahlquist DA, Gastroenterology 2002;122:Suppl A40; Chen WD et al., J Natl Cancer Inst 2005;97:1124–32; Zou HZ et al., Clin Cancer Res 2002;8:188–91; Itzkowitz SH et al., Clin Gastroenterol Hepatol 2007;5:111–7; Petko Z et al., Clin Cancer Res 2005;11:1203–9; Muller HM et al., Lancet 2004;363:1283–5; Leung WK ​​et al., Clin Chem 2004;50:2179–82; Ebert MP et al., Gastroenterology 2006;131:1418–30; Grady WM et al., Cancer Res 2001;61:900–2). Although some methylated genes have been found in most colorectal cancers, the yield of fluid-based assays remains suboptimal (Ahlquist DA et al., Gastroenterology 2002;122:Suppl A40; Chen WD et al., J Natl Cancer Inst 2005;97:1124–32; Zou H et al., Cancer Epidemiol Biomarkers Prev 2006;15:1115–9; Zou HZ, Clin Cancer Res 2002;8:188–91; Belinsky SA et al., Cancer Res 2006;66:3338–44; Itzkowitz SH et al., Clin Gastroenterol Hepatol 2007;5:111–7; Kann L et al., Clin Chem 2006;52:2299–302; Petko Z et al., Clin Cancer Res 2005;11:1203–9; Muller HM et al., Lancet 2004;363:1283–5; Leung WK ​​et al., Clin Chem 2004;50:2179–82; Ebert MP et al., Gastroenterology 2006;131:1418–30; Grady WM et al., Cancer Res 2001;61:900–2).

[0010] More precise, user-friendly, and widely distributable tools are needed to improve screening efficiency, acceptability, and accessibility. Summary of the invention

[0011] Provided herein are technologies related to detecting neoplasia, and particularly, but not exclusively, methods, compositions, and related uses of detecting pre-malignant and malignant colorectal cancer by analyzing blood and / or plasma samples from subjects (e.g., patients). When describing the technology herein, the section headings used are for organizational purposes only and should not be construed as limiting the subject matter in any way.

[0012] Provided herein is a panel of methylated DNA markers measured on tissues that achieve extremely high discrimination for colorectal cancer while remaining negative in normal colorectal tissues. The panel can be applied, for example, to blood- or body fluid-based tests and to colorectal cancer screening.

[0013] Markers and / or marker panels (e.g., chromosomal regions with annotations selected from the following: ANKRD13B; CHST2; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; CNNM1; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671) are identified in the study by comparing the methylation status of DNA markers from colorectal cancer samples with corresponding markers in normal (non-cancerous) samples.

[0014] As described herein, the technology provides a number of methylated DNA markers and subsets thereof (e.g., a collection of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more markers) with high discrimination for colon cancer. The experiment applies selection filters to candidate markers to identify markers that provide high signal-to-noise ratios and low background levels so as to provide high specificity and selectivity for cancer screening or diagnostic purposes. For example, as described below, a combination of 12 markers and carcinoembryonic antigen (CEA) protein produced a sensitivity of 67.4% (60 / 89 cancers) for all cancer plasma samples tested, with a specificity of 92.6%.

[0015] Thus, provided herein are techniques related to methods for screening for colon cancer in a sample obtained from a subject, the method comprising determining the amount of methylated marker DNA, e.g., to assess the methylation state of a marker in a sample obtained from a subject; when the methylation state of the marker is different from the methylation state of the marker determined in a subject without a tumor, the subject is identified as having colon cancer. In some embodiments, the marker comprises a chromosomal region having an annotation selected from the following: ANKRD13B; CHST2; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; CNNM1; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671. In some embodiments, the technique comprises measuring a plurality of markers, for example comprising measuring 2 to 20, preferably 2 to 14, more preferably 2 to 12 markers. For example, in some embodiments, the method comprises analyzing the methylation status of two or more markers selected from the following: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. In a preferred embodiment, the determination comprises detecting CEA protein.

[0016] The technology is not limited to assessing methylation status. In some embodiments, assessing the methylation status of a marker in a sample includes determining the methylation status of a base. In some embodiments, determining the methylation status of a marker in a sample includes determining the degree of methylation at multiple bases. Moreover, in some embodiments, the methylation status of a marker includes an increase in methylation of the marker relative to the normal methylation status of the marker, i.e., relative to the methylation status of the marker in DNA from a subject without neoplasia. In some embodiments, the methylation status of a marker includes a decrease in methylation of the marker relative to the normal methylation status of the marker. In some embodiments, the methylation status of a marker includes a different methylation pattern of the marker relative to the normal methylation status of the marker.

[0017] In some embodiments, the technology provides a method of generating a record reporting a colon tumor in a sample obtained from a subject, comprising the steps of:

[0018] a) determining a sample from a subject to obtain the amount of at least one methylation marker gene selected from the group consisting of ANKRD13B; CHST2; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; CNNM1; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671 in the sample obtained from the subject;

[0019] b) assaying the sample to obtain the amount of a reference marker in the sample;

[0020] c) comparing the amount of the at least one methylated marker gene with the amount of a reference marker (preferably a methylated reference marker) in the sample to determine the methylation state of the at least one marker gene in the sample; and

[0021] d) generating a record reporting the methylation status of the at least one marker gene in the sample.

[0022] The record of the methylation state of the reporting marker is not limited to any particular form of report, and may include, for example, an update to an electronic medical record, a printed report, or an electronic message. In some embodiments, laboratory data generated during the assay are included in the report, while in some embodiments, only a summary of the data or diagnostic results based on the determined methylation state of the at least one marker gene is included in the record.

[0023] In some embodiments, the sample is assayed for at least two markers, and preferably, the at least one methylation marker gene is selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. In a more preferred embodiment, the sample is assayed for a panel of markers comprising: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. In a preferred embodiment, the presence of CEA protein in a sample from a subject is assayed.

[0024] In some embodiments, the method for determining comprises obtaining a sample comprising DNA from a subject, and treating the DNA obtained from the sample with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues. In a preferred embodiment, the reagent comprises a bisulfite reagent.

[0025] The method is not limited to analyzing a methylation-marked region of a particular size, or analyzing the methylation status of the nucleotides of the number. In some embodiments, determining the methylation state of the marker DNA in the sample includes determining the methylation state of one base, while in other embodiments, the determination includes determining the degree of methylation at multiple bases. In some embodiments, the methylation state of the marker includes an increase or decrease in the methylation of the marker relative to the normal methylation state of the marker, while in some embodiments, the methylation state of the marker includes a different methylation pattern relative to the normal methylation state of the marker, for example, a different subset of methylated nucleotides in the methylated region of the marker.

[0026] The technology is not limited to a specific sample type. For example, in some embodiments, the sample is a tissue sample, a blood sample, a serum sample, or a sputum sample. In certain embodiments, the tissue sample comprises colon tissue.

[0027] The techniques are not limited to any particular method for assaying DNA samples. For example, 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 target capture. In certain preferred embodiments, the assay includes the use of flap endonuclease assays. In particularly preferred embodiments, the sample DNA and / or reference marker DNA are bisulfite converted, and the assay for determining the level of DNA methylation is achieved by the following techniques, including the use of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrophosphate sequencing, PCR-flap assay, flap endonuclease assay and / or bisulfite genomic sequencing PCR.

[0028] In some embodiments, the oligonucleotides in the mixture comprise a reporter molecule, and in preferred embodiments, the reporter molecule comprises a fluorophore. In some embodiments, the oligonucleotides comprise a flap sequence. In some embodiments, the mixture further comprises one or more of a FRET box, a FEN-1 endonuclease, and a thermostable DNA polymerase (preferably a bacterial DNA polymerase).

[0029] In some embodiments, the techniques used include detecting multiple markers and / or multiple regions of a single marker using an assay that reports the detection of multiple markers and / or multiple regions of a single marker to a single signal output, such as a single fluorescent dye. For example, in some embodiments, the assay is configured to report the cleavage of a flap endonuclease probe specific for multiple different target sites through a single FRET box.

[0030] Then, in some embodiments, the assay of the sample comprises preparing a reaction mixture comprising an amplification reagent for amplifying at least two methylation-marked DNAs, and a flap cleavage reagent for performing a flap endonuclease assay on the amplified marked DNAs, wherein the reagent comprises:

[0031] i) a first primer pair for generating a first amplified region of methylation marker DNA;

[0032] ii) a first probe comprising a) a sequence complementary to at least a portion of the first amplified region of the methylated marker DNA; and b) a flap portion having a first flap sequence that is substantially non-complementary to the first amplified region of the methylated marker DNA;

[0033] iii) a second primer pair for generating a second amplified region of methylation marker DNA;

[0034] iv) a second probe comprising a) a sequence complementary to at least a portion of the second region of the methylation marker DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is not substantially complementary to the second amplified region of the methylation marker DNA;

[0035] v) DNA polymerase; and

[0036] vi) Flap endonuclease.

[0037] In some embodiments, the first amplified region of the methylation marker DNA and the second amplified region of the methylation marker DNA are amplified from different regions of the same methylation marker gene, while in other embodiments, the first amplified region of the methylation marker DNA and the second amplified region of the methylation marker DNA are amplified from different methylation marker genes. In some preferred embodiments, amplifying the at least two methylation marker DNAs includes amplifying at least two methylation marker DNAs selected from the group consisting of: ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671.

[0038] In a preferred embodiment, amplifying the at least two methylation marker DNAs includes amplifying at least three methylation marker DNAs. In such embodiments, the reagents may preferably include a third primer pair for generating a third amplified region of the methylation marker DNA; and a third probe comprising a) a sequence complementary to at least a portion of the third amplified region of the methylation marker DNA; and b) a flap portion having the same first flap sequence, wherein the first flap sequence is substantially non-complementary to the third amplified region of the methylated DNA.

[0039] In some embodiments, a reference nucleic acid is also determined. In such embodiments, the reagents may also include a reference primer pair for generating an amplified region of the reference nucleic acid, and a reference probe comprising a) a sequence complementary to at least a portion of the amplified region of the reference nucleic acid; and b) a flap portion having a second flap sequence, wherein the second flap sequence is not substantially complementary to the amplified region of the reference nucleic acid or to the first FRET box; and a second FRET box comprising a sequence complementary to the second flap sequence.

[0040] The technology for detecting multiple nucleic acid sequences (e.g., multiple markers and / or multiple regions of a single marker) using an assay that reports the detection of multiple markers and / or multiple regions of a single marker to a single signal output (e.g., a single fluorescent dye) is not limited to analyzing the methylation of the sample types or markers discussed above, or detecting or determining the sample types or markers discussed above. For example, in some embodiments, the technology provides a method for characterizing any sample (e.g., from a subject), comprising detecting at least one target nucleic acid in a sample, wherein the detecting the at least one target nucleic acid in the sample comprises preparing a reaction mixture, the reaction mixture comprising an amplification reagent for producing at least two different amplified DNAs, and a flap cleavage reagent for performing a flap endonuclease assay on the at least two different amplified DNAs, wherein the reagent comprises:

[0041] i) a first primer pair for generating a first amplified DNA;

[0042] ii) a first probe comprising a) a sequence complementary to a region of the first amplified DNA; and b) a flap portion having a first flap sequence, the first flap sequence being substantially non-complementary to the first amplified DNA;

[0043] iii) a second primer pair for generating a second amplified DNA;

[0044] iv) a second probe comprising a) a sequence complementary to a region of the second amplified DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is not substantially complementary to the second amplified DNA;

[0045] v) a FRET box comprising a sequence complementary to the first flap sequence;

[0046] vi) DNA polymerase; and

[0047] vii) Flap endonuclease.

[0048] In some embodiments, the at least two different target DNAs may include at least two different marker genes or marker regions in the sample, and in some embodiments, the at least two different target DNAs include at least two different regions of a single marker gene in the sample. The nucleic acid that can be analyzed using the method disclosed herein is not limited to any particular type of nucleic acid, and can include any nucleic acid that can be used as a target for in vitro amplification (e.g., amplified by PCR). In some embodiments, one or more of the at least one target nucleic acid in the sample is RNA. As discussed above, the method is not limited to analyzing two markers or regions, but can be applied to, for example, three, four, five, six, seven (etc.) target sequences reported to the same FRET box. Further, it is possible to combine assays so that a variety of different target nucleic acids in the assay are reported to the first FRET box, a variety of different targets in the same assay are reported to the second FRET box, a variety of different targets in the same assay are reported to the third FRET box, and the like.

[0049] The technology also provides a kit. For example, in some embodiments, the kit includes a first primer pair for generating a first amplified DNA; a first probe comprising a) a sequence complementary to a region of the first amplified DNA; and b) a flap portion having a first flap sequence, wherein the first flap sequence is substantially non-complementary to the first amplified DNA; a second primer pair for generating a second amplified DNA; a second probe comprising a) a sequence complementary to a region of the second amplified DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is substantially non-complementary to the second amplified DNA; a FRET box comprising a sequence complementary to the first flap sequence; a DNA polymerase; and a flap endonuclease.

[0050] In certain preferred embodiments, the technology provides a kit comprising a) at least one oligonucleotide, wherein at least a portion of the oligonucleotide specifically hybridizes to a marker selected from the group consisting of: ANKRD13B; CHST2; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; CNNM1; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671, and b) at least one additional oligonucleotide, wherein at least a portion of the additional oligonucleotide specifically hybridizes to a reference nucleic acid. In preferred embodiments, the kit comprises an assay for detecting CEA protein. In some embodiments, the kit comprises at least two additional oligonucleotides, and in some embodiments, the kit further comprises a bisulfite reagent.

[0051] In certain embodiments, at least a portion of the oligonucleotides specifically hybridizes to at least one marker selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. In a preferred embodiment, the kit comprises at least 12 oligonucleotides, wherein each of the markers of the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

[0052] In a preferred embodiment, the one or more oligonucleotides provided in the kit are selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a nucleic acid probe, and an invasive oligonucleotide.

[0053] In some embodiments, any of the above kits further comprises a solid support, such as magnetic beads or particles. In a preferred embodiment, the solid support comprises one or more capture reagents, such as oligonucleotides complementary to the one or more marker genes.

[0054] The technology also provides compositions. For example, in some embodiments, the technology provides a composition comprising a mixture (e.g., a reaction mixture) comprising a first primer pair for producing a first amplified DNA; a first probe comprising a) a sequence complementary to a region of the first amplified DNA; and b) a flap portion having a first flap sequence, wherein the first flap sequence is substantially non-complementary to the first amplified DNA; a second primer pair for producing a second amplified DNA; a second probe comprising a) a sequence complementary to a region of the second amplified DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is substantially non-complementary to the second amplified DNA; a FRET box comprising a sequence complementary to the first flap sequence; a DNA polymerase; and a flap endonuclease. In a preferred embodiment, the composition further comprises a first amplified DNA and a second amplified DNA, wherein the first probe is substantially non-complementary to the second amplified DNA, and wherein the second probe is substantially non-complementary to the first amplified DNA. In some embodiments, the composition comprises a primer or probe complexed with DNA.

[0055] In some embodiments, the composition comprises a target nucleic acid selected from the group consisting of ANKRD13B; CHST2; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; CNNM1; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671, and a complex of an oligonucleotide that specifically hybridizes to the target nucleic acid. In a preferred embodiment, the mixture comprises a target nucleic acid selected from the group consisting of VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI, and a complex of an oligonucleotide that specifically hybridizes to the target nucleic acid. The oligonucleotides in the mixture include, but are not limited to, one or more of a capture oligonucleotide, a pair of nucleic acid primers, a hybridization probe, a hydrolysis probe, a flap assay probe, and an invasive oligonucleotide.

[0056] In some embodiments, the target nucleic acid in the mixture comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131 and 136.

[0057] In some embodiments, the mixture comprises a bisulfite-converted target nucleic acid comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, 132, and 137.

[0058] The present application further relates to the following embodiments:

[0059] 1. A method of characterizing a sample from a subject, comprising:

[0060] a) providing a sample from a subject;

[0061] b) determining the amount of at least one methylation marker DNA in the sample, wherein the methylation marker DNA is selected from the group of methylation marker genes consisting of: ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671;

[0062] b) assaying the sample to obtain the amount of a reference nucleic acid in the sample.

[0063] 2. The method according to embodiment 1, wherein the sample from the subject comprises a blood or plasma sample.

[0064] 3. A method according to embodiment 1 or embodiment 2, wherein the assay comprises obtaining a sample containing DNA from a subject and treating the DNA obtained from the sample with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues.

[0065] 4. The method according to embodiment 1, wherein the at least one methylation marker DNA is selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

[0066] 5. The method according to embodiment 1, wherein the at least one methylation-marked DNA comprises at least two methylation-marked DNAs.

[0067] 6. The method according to embodiment 1, wherein the at least one methylation marker DNA comprises the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

[0068] 7. The method according to embodiment 1, wherein the method further comprises determining the carcinoembryonic antigen (CEA) protein in the sample.

[0069] 8. A method according to embodiment 3, wherein the reagent comprises a bisulfite reagent.

[0070] 9. The method according to embodiment 1, wherein determining the amount of methylation marker DNA in the sample comprises determining the methylation status of a base in the methylation marker gene.

[0071] 10. The method according to embodiment 1, wherein determining the amount of the methylation marker in the sample comprises determining the degree of methylation at multiple bases in the methylation marker gene.

[0072] 11. A method according to any one of embodiment 1, wherein the amount of at least one methylation marker DNA indicates the methylation state of a methylation marker gene, wherein the methylation state of the methylation marker gene includes an increase or decrease in the amount of the methylation marker gene relative to the amount of the methylation marker gene in a normal sample.

[0073] 12. A method according to embodiment 1, wherein the amount of at least one methylated marker DNA indicates the methylation state of a methylated marker gene, wherein the methylation state includes that the methylation pattern of the marker gene is different from the methylation pattern of the methylated marker gene in a normal sample.

[0074] 13. A method according to embodiment 1, wherein the reference nucleic acid is a methylated reference DNA.

[0075] 14. A method according to embodiment 1, wherein the sample is a tissue sample or a plasma sample.

[0076] 15. The method of embodiment 14, wherein the sample is a tissue sample comprising colon tissue.

[0077] 16. A method according to embodiment 1, wherein the determination includes the use of polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation and / or target capture.

[0078] 17. A method according to embodiment 16, wherein the assay comprises using a flap endonuclease assay.

[0079] 18. A method according to embodiment 16, wherein the determination includes multiple amplification.

[0080] 19. A method according to embodiment 1, wherein the determination comprises converting the at least one methylation-tagged DNA and the reference nucleic acid with bisulfite.

[0081] 20. A method according to embodiment 1 or embodiment 19, wherein determining the amount of at least one methylation marker DNA comprises using one or more methods selected from the group consisting of: methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrophosphate sequencing, flap endonuclease assay, PCR-flap assay and bisulfite genomic sequencing PCR.

[0082] 21. A kit comprising:

[0083] a) at least one oligonucleotide, wherein at least a portion of the oligonucleotide specifically hybridizes to a marker selected from the group consisting of: ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671, and

[0084] b) at least one additional oligonucleotide, wherein at least a portion of the additional oligonucleotide specifically hybridizes to the reference nucleic acid.

[0085] 22. The kit according to embodiment 21, wherein the kit comprises at least two additional oligonucleotides.

[0086] 23. The kit of claim 21, wherein the kit further comprises bisulfite.

[0087] 24. The kit according to embodiment 21, wherein the kit further comprises a reagent for detecting CEA protein.

[0088] 25. A kit according to embodiment 21, wherein at least a portion of the oligonucleotide specifically hybridizes to at least one marker selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

[0089] 26. A kit according to embodiment 21, wherein the kit comprises at least 12 oligonucleotides, wherein each marker in the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI specifically hybridizes to at least one of the 12 oligonucleotides.

[0090] 27. The kit of embodiment 21, wherein the at least one 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.

[0091] 28. The kit of embodiment 27, wherein the kit further comprises a solid support.

[0092] 29. The kit of embodiment 28, wherein the solid support is magnetic beads.

[0093] 30. The kit of embodiment 29, wherein the solid support comprises one or more capture reagents.

[0094] 31. A kit according to embodiment 30, wherein the capture reagent is an oligonucleotide complementary to the one or more marker genes.

[0095] 32. A composition comprising a reaction mixture comprising a target nucleic acid selected from the group consisting of ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671, and a complex of an oligonucleotide that specifically hybridizes to the target nucleic acid.

[0096] 33. A composition according to embodiment 32, wherein the reaction mixture comprises a target nucleic acid selected from the group consisting of VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI and a complex of an oligonucleotide that specifically hybridizes to the target nucleic acid.

[0097] 34. The composition of embodiment 32, wherein the oligonucleotide is selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a hybridization probe, a hydrolysis probe, a flap assay probe, and an invasive oligonucleotide.

[0098] 35. A composition according to embodiment 32, wherein the target nucleic acid comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131 and 136.

[0099] 36. A composition according to embodiment 32, wherein the target nucleic acid is a bisulfite-converted target nucleic acid.

[0100] 37. A composition according to embodiment 36, wherein the bisulfite-converted target nucleic acid comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, 132 and 137.

[0101] 38. A composition according to any of embodiment 32, wherein the oligonucleotide comprises a reporter molecule.

[0102] 39. A composition according to embodiment 38, wherein the reporter molecule comprises a fluorophore.

[0103] 40. The composition of any one of embodiments 32, wherein the oligonucleotide comprises a flap sequence.

[0104] 41. The composition of embodiment 32, further comprising one or more of a FRET box, a FEN-1 endonuclease, and / or a thermostable DNA polymerase.

[0105] 42. A composition according to embodiment 41, wherein the thermostable DNA polymerase is a bacterial DNA polymerase.

[0106] 43. The method of embodiment 1, wherein the assay comprises preparing a reaction mixture comprising an amplification reagent for amplifying at least two methylation-marked DNAs, and a flap-cleavage reagent for performing a flap endonuclease assay on the amplified marked DNAs, wherein the reagent comprises:

[0107] a first primer pair for generating a first amplified region of methylation-marked DNA;

[0108] a first probe comprising a) a sequence complementary to at least a portion of the first amplified region of the methylated marker DNA; and b) a flap portion having a first flap sequence that is substantially non-complementary to the first amplified region of the methylated marker DNA;

[0109] a second primer pair for generating a second amplified region of methylation-marked DNA;

[0110] a second probe comprising a) a sequence complementary to at least a portion of the second region of the methylation marker DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is not substantially complementary to the second amplified region of the methylation marker DNA;

[0111] a first FRET box comprising a sequence complementary to the first flap sequence;

[0112] DNA polymerase; and

[0113] Flap endonuclease.

[0114] 44. A method according to embodiment 43, wherein the first amplified region of methylation marker DNA and the second amplified region of methylation marker DNA are amplified from different regions of the same methylation marker gene.

[0115] 45. A method according to embodiment 43, wherein the first amplified region of methylation marker DNA and the second amplified region of methylation marker DNA are amplified from different methylation marker genes.

[0116] 46. ​​A method according to embodiment 43, wherein amplifying the at least two methylation marker DNAs includes amplifying regions of at least two methylation marker genes selected from the group consisting of: ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671.

[0117] 47. A method according to embodiment 43, wherein amplifying the at least two methylation-marked DNAs includes amplifying at least three methylation-marked DNAs.

[0118] 48. The method of embodiment 47, wherein the reagent further comprises:

[0119] a third primer pair for generating a third amplified region of methylation-marked DNA; and

[0120] A third probe comprising a) a sequence complementary to at least a portion of the third amplified region of the methylated marker DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is substantially non-complementary to the third amplified region of the methylated DNA.

[0121] 49. The method according to embodiment 47, wherein the reagent further comprises:

[0122] a reference primer pair for generating an amplified region of a reference nucleic acid; and

[0123] a reference probe comprising a) a sequence complementary to at least a portion of the amplified region of the reference nucleic acid; and b) a flap portion having a second flap sequence, wherein the second flap sequence is not substantially complementary to the amplified region of the reference nucleic acid or to the first FRET box; and

[0124] A second FRET cassette comprises a sequence complementary to the second flap sequence.

[0125] 50. A method of characterizing a sample from a subject, comprising detecting at least one target nucleic acid in the sample, wherein the detecting the at least one target nucleic acid in the sample comprises preparing a reaction mixture comprising amplification reagents for producing at least two different amplified DNAs, and flap cleavage reagents for performing a flap endonuclease assay on the at least two different amplified DNAs, wherein the reagents comprise:

[0126] a first primer pair for generating a first amplified DNA;

[0127] a first probe comprising a) a sequence complementary to a region of the first amplified DNA; and b) a flap portion having a first flap sequence that is substantially non-complementary to the first amplified DNA;

[0128] a second primer pair for producing a second amplified DNA;

[0129] a second probe comprising a) a sequence complementary to a region of the second amplified DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is not substantially complementary to the second amplified DNA;

[0130] a FRET box comprising a sequence complementary to the first flap sequence;

[0131] DNA polymerase; and

[0132] Flap endonuclease.

[0133] 51. A method according to embodiment 50, wherein the at least two different target DNAs comprise at least two different marker genes or marker regions in the sample.

[0134] 52. A method according to embodiment 50 or embodiment 51, wherein the at least two different target DNAs comprise at least two different regions of a single marker gene in the sample.

[0135] 53. A method according to embodiment 50, wherein one or more of the at least one target nucleic acid in the sample is RNA.

[0136] 54. A kit comprising:

[0137] a first primer pair for generating a first amplified DNA;

[0138] a first probe comprising a) a sequence complementary to a region of the first amplified DNA; and b) a flap portion having a first flap sequence that is substantially non-complementary to the first amplified DNA;

[0139] a second primer pair for producing a second amplified DNA;

[0140] a second probe comprising a) a sequence complementary to a region of the second amplified DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is not substantially complementary to the second amplified DNA;

[0141] a FRET box comprising a sequence complementary to the first flap sequence;

[0142] DNA polymerase; and

[0143] Flap endonuclease.

[0144] 55. The kit of claim 54, wherein the kit further comprises bisulfite.

[0145] 56. A method of screening for colon tumors in a sample obtained from a subject, the method comprising:

[0146] a) determining the amount of at least one methylation marker DNA in a sample from a subject, the methylation marker DNA being selected from the group of methylation marker genes consisting of: ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671;

[0147] b) assaying the sample to obtain the amount of a reference nucleic acid in the sample;

[0148] c) comparing the amount of the at least one methylated marker DNA with the amount of a reference marker nucleic acid in the sample to determine the methylation status of the at least one marker DNA in the sample.

[0149] 57. A method according to embodiment 56, wherein the sample from the subject comprises a blood or plasma sample.

[0150] 58. A method according to embodiment 56 or embodiment 57, wherein the assay comprises obtaining a sample comprising DNA from a subject and treating the DNA obtained from the sample with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues.

[0151] 59. The method of any one of embodiments 1-3 and 56-58, wherein the at least one methylation marker DNA is selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

[0152] 60. The method according to any one of embodiments 1-4 and 54-57, wherein the at least one methylation marker DNA comprises at least two methylation marker DNAs.

[0153] 61. A method according to embodiment 60, wherein the at least one methylation marker DNA includes the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

[0154] 62. The method of any one of embodiments 1-6 and 56-61, wherein the method further comprises determining carcinoembryonic antigen (CEA) protein in the sample.

[0155] 63. A method according to embodiment 58, wherein the reagent comprises a bisulfite reagent.

[0156] 64. A method according to any one of embodiments 1-8 and 56-63, wherein determining the amount of methylation marker DNA in the sample includes determining the methylation status of a base in the methylation marker gene.

[0157] 65. A method according to any one of embodiments 1-8 and 56-63, wherein determining the amount of methylation marker in the sample includes determining the degree of methylation at multiple bases in the methylation marker gene.

[0158] 66. A method according to any one of embodiments 1-10 and 56-65, wherein the amount of at least one methylation marker DNA indicates the methylation state of a methylation marker gene, wherein the methylation state of the methylation marker gene includes an increase or decrease in the amount of the methylation marker gene relative to the amount of the methylation marker gene in a normal sample.

[0159] 67. A method according to any one of embodiments 1-11 and 56-66, wherein the amount of at least one methylated marker DNA indicates the methylation state of a methylated marker gene, wherein the methylation state includes that the methylation pattern of the marker gene is different relative to the methylation pattern of the methylated marker gene in a normal sample.

[0160] 68. The method of any one of embodiments 1-12 and 56-67, wherein the reference nucleic acid is a methylated reference DNA.

[0161] 69. The method of any one of embodiments 1, 3-13, 56, and 58-68, wherein the sample is a tissue sample.

[0162] 70. The method of embodiment 59, wherein the tissue sample comprises colon tissue.

[0163] 71. A method according to any one of embodiments 1-15 and 56-70, wherein the determining comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture.

[0164] 72. A method according to embodiment 71, wherein the assay comprises using a flap endonuclease assay.

[0165] 73. A method according to embodiment 71, wherein the determination includes multiple amplification.

[0166] 74. A method according to any one of embodiments 1-18 and 56-73, wherein the determining comprises converting the at least one methylation-tagged DNA and the reference nucleic acid with bisulfite.

[0167] 75. A method according to embodiment 74, wherein determining the amount of at least one methylation marker DNA includes using one or more methods selected from the group consisting of: methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrophosphate sequencing, flap endonuclease assay, PCR-flap assay and bisulfite genomic sequencing PCR.

[0168] 76. The kit of any one of embodiments 21-27, wherein the kit further comprises a solid support.

[0169] 77. The kit of embodiment 76, wherein the solid support is magnetic beads.

[0170] 78. The kit of embodiment 77, wherein the solid support comprises one or more capture reagents.

[0171] 79. A kit according to embodiment 78, wherein the capture reagent is an oligonucleotide complementary to the one or more marker genes.

[0172] 80. A composition according to any one of embodiments 32-37, wherein the oligonucleotide comprises a reporter molecule.

[0173] 81. A composition according to embodiment 80, wherein the reporter molecule comprises a fluorophore.

[0174] 82. The composition of any one of embodiments 32-39 and 80-81, wherein the oligonucleotide comprises a flap sequence.

[0175] 83. The composition of any one of embodiments 32-40 and 80-82, further comprising one or more of a FRET box, a FEN-1 endonuclease, and a thermostable DNA polymerase.

[0176] 84. A composition according to embodiment 83, wherein the thermostable DNA polymerase is a bacterial DNA polymerase.

[0177] 85. The method of any one of embodiments 1-20 and 56-75, wherein the assay comprises preparing a reaction mixture comprising an amplification reagent for amplifying at least two methylated marker DNAs, and a flap cleavage reagent for performing a flap endonuclease assay on the amplified marker DNAs, wherein the reagents comprise:

[0178] a first primer pair for generating a first amplified region of methylation-marked DNA;

[0179] a first probe comprising a) a sequence complementary to at least a portion of the first amplified region of the methylated marker DNA; and b) a flap portion having a first flap sequence that is substantially non-complementary to the first amplified region of the methylated marker DNA;

[0180] a second primer pair for generating a second amplified region of methylation-marked DNA;

[0181] a second probe comprising a) a sequence complementary to at least a portion of the second region of the methylation marker DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is not substantially complementary to the second amplified region of the methylation marker DNA;

[0182] a first FRET box comprising a sequence complementary to the first flap sequence;

[0183] DNA polymerase; and

[0184] Flap endonuclease.

[0185] 86. A method according to embodiment 85, wherein the first region of methylation marker DNA and the second region of methylation marker DNA are different regions of the same methylation marker DNA.

[0186] 87. A method according to embodiment 85, wherein the first region of methylation marker DNA and the second region of methylation marker DNA are regions of different methylation marker DNA.

[0187] 88. A method according to any one of embodiments 85-87, wherein amplifying the at least two methylation marker DNAs includes amplifying at least two methylation marker DNAs selected from the group consisting of: ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671.

[0188] 89. A method according to any one of embodiments 85-88, wherein amplifying the at least two methylation-marked DNAs includes amplifying at least three methylation-marked DNAs.

[0189] 90. The method of embodiment 89, wherein the reagent further comprises:

[0190] a third primer pair for amplifying a third region of the methylation marker DNA; and

[0191] A third probe comprising a) a sequence complementary to at least a portion of the third region of the methylated marker DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is substantially non-complementary to the third region of the methylated DNA.

[0192] 91. The method of embodiment 89 or embodiment 90, wherein the reagent further comprises:

[0193] a reference primer pair for amplifying a reference nucleic acid; and

[0194] A reference probe comprising a) a sequence complementary to at least a portion of an amplified reference nucleic acid; and b) a flap portion having a second flap sequence, wherein the second flap sequence is substantially non-complementary to the amplified reference nucleic acid; and

[0195] A second FRET cassette comprises a sequence complementary to the second flap sequence.

[0196] 92. A method according to any one of embodiments 50-52, wherein one or more of the at least one target nucleic acid in the sample is RNA.

[0197] 93. A composition comprising:

[0198] a first primer pair for generating a first amplified DNA;

[0199] a first probe comprising a) a sequence complementary to a region of the first amplified DNA; and b) a flap portion having a first flap sequence that is substantially non-complementary to the first amplified DNA;

[0200] a second primer pair for producing a second amplified DNA;

[0201] a second probe comprising a) a sequence complementary to a region of the second amplified DNA; and b) a flap portion having the first flap sequence, wherein the first flap sequence is not substantially complementary to the second amplified DNA;

[0202] a FRET box comprising a sequence complementary to the first flap sequence;

[0203] DNA polymerase; and

[0204] Flap endonuclease.

[0205] 94. A composition according to embodiment 93, comprising the first amplified DNA and the second amplified DNA, wherein the first probe is substantially non-complementary to the second amplified DNA, and wherein the second probe is substantially non-complementary to the first amplified DNA.

[0206] 95. The composition of embodiment 93 or embodiment 94, further comprising DNA isolated from a sample from a subject.

[0207] 96. A composition according to embodiment 95, wherein the subject is a patient suffering from or suspected of suffering from a tumor.

[0208] 97. A composition according to embodiment 96, wherein the tumor is a colorectal tumor.

[0209] 98. A composition according to any of embodiments 95-97, wherein the sample is selected from the group consisting of a stool sample, a tissue sample, and a blood sample, and a plasma sample.

[0210] definition

[0211] To facilitate understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0212] Throughout the specification and claims, unless the context clearly states otherwise, the following terms adopt the meanings clearly associated herein. As used herein, the phrase "in one embodiment" does not necessarily refer to the same embodiment, but may refer to the same embodiment. In addition, as used herein, the phrase "in another embodiment" does not necessarily refer to a different embodiment, but may refer to a different embodiment. Therefore, as described below, various embodiments of the present invention can be easily combined without departing from the scope or spirit of the present invention.

[0213] In addition, as used herein, unless the context clearly dictates otherwise, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or". Unless the context clearly dictates otherwise, the term "based on" is not exclusive and allows for being based on additional factors that are not described. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural references. The meaning of "in..." includes "in..." and "on...".

[0214] The conjunction "consisting essentially of" as used in the claims of this application limits the scope of the claim to the specified materials or steps of the claimed invention "and those materials or steps which do not materially affect one or more of the basic and novel characteristics of the claimed invention," as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition "consisting essentially of the recited elements" may contain a level of unrecited contaminants such that, although present, the contaminants do not alter the function of the recited composition as compared to the pure composition, i.e., a composition "consisting of the recited components."

[0215] As used herein, "methylation" refers to methylation of cytosine at position C5 or N4 of cytosine, N6 position of adenine, or other types of nucleic acid methylation. The DNA amplified in vitro is usually unmethylated because typical in vitro DNA amplification methods do not retain the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to the amplified DNA that is unmethylated or methylated of the original template, respectively.

[0216] Thus, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, whereas a methyl moiety is not present in recognized typical nucleotide bases. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide while 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at position 5 of its pyrimidine ring; however, for purposes herein, thymine is not considered a methylated nucleotide when present in DNA, as thymine is a typical nucleotide base of DNA.

[0217] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule containing one or more methylated nucleotides.

[0218] As used herein, the "methylation state," "methylation pattern," and "methylation status" of a nucleic acid molecule refers to the presence or absence of one or more methylated nucleotide bases in a 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.

[0219] The methylation state of a particular nucleic acid sequence (e.g., a genetic marker or DNA region as described herein) can indicate the methylation state of each base in the sequence, or can indicate the methylation state of a subset of bases within the sequence (e.g., one or more cytosines), or can indicate information about the methylation density of a region within the sequence, with or without providing precise information about the location within the sequence where methylation occurs.

[0220] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a specific locus in a nucleic acid molecule. For example, when the nucleotide present at the 7th nucleotide in a nucleic acid molecule is 5-methylcytosine, the methylation state of the cytosine at the 7th nucleotide in the nucleic acid molecule is methylated. Similarly, when the nucleotide present at the 7th nucleotide in a nucleic acid molecule is cytosine (rather than 5-methylcytosine), the methylation state of the cytosine at the 7th nucleotide in the nucleic acid molecule is unmethylated.

[0221] The methylation status can optionally be represented or indicated by a "methylation value" (e.g., representing a methylation frequency, score, ratio, percentage, etc.). The methylation value 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 the amplification profile after a bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Thus, the value (e.g., methylation value) represents the methylation status and can be used as a quantitative indicator of the methylation status at multiple copies of a locus. This is particularly useful when it is necessary to compare the methylation status of a sequence in a sample to a threshold or reference value.

[0222] As used herein, "methylation frequency" or "methylation percentage (%)" refers to the number of instances where a molecule or locus is methylated relative to the number of instances where the molecule or locus is not methylated.

[0223] Therefore, methylation state describes the methylation state of nucleic acid (e.g., genomic sequence). In addition, methylation state refers to the characteristics of the nucleic acid segment at the specific genomic locus associated with methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residue in the DNA sequence is methylated, the position of the methylated C residue, the frequency or percentage of methylated C in any specific region of nucleic acid, and the allele difference of methylation caused by the difference of, for example, allele origin. The terms "methylation state", "methylation pattern" and "methylation status" also refer to the relative concentration, absolute concentration or pattern of methylated C or unmethylated C in any specific region of nucleic acid in a biological sample. For example, if one or more cytosine (C) residues in a nucleic acid sequence are methylated, it can be referred to as "hypermethylated" or have "increased methylation", and if one or more cytosine (C) residues in a DNA sequence are not methylated, it can be referred to as "hypomethylated" or have "reduced methylation". Similarly, if one or more cytosine (C) residues in a nucleic acid sequence are methylated compared to another nucleic acid sequence (for example, from different regions or from different individuals, etc.), the sequence is considered to be hypermethylated or has the methylation increased compared to other nucleic acid sequences. Alternatively, if one or more cytosine (C) residues in a DNA sequence are unmethylated compared to another nucleic acid sequence (for example, from different regions or from different individuals, etc.), the sequence is considered to be hypomethylated or has the methylation reduced compared to other nucleic acid sequences. In addition, the term "methylation pattern" as used herein refers to the concentrated sites of methylated and unmethylated nucleotides on the nucleic acid region. When the number of methylated and unmethylated nucleotides is identical or similar in the entire region but the position of methylated and unmethylated nucleotides is different, two nucleic acids can have identical or similar methylation frequencies or methylation percentages but have different methylation patterns. When a sequence is different in the degree of methylation (for example, one has the methylation increased or decreased relative to another), frequency or pattern, the sequence is referred to as "differentially methylated" or has "methylation differences". 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 of nucleic acid methylation in a cancer-negative sample. It can also refer to the difference in level or pattern between patients whose cancer relapsed after surgery and those who did not. For example, differential methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers once the correct cutoff or predictive signature is defined.

[0224] Methylation state frequency can be used to describe individual populations or samples from a single individual. For example, a methylation state frequency of 50% of the nucleotide loci is methylated in 50% of the cases and unmethylated in 50% of the cases. For example, such frequencies can be used to describe the degree of methylation of nucleotide loci or nucleic acid regions in individual populations or nucleic acid sets. Therefore, when the methylation in a first nucleic acid molecule population or library is different from the methylation in a second nucleic acid molecule population or library, the methylation state frequency of the first population or library will be different from the methylation state frequency of the second population or library. Such frequencies can also be used, for example, to describe the degree of methylation of nucleotide loci or nucleic acid regions in a single individual. For example, such frequencies can be used to describe the degree of methylation or unmethylation of a group of cells from a tissue sample in a nucleotide locus or nucleic acid region.

[0225] As used herein, "nucleotide locus" refers to the position of a nucleotide in a nucleic acid molecule. The nucleotide locus of a methylated nucleotide refers to the position of a methylated nucleotide in a nucleic acid molecule.

[0226] Typically, methylation of human DNA occurs on dinucleotide sequences including adjacent guanine and cytosine (also referred to as CpG dinucleotide sequences), where cytosine is located 5' to guanine. Most cytosines within CpG dinucleotides are methylated in the human genome, but some remain unmethylated in specific CpG dinucleotide-rich genomic regions (called CpG islands) (see, e.g., Antequera et al. (1990) Cell 62:503–514).

[0227] As used herein, "CpG island" refers to a G:C-rich region of genomic DNA containing an increased number of CpG dinucleotides relative to total genomic DNA. The length of a CpG island can be at least 100, 200 or more base pairs, wherein the G:C content of the region is at least 50% and the ratio of the CpG observation frequency exceeding the expected frequency is 0.6; in some cases, the length of a CpG island can be at least 500 base pairs, wherein the G:C content of the region is at least 55%) and the ratio of the CpG observation frequency exceeding the expected frequency is 0.65. The CpG observation frequency exceeding the expected frequency can be calculated according to the method provided in Gardiner-Garden et al. (1987) J.Mol.Biol.196:261–281. For example, the observed frequency of CpGs exceeding the expected frequency can be calculated according to the formula R = (A x B) / (C x D), where R is the ratio of the observed frequency of CpGs exceeding 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. The methylation status is usually determined in CpG islands, such as at promoter regions. However, it is understood that other sequences in the human genome are susceptible to DNA methylation, 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).

[0228] As used herein, "methylation-specific reagents" refer to reagents that modify the nucleotides of nucleic acid molecules as the methylation state of the nucleic acid molecules changes, or methylation-specific reagents refer to compounds or compositions or other agents that can change the nucleotide sequence of nucleic acid molecules in a manner that reflects the methylation state of the nucleic acid molecules. Methods for treating nucleic acid molecules with such reagents may include contacting the nucleic acid molecules with the reagents, and if necessary, adding additional steps to achieve the desired changes in the nucleotide sequence. Such methods can be applied in a manner that unmethylated nucleotides (e.g., each unmethylated cytosine) are modified to different nucleotides. For example, in some embodiments, such reagents can deaminize unmethylated cytosine nucleotides to produce deoxyuracil residues. An exemplary reagent is a bisulfite reagent.

[0229] The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, acid sulfite or a combination thereof, which can be used to distinguish methylated and unmethylated CpG dinucleotide sequences as disclosed herein. The treatment methods are known in the art (e.g., PCT / EP2004 / 011715 and WO 2013 / 116375, each of which is incorporated 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-alkylene glycol 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 of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chroman derivative, such as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or trihydroxybenzone acid and its derivatives, such as gallic acid (see: PCT / EP2004 / 011715, which is incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction comprises treatment with ammonium bisulfite, for example, as described in WO 2013 / 116375.

[0230] Alteration of a nucleic acid nucleotide sequence by a methylation-specific agent can also produce a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide.

[0231] 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.

[0232] As used herein, the "sensitivity" of a given marker (or a set of markers used together) refers to the percentage of samples reporting DNA methylation values ​​above a threshold value for distinguishing tumor and non-tumor samples. In some embodiments, a positive is defined as a histologically confirmed neoplasia reporting a DNA methylation value above a threshold value (e.g., a range associated with a disease), while a false negative is defined as a histologically confirmed neoplasia reporting a DNA methylation value below a threshold value (e.g., a range associated with no disease). Therefore, the sensitivity value reflects the probability that the DNA methylation measurement value of a given marker obtained from a known diseased sample is within the range of disease-related measurements. As defined herein, the clinical relevance of the calculated sensitivity value represents an estimate of the probability that a given marker will detect the presence of the condition when applied to a subject with a clinical condition.

[0233] As used herein, the "specificity" of a given marker (or a set of markers used together) refers to the percentage of non-tumor samples that report DNA methylation values ​​below a threshold that distinguishes tumor from non-tumor samples. In some embodiments, a negative is defined as a histologically confirmed non-tumor sample that reports a DNA methylation value below a threshold (e.g., a range associated with no disease), while a false positive is defined as a histologically confirmed non-tumor sample that reports a DNA methylation value above a threshold (e.g., a range associated with a disease). Thus, the specificity value reflects the probability that a DNA methylation measurement of a given marker obtained from a known non-tumor sample is within the range of non-disease-related measurements. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the probability that a given marker will detect the absence of a clinical condition when applied to a patient without the condition.

[0234] As used herein, "selected nucleotide" refers to one of the four commonly occurring nucleotides in nucleic acid molecules (C, G, T and A for DNA and C, G, U and A for RNA), and may include methylated derivatives of commonly occurring nucleotides (e.g., when C is the selected nucleotide, both methylated and unmethylated C are included in the meaning of the selected nucleotide), while a methylated selected nucleotide specifically refers to a commonly methylated nucleotide and an unmethylated selected nucleotide specifically refers to a nucleotide that normally exists in an unmethylated form.

[0235] The term "methylation-specific restriction enzyme" or "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids based on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cuts if the recognition site is not methylated or hemimethylated, if the recognition site is methylated, the cutting will not occur or will be performed with significantly reduced efficiency. In the case of a restriction enzyme that specifically cuts if the recognition site is methylated, if the recognition site is not methylated, the cutting will not occur or will be performed with significantly reduced efficiency. Preferred are methylation-specific restriction enzymes whose recognition sequences contain CG dinucleotides (e.g., recognition sequences such as CGCG or CCCGGG). For some embodiments, it is further preferred that the restriction enzyme does not cut if the cytosine in the dinucleotide is methylated at carbon atom C5.

[0236] The term "primer" refers to an oligonucleotide that can serve as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid template strand (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase, and at a suitable temperature and pH), whether naturally occurring, such as a nucleic acid fragment from a restriction digest, or synthetically produced. Primers are preferably single-stranded to obtain maximum amplification efficiency, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands and then used to prepare an extension product. Preferably, the primer is an oligodeoxynucleotide. The primer must be long enough to initiate the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer depends on many factors, including the use of temperature, primer source, and method.

[0237] The term "probe" refers to an oligonucleotide (e.g., a nucleotide sequence) that can hybridize with another target oligonucleotide, whether naturally occurring (e.g., in a purified restriction digest), or produced by synthesis, recombination, or by PCR amplification. The probe can be single-stranded or double-stranded. The probe can be used to detect, identify, and separate specific gene sequences (e.g., "capture probes"). It is contemplated that in some embodiments, any probe used in the present invention can 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. It is not intended to limit the present invention to any particular detection system or marker.

[0238] As used herein, the term "target" refers to a nucleic acid that is attempted to be sorted from other nucleic acids, e.g., by probe binding, amplification, separation, capture, etc. For example, when used with respect to polymerase chain reaction, "target" refers to the region of nucleic acid to which primers used for polymerase chain reaction bind, while when used in an assay in which target DNA is not amplified, such as in some embodiments of an invasive cleavage assay, the target comprises a site at which the probe and invasive oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasive cleavage structure, such that the presence of the target nucleic acid can be detected. A "segment" is defined as a region of nucleic acid within a target sequence.

[0239] As used herein, the term "marker" refers to a substance (e.g., a nucleic acid, or a nucleic acid region, or a protein) that can be used to distinguish abnormal cells (e.g., cancer cells) from normal cells, for example based on the presence, absence, or condition (e.g., methylation status) of the marker substance.

[0240] As used herein, the term "tumor" refers to any new and abnormal growth of tissue. Thus, a tumor may be a premalignant tumor or a malignant tumor.

[0241] As used herein, the term "tumor-specific marker" refers to any biological material or element that can be used to indicate the presence of a tumor. Examples of biological materials include, but are not limited to, nucleic acids, polypeptides, carbohydrates, fatty acids, cell components (e.g., cell membranes and mitochondria) and whole cells. In some cases, a marker is a specific nucleic acid region (e.g., a gene, an intragenic region, a specific locus, etc.). A nucleic acid region as a marker can be referred to as, for example, a "marker gene," "marker region," "marker sequence," "marker locus," etc.

[0242] The term "sample" is used in its broadest sense. In a sense, it can refer to animal cells or tissues. In another sense, it refers to specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and cover fluids, solids, tissues and gases. Environmental samples include environmental materials, such as surface materials, soil, water and industrial samples. These examples should not be interpreted as limiting the sample types applicable to the present invention.

[0243] As used herein, the term "patient" or "subject" refers to an organism to be subjected to various tests provided by the technology. The term "subject" includes animals, preferably mammals, including humans. In preferred embodiments, the subject is a primate. In even more preferred embodiments, the subject is a human. Further with respect to diagnostic methods, preferred subjects are vertebrate subjects. Preferred vertebrates are warm-blooded animals; preferred warm-blooded vertebrates are mammals. Preferred mammals are most preferably humans. As used herein, the term "subject" includes human and animal subjects. Therefore, veterinary therapeutic uses are provided herein. Thus, the present technology provides for mammals such as humans, as well as mammals of great significance due to endangerment, such as Siberian tigers; animals of great economic significance, such as those raised on farms for human consumption; and / or animals of great social significance to humans, such as animals raised as pets or in zoos. Examples of such animals include, but are not limited to, carnivores such as cats and dogs; pigs, including pigs, hogs, and wild boars; ruminants and / or ungulates, such as cattle, bulls, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Therefore, diagnosis and treatment of livestock are also provided, including, but not limited to, domestic pigs, ruminants, ungulates, horses (including race horses), and the like. The subject matter disclosed in the present invention also includes a system for diagnosing colon cancer in a subject. The system can be provided, for example, as a commercial kit, which can be used to screen for the risk of colon cancer or diagnose colon cancer in a subject from whom a biological sample has been collected. An exemplary system provided according to the present technology includes evaluating the methylation status of the markers described herein.

[0244] In the context of nucleic acids, the term "amplify" or "amplify" refers to the production of multiple copies of a polynucleotide or a portion of a polynucleotide, usually starting from a small amount of polynucleotide (e.g., a single polynucleotide molecule), wherein the amplification product or amplicon is generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The production of multiple DNA copies from several copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5,494,810; which is incorporated herein by reference in its entirety) is a form of amplification. Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611; incorporated herein by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408; incorporated herein by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594; incorporated herein by reference in its entirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671; each incorporated herein by reference in its entirety), inter-sequence-specific PCR, inverse PCR (see, e.g., Triglia et al. (1988) Nucleic Acids Res., 16:8186; incorporated herein by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R.et al., Nucleic Acids Research, 25:1854-1858 (1997); U.S. Pat. No. 5,508,169; each of which is incorporated by reference in its entirety), methylation-specific PCR (see, e.g., Herman et al., (1996) PNAS 93(13)9821-9826; incorporated herein by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten et al., (2002) Nucleic Acids Research 30(12):e57; incorporated herein by reference in its entirety), multiplex PCR (see, e.g., 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 which is incorporated herein by reference in its entirety), nested PCR, overlap-extension PCR (see, e.g., Higuchi et al., (1988) Nucleic Acids Research 16 (15) 7351-7367; incorporated herein by reference in its entirety), real-time PCR (see, e.g., Higuchi et al., (1992) Biotechnology 10:413-417; Higuchi et al., (1993) Biotechnology 11:1026-1030; each of which is incorporated herein by reference in its entirety), reverse transcription PCR (see, e.g., Bustin, SA (2000) J. Molecular Endocrinology 25:169-193; incorporated herein by reference in its entirety), solid phase PCR, thermal asymmetric staggered PCR, and touchdown PCR (see, e.g., 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 which is incorporated herein by reference in its entirety). Polynucleotide amplification can also be accomplished using digital PCR (see, e.g., 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 which is incorporated herein by reference in its entirety).

[0245] The term "polymerase chain reaction" ("PCR") refers to the method of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188 to K.B. Mullis, which describes a method for increasing the concentration of a target sequence segment in a genomic or other DNA or RNA mixture without cloning or purification. This process of amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by a series of precise thermal cycles in the presence of a 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 the primers are then annealed to their complementary sequences within the target molecule. After annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute one "cycle"; there can be many "cycles") to obtain a high concentration 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 with respect to each other, and therefore the length is a controllable parameter. Due to the repetitive aspect of the process, the method is referred to as "polymerase chain reaction" ("PCR"). Because the desired amplified segments of the target sequence become the predominant sequence in the mixture (in terms of concentration), they are referred to as "PCR amplified" and are "PCR products" or "amplicons". Those skilled in the art will appreciate that the term "PCR" encompasses many variations of the originally described method, using, for example, real-time PCR, nested PCR, reverse transcription-PCR (RT-PCR), single primers and arbitrarily primed PCR, etc.

[0246] As used herein, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays (Hologic, Inc.) and, for example, in U.S. Pat. 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 US2009 / 0253142, each of which is incorporated herein by reference in its entirety for all purposes); enzymatic mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, which are incorporated herein by reference in their entirety); polymerase chain reaction (PCR) as described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, which are incorporated herein by reference in their entireties); rolling circle replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960, and 6,235,502, which are incorporated herein by reference in their entireties); NASBA (e.g., U.S. Pat. No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, which is incorporated herein by reference in its entirety); electronic sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, which are incorporated herein by reference in their entirety); ring probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, which are incorporated herein by reference in their entirety); Dade Behring signal amplification method (e.g., U.S. Pat. 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 entireties); ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization method (e.g., U.S. Pat. No. 5,288,609, which is incorporated herein by reference in its entirety).

[0247] In some embodiments, the target nucleic acid is amplified (e.g., by PCR), and the amplified nucleic acid is detected simultaneously using an invasive cleavage assay. The assay configured to perform a detection assay (e.g., an invasive cleavage assay) in combination with an amplification assay is described in U.S. Patent No. 9,096,893, which is incorporated herein by reference in its entirety for all purposes. Additional amplification plus an invasive cleavage detection configuration (referred to as the QuARTS method) is described in, for example, U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, each of which is incorporated herein by reference 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 nucleic acid and the downstream nucleic acid are annealed to a continuous 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. Overlap occurs when one or more bases from an upstream nucleic acid and a downstream nucleic acid occupy the same position relative to a target nucleic acid base, regardless of whether the one or more overlapping bases of the upstream nucleic acid are complementary to the target nucleic acid, and regardless of whether those bases are natural bases or non-natural bases. In some embodiments, the 3' portion of the upstream nucleic acid that overlaps the downstream duplex is a non-basic chemical moiety, such as an aromatic ring structure, for example, as disclosed in U.S. Patent No. 6,090,543, which is incorporated herein by reference in its entirety. In some embodiments, one or more nucleic acids can be attached to each other, for example, by covalent bonds such as nucleic acid stem-loops, or by non-nucleic acid chemical bonds (e.g., polycarbon chains). As used herein, the term "flap endonuclease assay" includes the "INVADER" invasive cleavage assay and QuARTS assay as described above.

[0248] The term "probe oligonucleotide" or "flap oligonucleotide" when used with respect to a flap assay refers to an oligonucleotide that interacts with a target nucleic acid in the presence of an invasive oligonucleotide to form a cleavage structure.

[0249] The term "invasive oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid at a position adjacent to the hybridization region between the probe and the target nucleic acid, wherein the 3' end of the invasive oligonucleotide comprises a portion (e.g., a chemical moiety or one or more nucleotides) that overlaps the hybridization region between the probe and the target. The 3' terminal nucleotide of the invasive oligonucleotide may or may not base pair with a nucleotide in the target. In some embodiments, the invasive oligonucleotide contains a sequence at its 3' end that is substantially identical to the sequence at the 5' end of the probe oligonucleotide portion that anneals to the target strand.

[0250] As used herein, the term "flap endonuclease" or "FEN" refers to a class of nucleolytic enzymes, typically 5' nucleases, that act as structure-specific endonucleases on DNA structures having duplexes that contain a single-stranded 5' overhang or flap on one of the strands that is displaced by the other strand of nucleic acid (e.g., such that there are overlapping nucleotides at the junction between single-stranded and double-stranded DNA). FENs catalyze the hydrolytic cleavage of the phosphodiester bond at the junction of single-stranded and double-stranded DNA, releasing the overhang or flap. Flap endonucleases are reviewed by Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336) and Liu et al. (Annu. Rev. Biochem. 2004 73:589-615; which are incorporated herein by reference in their entirety). FENs can be separate enzymes, multi-subunit enzymes, or can exist as an activity of another enzyme or protein complex (e.g., DNA polymerase).

[0251] The flap endonuclease can be thermostable. For example, the FEN-1 flap endonuclease from archival thermophile is a typical thermostable one. As used herein, the term "FEN-1" refers to a non-polymerase flap endonuclease from a eukaryotic or archaeal organism. See, e.g., WO 02 / 070755 and Kaiser MW et al. (1999) J. Biol. Chem., 274: 21387, which are incorporated herein by reference in their entirety for all purposes.

[0252] As used herein, the term "cleaved flap" refers to a single stranded oligonucleotide that is a cleavage product of a flap assay.

[0253] When used with respect to a flap cleavage reaction, the term "cassette" refers to an oligonucleotide or combination of oligonucleotides that is configured to produce a detectable signal in response to cleavage of a flap or probe oligonucleotide (e.g., in a primary or first cleavage structure formed in a flap cleavage assay). In a preferred embodiment, the cassette hybridizes to a non-target cleavage product produced by cleavage of the flap oligonucleotide to form a second overlapping cleavage structure, such that the cassette can then be cleaved by the same enzyme, such as FEN-1 endonuclease.

[0254] In some embodiments, the box is a single oligonucleotide comprising a hairpin portion (i.e., a region in which a portion of the box oligonucleotide hybridizes with a second portion of the same oligonucleotide under reaction conditions to form a duplex). In other embodiments, the box comprises at least two oligonucleotides comprising complementary portions that can form a duplex under reaction conditions. In a preferred embodiment, the box comprises a marker, such as a fluorophore. In a particularly preferred embodiment, the box comprises a labeled half family that produces a FRET effect. In such embodiments, the box can be referred to as a "FRET box". See, for example, also U.S. Patent Application Serial No. 62 / 249,097 filed October 30, 2015, U.S. Patent Application Serial No. 15 / 335,096 filed October 26, 2016, and International Application Serial No. PCT / US16 / 58875 filed October 26, 2016, each of which is incorporated herein by reference in its entirety for all purposes.

[0255] As used herein, the phrase "substantially non-complementary" as used with respect to a probe flap or arm means that the flap portion is sufficiently non-complementary to be unable to selectively hybridize to a nucleic acid sequence, such as a target nucleic acid or amplified DNA, under specified annealing conditions or stringent conditions, encompassing the terms "substantially non-complementary" and "completely non-complementary."

[0256] The term "complementary" is used herein to mean that primers or probes are sufficiently complementary to selectively hybridize to a target nucleic acid sequence under specified annealing conditions or stringent conditions, for example, and encompasses the terms "substantially complementary" and "fully complementary".

[0257] As used herein, the term "FRET" refers to fluorescence resonance energy transfer, which is a process in which a moiety (e.g., a fluorophore) transfers energy, for example, between itself, or transfers energy from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some cases, FRET involves an excited donor fluorophore transferring energy to a low-energy acceptor fluorophore through a short-range (e.g., about 10 nM or less) dipole-dipole interaction. In other cases, FRET involves a loss of fluorescence energy from the donor and an increase in fluorescence in the acceptor fluorophore. In still other forms of FRET, energy can be transferred from an excited donor fluorophore to a non-fluorescent molecule (e.g., a "dark" quencher molecule). FRET is known to those skilled in the art and has been described (see, e.g., Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res 573, 103-110, each of which is herein incorporated by reference in its entirety).

[0258] In an exemplary flap detection assay, an invasive oligonucleotide and a flap oligonucleotide are hybridized with a target nucleic acid to produce a first complex having an overlap as described above. An unpaired "flap" is included at the 5' end of the flap oligonucleotide. The first complex is a substrate for a flap endonuclease, such as FEN-1 endonuclease, which cuts the flap oligonucleotide to release the 5' flap portion. In a secondary reaction, the released 5' flap product acts as an invasive oligonucleotide on the FRET box to again produce a structure recognized by the flap endonuclease, thereby causing the FRET box to be cut. When the fluorophore and quencher are separated due to the cutting of the FRET box, a detectable fluorescent signal is generated that is higher than the background fluorescence.

[0259] The term "real time" as used herein for the detection of nucleic acid amplification or signal amplification refers to the accumulation of product or signal in the detection or measurement reaction while the reaction is ongoing, such as during incubation or thermal cycling. Such detection or measurement can be performed continuously, or can be performed at multiple discrete points in the process of amplified reaction progress, or can be a combination. For example, in polymerase chain reaction, detection (for example, fluorescence detection) can be performed continuously during the whole or part of thermal cycle, or can be performed instantaneously at one or more points during one or more cycles. In some embodiments, the real-time detection of PCR or QuARTS reactions is realized by measuring fluorescence level in each of multiple cycles or at the same point in each cycle (for example, time point in cycle or temperature step in cycle). The real-time detection of amplification can also be referred to as the detection of "during amplified reaction".

[0260] As used herein, the term "quantitative amplification data set" refers to data obtained during quantitative amplification of a target sample, such as target DNA. In the case of quantitative PCR or QuARTS assays, the quantitative amplification data set is a collection of fluorescence values ​​obtained during amplification, such as during multiple or all thermal cycles. The quantitatively amplified data is not limited to data collected at any specific point in the reaction, and fluorescence can be measured at discrete points in each cycle or continuously throughout each cycle.

[0261] The abbreviations "Ct" and "Cp" used herein for data collected during real-time PCR and PCR+INVADER assays refer to the cycle at which a signal (e.g., a fluorescent signal) exceeds a predetermined threshold indicating a positive signal. Various methods have been used to calculate a threshold value used as a determinant of signal and concentration, and the value is generally expressed as a "crossing threshold" (Ct) or "crossing point" (Cp). Cp values ​​or Ct values ​​can be used in embodiments of the methods provided herein to analyze real-time signals to determine the percentage of variants and / or non-variant components in an assay or sample.

[0262] As used herein, the term "control" when used with respect to nucleic acid detection or analysis refers to a nucleic acid with known characteristics (e.g., known sequence, known number of copies per cell) for comparison with an experimental target (e.g., a nucleic acid of unknown concentration). The control can be an endogenous, preferably an invariant gene against which the test or target nucleic acid in the assay can be standardized. Such standardization controls for sample-to-sample variations that may occur in aspects such as sample handling, assay efficiency, and allows accurate sample-to-sample data comparisons. Genes that can be used to standardize nucleic acid detection assays for human samples include, for example, β-actin, ZDHHC1, and B3GALT6 (see, e.g., U.S. Patent Application Serial Nos. 14 / 966,617 and 62 / 364,082, each of which is incorporated herein by reference.

[0263] Controls can also be external. For example, in quantitative assays such as qPCR, QuARTS, "calibrators" or "calibration controls" are known sequences (e.g., sequences identical to a portion of an experimental target nucleic acid), and nucleic acids of known concentration or a range of concentrations (e.g., control targets for serial dilutions used to generate calibration curves in quantitative PCR). Typically, calibration controls are analyzed using the same reagents and reaction conditions used on experimental DNA. In certain embodiments, the measurement of calibrators is performed simultaneously with the experimental assay, such as in the same thermal cycler. In a preferred embodiment, a variety of calibrators may be included in a single plasmid so that different calibrator sequences are easily provided in equimolar amounts. In a particularly preferred embodiment, plasmid calibrators are digested, for example, with one or more restriction enzymes to release the calibrator portion from the plasmid vector. See, for example, WO 2015 / 066695, which is included herein by reference. In some embodiments, calibrator DNA is synthetic, for example, as described in U.S. Patent Application Serial No. 15 / 105,178, which is incorporated herein by reference.

[0264] As used herein, "ZDHHC1" refers to a gene encoding a protein characterized as DHHC-type zinc finger-containing protein 1, located in human DNA on Chr 16 (16q22.1), and belonging to the DHHC palmitoyltransferase family.

[0265] As used herein, the term "process control" refers to an exogenous molecule, such as an exogenous nucleic acid added to a sample prior to extracting target DNA, which can be measured after extraction to assess the efficiency of the process and to determine success or failure modes. The nature of the process control nucleic acid used generally depends on the type of assay and the material being measured. For example, if the assay used is for detecting and / or quantifying double-stranded DNA or mutations therein, a double-stranded DNA process control is typically incorporated into the sample prior to extraction. Similarly, for assays monitoring mRNA or microRNA, the process control used is typically an RNA transcript or synthetic RNA. See, for example, U.S. patent application serial number 62 / 364,049 filed on July 19, 2016, which is incorporated herein by reference and describes the use of zebrafish DNA as a process control for human samples.

[0266] As used herein, the term "zebrafish DNA" is distinct from bulk "fish DNA" (e.g., purified salmon DNA), and refers to DNA isolated from zebrafish (Danio rerio) or produced in vitro (e.g., enzymatically, synthetically) having a nucleotide sequence found in DNA from zebrafish. In a preferred embodiment, the zebrafish DNA is methylated DNA added by a sample processing step as a detectable control DNA (e.g., a process control for verifying DNA recovery). In particular, zebrafish DNA comprising at least a portion of the RASSF1 gene can be used, for example, as a process control for human samples, as described in U.S. Patent Application Serial No. 62 / 364,049.

[0267] As used herein, the term "fish DNA" is distinct from zebrafish DNA and refers to a large volume of (e.g., genomic) DNA isolated from fish, for example, as described in U.S. Pat. No. 9,212,392. Large volumes of purified fish DNA are commercially available, for example, in the form of cod and / or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB / Affymetrix).

[0268] As used herein, the terms "particles" and "beads" are used interchangeably, and the terms "magnetic particles" and "magnetic beads" are used interchangeably and refer to particles or beads that respond to a magnetic field. Typically, magnetic particles include materials that do not have a magnetic field but form a magnetic dipole when exposed to a magnetic field, for example, a material that can be magnetized in the presence of a magnetic field but does not itself have magnetic properties in the absence of a magnetic field. The term "magnetism" as used in this context includes materials with paramagnetism or superparamagnetic materials. The term "magnetism" as used herein also encompasses temporary temporary materials, such as ferromagnetic or ferrimagnetic materials with a low Curie temperature, provided that such temporary magnetic materials have paramagnetism in the temperature range of separation of biological materials using a silica magnetic material containing such materials according to the method of the present invention.

[0269] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for performing the assay, etc.) to be stored, transported, or delivered from one location to another. For example, a kit includes one or more housings (e.g., boxes) containing relevant reaction reagents and / or support materials. As used herein, the term "fragmented kit" refers to a delivery system comprising two or more independent containers, each of which contains a sub-portion of a total kit assembly. The container can be delivered to an intended recipient together or individually. For example, a first container can contain an enzyme for assaying, and a second container contains an oligonucleotide.

[0270] As used herein, the term "system" refers to a collection of items used for a specific purpose. In some embodiments, the items include instructions for use, such as information provided on, for example, the item, on paper, or on a recordable medium (e.g., a DVD, CD, flash drive, etc.). In some embodiments, the instructions direct the user to an online location, e.g., a website.

[0271] As used herein, the term "information" refers to a collection of any facts or data. With respect to information stored or processed using one or more computer systems (including but not limited to the Internet), the term refers to any data stored in any format (e.g., analog, digital, optical format, etc.). As used herein, the term "information related to a subject" refers to facts or data related to a subject (e.g., a human, plant, or animal). The term "genomic information" refers to information related to a genome, including but not limited to nucleic acid sequences, genes, methylation percentages, allele frequencies, RNA expression levels, protein expression, phenotypes associated with genotypes, etc. "Allele frequency information" refers to facts or data related to allele frequencies, including but not limited to allele identity, the statistical correlation between the presence of an allele and the characteristics of a subject (e.g., a human subject), the presence or absence of an allele in an individual or population, the probability percentage that an allele is present in an individual with one or more specific characteristics, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0272] Figure 1 Schematic diagram of the marker target region in unconverted and bisulfite converted form is shown. Flap assay primers and probe for detection of bisulfite converted target DNA are shown.

[0273] Figure 2 Tables of nucleic acid sequences and corresponding SEQ ID NOs are provided.

[0274] Figure 3 A table showing data and results from the assays of Example 2 is provided.

[0275] Figure 4 A table showing data and results from the assays of Example 2 is provided.

[0276] Figure 5 A schematic diagram is provided showing a combined PCR-invasive cleavage assay ("PCR-flap assay") (e.g., QuARTS assay), in which three different regions of a target nucleic acid (e.g., a methylation marker) are amplified by primer pairs specific to each different region and in the presence of different flap probes, each flap probe being specific to one of the different regions, but each flap probe having the same flap arm sequence. Flap release during each PCR-flap assay is reported to the same FRET box to generate a fluorescent signal from the same fluorophore. DETAILED DESCRIPTION

[0277] Provided herein are techniques related to the selection and use of nucleic acid markers for use in assays for detecting and quantifying DNA (e.g., methylated DNA). In particular, the techniques relate to the use of methylation assays to detect colon cancer.

[0278] In this detailed description of various embodiments, for the purpose of explanation, many specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced with or without these specific details. In other cases, structures and devices are shown in block diagram form. In addition, those skilled in the art can readily appreciate that the specific order of presentation and execution methods is illustrative, and it is contemplated that the order may be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

[0279] In some embodiments, the analysis of target DNA includes analyzing multiple different DNAs in a single reaction. Typical instruments for real-time detection of amplification reactions allow for simultaneous detection and quantification of only 3-5 fluorescent dyes. This is mainly because when many dyes with overlapping excitation and / or emission spectra are used together, the spectral overlap between fluorophores makes it difficult to distinguish one dye from another. When detecting a specific disease from a biological specimen requires a group containing more than about 5 different markers, this presents a challenge, especially when the size of the sample is limited and the marker is present at a low level, this situation often requires the use of the entire sample in a single amplification run.

[0280] In some embodiments, the methods described herein allow for the detection of multiple different markers in the same sample by having each sample produce results from the same dye. In embodiments described in detail herein, a multiplex flap cleavage assay (e.g., a QuARTS flap endonuclease assay) for multiple different markers produces an initial cleavage product that uses the same FRET box to produce a fluorescent signal.

[0281] In a preferred embodiment, the combined assay includes several different probe oligonucleotides, each having a portion that hybridizes to a different target nucleic acid, but all having substantially the same 5' arm sequence. The cleavage of the probes in the presence of their respective target nucleic acids all releases the same 5' arm, and then all released arms are combined with a FRET box having the same flap binding sequence and the same dye to generate a fluorescent signal by endonuclease cleavage of the FRET box. In other embodiments, probes for different targets may have different flap arms that report to different FRET boxes, wherein the different FRET boxes all use the same reporting fluorophore.

[0282] Combining assays in this way has several advantages. For example, if any one If a target sequence is associated with a condition (e.g., a disease state such as colorectal cancer), the sample can provide a result without the need to separate the sample into multiple different assays, and further, if more than one target sequence provides such a result, aggregating these signals into a single dye channel can provide a stronger signal than background, providing more certainty for the assay result. During the development of the methods described herein, it was surprisingly discovered that combining a large number of primers and flap assay probes for detecting multiple different target sequences together with a common FRET box in a single amplification plus flap cleavage assay reaction does not increase the background signal in no-target controls or negative samples.

[0283] In some embodiments, the different target sequences reported to a single FRET box and a single dye channel may not be from different marker genes or regions, but may be from different regions within a single marker (e.g., a single methylated marker gene). As described in Example 4, configuring the assay to detect multiple regions of a single marker gene in an assay where all regions, such as a single FRET box, report to a single dye increases the level of detectable signal from the target gene copies present in the reaction.

[0284] In other embodiments, the different target sequences to be detected can be a mixture of multiple regions of one marker together with one or more regions of one or more different markers. The different target sequences can include any combination of methylation markers, mutation markers, deletions, insertions, or any other form of nucleic acid variants detectable in an assay such as the QuARTS amplification / flap cleavage assay.

[0285] In some embodiments, the marker is a region with 100 or fewer bases, the marker is a region with 500 or fewer bases, the marker is a region with 1000 or fewer bases, the marker is a region with 5000 or fewer bases, or, in some embodiments, the marker is one base. In some embodiments, the marker is in a high CpG density promoter.

[0286] The technology is not limited by the sample type. For example, in some embodiments, the sample is a stool sample, a tissue sample, sputum, a blood sample (e.g., plasma, serum, whole blood), a feces or urine sample.

[0287] In addition, the technology is not limited to the method for determining methylation status. In some embodiments, the determination includes the use of methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, chip or array hybridization, methylation-specific nuclease, separation based on quality or target capture. In some embodiments, the determination includes the use of methylation-specific oligonucleotides. In some embodiments, the technology uses large-scale parallel sequencing (e.g., next-generation sequencing) to determine methylation status, such as sequencing-by-synthesis, real-time (e.g., single molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc.

[0288] The technology provides reagents for detecting differentially methylated regions (DMRs). In some embodiments, oligonucleotides comprising sequences complementary to chromosomal regions are provided, and kit embodiments are provided, such as kits comprising bisulfite reagents and control nucleic acids, the control nucleic acids comprising chromosomal regions with annotations selected from the following: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI and having a methylation state associated with a subject who does not suffer from cancer (e.g., colon cancer). In some embodiments, the kit comprises a bisulfite reagent and an oligonucleotide as described herein. In some embodiments, the kit comprises a bisulfite reagent and a control nucleic acid, the control nucleic acid comprising a sequence from such a chromosomal region and having a methylation state associated with a subject with colon cancer.

[0289] The technology is related to embodiments of compositions (e.g., reaction mixtures). In some embodiments, a composition comprising a nucleic acid and a bisulfite reagent is provided, wherein the nucleic acid comprises a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. Some embodiments provide a composition comprising a nucleic acid and an oligonucleotide as described herein, wherein the nucleic acid comprises a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. Some embodiments provide a composition comprising a nucleic acid comprising a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI and a methylation-sensitive restriction enzyme. Some embodiments provide a composition comprising a nucleic acid comprising a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI and a polymerase.

[0290] Additional related method embodiments are provided for screening for tumors (e.g., colon cancer) in a sample obtained from a subject, for example, a method comprising the steps of: determining the methylation status of a marker in the sample, wherein the marker comprises bases in a chromosomal region having an annotation selected from the following: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI; comparing the methylation status of the marker in the sample from the subject with the methylation status of the marker in a normal control sample from a subject who does not have colon cancer; and determining a confidence interval and / or p-value for the difference in methylation status between the subject sample and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Some embodiments of the method provide the following steps: reacting a nucleic acid with a bisulfite reagent to produce a bisulfite-reacted nucleic acid, the nucleic acid comprising a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid comprising a chromosomal region from a subject who does not have colon cancer to identify differences between the two sequences; and identifying the subject as having a tumor when a difference exists.

[0291] The technology provides a system for screening colon cancer in a sample obtained from a subject. Exemplary embodiments of the system include, for example, a system for screening colon cancer in a sample obtained from a subject, the system including an analysis component configured to determine the methylation state of the sample, a software component configured to compare the methylation state of the sample with the methylation state of a control sample or a reference sample recorded in a database, and an alarm component configured to alert a user to a cancer-related methylation state. In some embodiments, an alarm is determined by a software component that receives results from multiple assays (e.g., determining the methylation state of multiple markers, the markers, for example, have a chromosomal region with annotations selected from the following: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI and calculating the value or result to be reported based on multiple results. Some embodiments provide a database of weighted parameters associated with each chromosomal region with annotations selected from the following: VAV 3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI, the database provided herein is for calculating values ​​or results and / or alerts for reporting to a user (e.g., such as a doctor, nurse, clinician, etc.). In some embodiments, all results from multiple assays are reported and in some embodiments, one or more results are utilized to provide a composite score, value or result indicating a subject's risk of colon cancer based on one or more results from multiple assays.

[0292] In some embodiments of the system, the sample comprises a nucleic acid comprising a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. In some embodiments, the system further comprises a component for isolating nucleic acid, a component for collecting a sample, such as a component for collecting a stool sample. In some embodiments, the system comprises a nucleic acid sequence comprising a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. In some embodiments, the database comprises nucleic acid sequences from subjects who do not have colon cancer. Also provided are nucleic acids, e.g., sets of nucleic acids, each having a sequence comprising a chromosomal region having an annotation selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

[0293] Related system embodiments include a nucleic acid set as described and a database of nucleic acid sequences related to the nucleic acid set. Some embodiments also include a bisulfite reagent. And, some embodiments also include a nucleic acid sequencer.

[0294] In certain embodiments, a method for characterizing a sample obtained from a human subject is provided, comprising a) obtaining a sample from the human subject; b) determining the methylation state of one or more markers in the sample, wherein the markers comprise bases in an annotated chromosomal region having a marker selected from the following group: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI; c) comparing the methylation state of the determined markers with the methylation state of the markers determined in a subject without a tumor.

[0295] In some embodiments, the technology is related to the presence and methylation state of one or more markers identified in biological samples herein. These markers include one or more differentially methylated regions (DMRs) as discussed herein. Methylation state is assessed in the embodiments of the technology. Thus, the technology provided herein is not limited to the method for measuring gene methylation state. For example, in some embodiments, methylation state is measured by genome scanning method. For example, one method involves restriction marker genome scanning (Kawai et al. (1994) Mol. Cell. Biol. 14: 7421–7427) and another example involves methylation-sensitive arbitrary primer PCR (Gonzalgo et al. (1997) Cancer Res. 57: 594-599). In some embodiments, by digesting genomic DNA with methylation-sensitive restriction enzymes, Southern analysis (digestion-Southern method) is then performed on the target region to monitor the change of methylation pattern at specific CpG sites. In some embodiments, analyzing changes in methylation patterns involves a PCR-based process that involves digesting genomic DNA with a methylation-sensitive restriction enzyme prior to PCR amplification (Singer-Sam et al. (1990) Nucl. Acids Res. 18: 687). In addition, other techniques that utilize bisulfite treatment of DNA as a starting point for methylation analysis have been reported. These techniques include methylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93: 9821–9826) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby (1996) Nucl. Acids Res. 24: 5058–5059; and Xiong and Laird (1997) Nucl. Acids Res. 25: 2532-2534). PCR techniques have been developed for detecting gene mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1143–1147) and quantifying allele-specific expression (Szabo and Mann (1995) Genes Dev. 9: 3097–3108; and Singer-Sam et al. (1992) PCR Methods Appl. 1: 160-163). Such techniques use internal primers that anneal to the PCR-generated template and terminate immediately 5' of the single nucleotide to be assayed. Methods using the "quantitative Ms-SNuPE assay" as described in U.S. Pat. No. 7,037,650 are used in some embodiments.

[0296] When evaluating methylation status, the methylation status is often expressed as the fraction or percentage of a single DNA strand methylated at a specific site (e.g., at a single nucleotide, at a specific region or locus, at a longer target sequence, e.g., up to 100-bp, 200-bp, 500-bp, 1000-bp or longer subsequence of DNA) relative to the total DNA population containing the specific site in the sample. Traditionally, the amount of unmethylated nucleic acid is determined by PCR using a calibrator. Then, a known amount of DNA is treated with bisulfite and the resulting methylation-specific sequence is determined using real-time PCR or other exponential amplification, such as QuARTS assay (e.g., as provided in U.S. Patents 8,361,720, 8,715,937, 8,916,344 and 9,212,392).

[0297] For example, in some embodiments, the method includes generating a standard curve of an unmethylated target by using an external standard. The standard curve is composed of at least two points, and the real-time Ct value of unmethylated DNA is associated with a known quantitative standard. Then, a second standard curve of the methylated target is constructed by at least two points and an external standard. The second standard curve associates the Ct of methylated DNA with a known quantitative standard. Next, the Ct values ​​of the test samples of methylated and unmethylated populations are determined, and the genome equivalent of DNA is calculated by the standard curve generated by the first two steps. The methylation percentage at the target site is calculated according to the amount of methylated DNA relative to the total amount of DNA in the population, such as (the number of methylated DNA) / (the number of methylated DNA+the number of unmethylated DNA)×100.

[0298] Also provided herein are compositions and kits for implementing the methods. For example, in some embodiments, reagents (e.g., primers, probes) specific to one or more markers are provided separately or in sets (e.g., sets of primer pairs for amplifying multiple markers). Other reagents for performing detection assays (e.g., enzymes, buffers, positive and negative controls for performing QuARTS, PCR, sequencing, bisulfite or other assays) can also be provided. In some embodiments, a kit containing one or more reagents necessary, sufficient or useful for performing the method is provided. A reaction mixture containing the reagent is also provided. A master mixed reagent set containing multiple reagents is also provided, and the reagents can be added to each other and / or added to the test sample to complete the reaction mixture.

[0299] Methods for isolating DNA suitable for these assay techniques are known in the art. Specifically, some embodiments include isolating nucleic acids as described in U.S. Patent 9,000,146, which is incorporated herein by reference in its entirety.

[0300] Genomic DNA can be separated by any means, including the use of commercially available test kits. In short, where the target DNA is encapsulated by the cell membrane, enzymatic, chemical or mechanical methods must be utilized to destroy and crack the biological sample. Proteins and other contaminants in the DNA solution can then be removed, for example, by digestion with proteinase K. The genomic DNA is then recovered from the solution. This can be done by a variety of methods, including salting out, organic extraction, or DNA is bound to a solid support. The selection of the method will be affected by several factors including time, cost and required DNA quantity. All clinical sample types containing tumor material or pre-neoplastic material (pre-neoplastic matter) are suitable for use in this method, such as cell lines, tissue sections, biopsies, paraffin-embedded tissues, body fluids, feces, colon effluent, urine, plasma, serum, whole blood, separated blood cells, cells separated from blood and combinations thereof.

[0301] The technology is not limited to methods for preparing samples and providing nucleic acids for testing. For example, in some embodiments, DNA is isolated from a stool sample or a blood or plasma sample using direct gene capture, e.g., as described in detail in U.S. Pat. Nos. 8,808,990 or 9,000,146, or by related methods.

[0302] The technology involves analyzing any sample associated with colon cancer. For example, in some embodiments, the sample comprises tissue and / or biological fluid obtained from a patient. In some embodiments, the sample comprises secretions. In some embodiments, the sample comprises sputum, blood, serum, plasma, gastric secretions, colon tissue samples, colon cells, or colon DNA recovered from feces. In some embodiments, the subject is a human. Such samples can be obtained by many methods known in the art, such as those apparent to the skilled person.

[0303] I. Methylation Assays for Detection of Colon Cancer

[0304] Candidate methylated DNA markers were identified by unbiased full methylation profiling of selected colon cancer cases and colon control tissues. Top candidate markers were further evaluated in 89 cancer samples and 95 normal plasma samples. DNA extracted from patient tissue samples was bisulfite treated and then assayed by quantitative allele-specific real-time target and signal amplification (QuARTS amplification) to determine candidate markers and reference genes (e.g., β-actin or B3GALT6) as normalization genes. QuARTS assay chemistry yields high discrimination for methylation marker selection and screening.

[0305] In receiver operating characteristic analysis of single marker candidates, the area under the curve (AUC) ranged from 0.63 to 0.75. At 92.6% specificity, a combined panel of 12 methylation markers (VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI) plus measurement of CEA protein yielded a sensitivity of 67.4% in all stages of colon cancer.

[0306] II. Methylation Detection Assays and Kits

[0307] The markers described herein can be used in a variety of methylation detection assays. The most commonly used method for analyzing the presence of 5-methylcytosine in nucleic acids is based on the bisulfite method described by Frommer et al. for detecting 5-methylcytosine in DNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827–31, which is expressly incorporated herein by reference as a whole for all purposes) or its variants. The bisulfite method for mapping 5-methylcytosine is based on the observation that cytosine, rather than 5-methylcytosine, reacts with acidic sulfite ions (also referred to as bisulfite). The reaction is generally carried out according to the following steps: First, cytosine reacts with bisulfite to form sulfonated cytosine. Next, the sulfonation reaction intermediate is spontaneously deaminated to produce sulfonated uracil. Finally, sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil bases pair with adenine (thus behaving similarly to thymine), whereas 5-methylcytosine bases pair with guanine (thus behaving similarly to cytosine). This enables the identification of methylated cytosines from unmethylated cytosines by, for example, bisulfite genomic 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, for example, in U.S. Pat. No. 5,786,146, or using an assay involving sequence-specific probe cleavage, such as the QuARTS flap endonuclease assay (see, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with novel methylation specific technology" Clin Chem 56:A199; and U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392).

[0308] Some conventional techniques are related to such a method, which includes enclosing the DNA to be analyzed in an agarose matrix to prevent DNA diffusion and renaturation (bisulfite reacts only with single-stranded DNA), and replacing the 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 in a single cell can be analyzed, which illustrates the practicality and sensitivity of the method. Rein, T. et al. (1998) Nucleic Acids Res. 26: 2255 provides an overview of conventional methods for detecting 5-methylcytosine.

[0309] Bisulfite techniques generally involve amplifying short, specific fragments of known nucleic acids after bisulfite treatment, followed by assaying the products for analysis of individual cytosine positions by sequencing (Olek and Walter (1997) Nat. Genet. 17: 275–6) or primer extension reactions (Gonzalgo and Jones (1997) Nucleic Acids Res. 25: 2529–31; WO 95 / 00669; U.S. Pat. No. 6,251,594). Some methods use 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 methylation detection of single 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; WO9746705; WO 9515373).

[0310] Various methylation assay procedures can be used in conjunction with bisulfite treatment according to the present technology. These assays allow determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays involve, among other techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-sensitive restriction enzymes.

[0311] For example, genomic sequencing has been simplified by using bisulfite treatment to analyze methylation patterns and 5-methylcytosine distribution (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-1831). In addition, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA can be used to assess methylation status, for example, as described in Sadri and Hornsby (1997) Nucl. Acids Res. 24: 5058-5059 or as embodied in a method called COBRA (combined bisulfite restriction analysis) (Xiong and Laird (1997) Nucleic Acids Res. 25: 2532-2534).

[0312] COBRA TM Analysis is a quantitative methylation assay that can be used to determine the DNA methylation level at a specific locus in a small amount of genomic DNA (Xiong and Laird, Nucleic Acids Res. 25: 2532-2534, 1997). In short, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. According to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89: 1827-1831, 1992, methylation-dependent sequence differences are first introduced into genomic DNA by standard bisulfite treatment). The bisulfite-converted DNA is then PCR amplified using primers specific to the target CpG island, followed by restriction endonuclease digestion, gel electrophoresis, and detection using a specific labeled hybridization probe. The methylation level in the original DNA sample is represented by the relative amount of digested and undigested PCR products in a linear quantitative manner over a wide range of DNA methylation levels. Additionally, the technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0313] For COBRA TM Typical reagents for the assay (e.g., as may be found in a typical COBRA-based TM The reagents (found in the kit) 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.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; kinase labeling kits for oligonucleotide probes; and labeled nucleotides. In addition, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffer; and DNA recovery components.

[0314] Assays such as the following are used alone or in combination with one or more of these methods: "MethyLight TM "(Real-time PCR based on fluorescence) (Eads et al., Cancer Res. 59: 2302-2306, 1999), Ms-SNuPE TM (methylation-sensitive single nucleotide primer extension) reaction (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; U.S. Pat. No. 5,786,146), and methylated CpG island amplification ("MCA"; Toyota et al., Cancer Res. 59:2307-12, 1999).

[0315] “HeavyMethyl TM The assay technology is a quantitative method to assess methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") covering CpG positions between or covered by amplification primers enable methylation-specific selective amplification of nucleic acid samples.

[0316] The term "Heavy Methyl TM MethyLight TM "Determination refers to HeavyMethyl TM MethyLight TM Assay, which is MethyLight TM Assay variants in which MethyLight TM The assay is combined with a methylation-specific blocking probe covering the CpG position between the amplification primers. TM The assay can also be used in combination with methylation-specific amplification primers.

[0317] Uses for HeavyMethyl TM Typical reagents for the assay (e.g., as can be found in typical MethyLight-based TMThe kit may include, but is not limited to: PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, or bisulfite-treated DNA sequences or CpG islands, etc.); blocking oligonucleotides; optimized PCR buffers and deoxynucleotides; and Taq polymerase.

[0318] MSP (methylation-specific PCR) allows the assessment of the methylation status of almost any group 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; U.S. Pat. No. 5,786,146). Briefly, DNA is modified with sodium bisulfite, which converts unmethylated cytosines to uracils without converting methylated cytosines to uracils, and the products are subsequently amplified with primers that are specific for methylated DNA relative to unmethylated DNA. MSP requires only small amounts of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents for MSP analysis (e.g., as may be found in a typical MSP-based kit) may include, but are not limited to: methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; and specific probes.

[0319] MethyLight TM The assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR that requires no further manipulation after the PCR step (e.g., ) (Eads et al., Cancer Res. 59:2302-2306, 1999). In brief, MethyLight TM The method starts with a mixed sample of genomic DNA, which is converted into a mixed pool of methylation-dependent sequence differences in a sodium bisulfite reaction according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction, for example, with PCR primers that overlap with known CpG dinucleotides. Sequence identification occurs both at the level of the amplification process and at the level of the fluorescence detection process.

[0320] MethyLight TMThe assay is used as a quantitative test for methylation patterns in nucleic acids, such as genomic DNA samples, where sequence discrimination occurs at the probe hybridization level. In the quantitative format, the PCR reaction provides methylation-specific amplification in the presence of a fluorescent probe that overlaps a specific putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primer nor the probe covers any CpG dinucleotide. Alternatively, the methylation site is amplified by using a control oligonucleotide that does not cover a known methylation site (e.g., a fluorescent-based HeavyMethyl TM Qualitative testing of genomic methylation can be achieved by using either PCR or MSP techniques (such as PCR amplification and MSP).

[0321] MethyLight TM The method and any suitable probe (e.g., Probe, For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and used probe (e.g., together with MSP primers) and / or HeavyMethyl blocking oligonucleotides and The probe performs one of two PCR reactions on the double-stranded genomic DNA. The probe is dual-labeled with a fluorescent "reporter" and a "quencher" molecule and is designed to be specific for regions with relatively high GC content, so that it melts during PCR cycles at a temperature approximately 10°C higher than that of the forward or reverse primer. This allows The probe remains fully hybridized during the PCR annealing / extension step. As the Taq polymerase enzymatically synthesizes new strands during PCR, it eventually reaches the annealed The Taq polymerase 5' to 3' endonuclease activity will then The probe is digested and replaced The probe releases fluorescent reporter molecules so that the unquenched signal can be quantitatively detected using a real-time fluorescence detection system.

[0322] Use for MethyLight TM Typical reagents for the assay (e.g., as can be found in typical MethyLight-based TM The primers (found in the kit) 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 probe; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0323] QM TM(Quantitative methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, in which sequence discrimination occurs at the probe hybridization level. In this quantitative format, the PCR reaction provides unbiased amplification in the presence of a fluorescent probe that overlaps a specific putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primer nor the probe covers any CpG dinucleotide. Alternatively, by using a control oligonucleotide (fluorescence-based HeavyMethyl) that does not cover a known methylation site, the PCR reaction is performed. TM Qualitative testing of genomic methylation can be achieved by using either PCR or MSP techniques (such as PCR amplification and MSP).

[0324] QM TM The method can be used with any suitable probe, e.g. Probe, The probe is used together in the amplification process. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and Probe. The probe is dual-labeled with a fluorescent "reporter" and a "quencher" molecule and is designed to be specific for regions with relatively high GC content, so that it melts during PCR cycles at a temperature approximately 10°C higher than that of the forward or reverse primer. This allows The probe remains fully hybridized during the PCR annealing / extension step. As the Taq polymerase enzymatically synthesizes new strands during PCR, it eventually reaches the annealed The Taq polymerase 5' to 3' endonuclease activity will then The probe is digested and replaced The probe releases the fluorescent reporter molecule so that the unquenched signal can be quantitatively detected using a real-time fluorescence detection system. TM Typical reagents for the assay (e.g., as may be found in a typical QM-based TM The primers (found in the kit) 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 probe; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0325] Ms-SNuPE TMThe technique is a quantitative method for evaluating methylation differences at specific CpG sites based on bisulfite treatment of DNA and subsequent single nucleotide primer extension (Gonzalgo and Jones, Nucleic Acids Res. 25: 2529-2531, 1997). In brief, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine into uracil while keeping 5-methylcytosine unchanged. The desired target sequence is then amplified using PCR primers specific for bisulfite-converted DNA, and the resulting product is separated and used as a template for methylation analysis at the target CpG site. A small amount of DNA (e.g., microdissected pathology sections) can be analyzed and it avoids the use of restriction enzymes to determine the methylation status at the CpG site.

[0326] For Ms-SNuPE TM Typical reagents for the assay (e.g., as may be found in a typical Ms-SNuPE-based TM The kits (found in the kit) 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.); optimized PCR buffers and deoxynucleotides; gel extraction kits; positive control primers; Ms-SNuPE for specific loci; TM Primer; reaction buffer (for Ms-SNuPE reaction); and labeled nucleotides. In addition, the bisulfite conversion reagent may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagent or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery component.

[0327] Reduced representation bisulfite sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracils, followed by restriction enzyme digestion (e.g., with an enzyme that recognizes sites containing CG sequences (such as MspI)) and complete sequencing of the fragments after coupling to an aptamer ligand. The choice of restriction enzymes enriches fragments for CpG-dense regions, reducing the number of redundant sequences that may be mapped 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 for promoters, CpG islands, and other genomic features, so that the sample has a high frequency of restriction enzyme sites in these regions, thereby providing an assay for evaluating the methylation state of one or more genomic loci.

[0328] A typical protocol for RRBS includes the following steps: digestion of nucleic acid samples with restriction enzymes such as MspI, filling in overhangs and A-tails, ligation of adapters, bisulfite conversion, and PCR. See, e.g., et al. (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" Nat Methods 7:133–6; Meissner et al. (2005) "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis" Nucleic Acids Res. 33:5868–77.

[0329] In some embodiments, the methylation status is evaluated using a quantitative allele-specific real-time target and signal amplification (QuARTS) assay. Three reactions occur sequentially in each QuARTS assay, including amplification (reaction 1) and target probe cleavage (reaction 2) in the primary reaction; and FRET cleavage and fluorescence signal generation (reaction 3) in the secondary reaction. When the target nucleic acid is amplified with specific primers, a specific detection probe with a flap sequence is loosely bound to the amplicon. The specific invasive oligonucleotide present at the target binding site releases the flap sequence by cleaving between the detection probe and the flap sequence, such as a 5' nuclease, such as a FEN-1 endonuclease. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET box. Therefore, the flap sequence acts as an invasive oligonucleotide on the FRET box and achieves cleavage between the FRET box fluorophore and the quencher, thereby generating a fluorescent signal. The cleavage reaction can cleave multiple probes for each target, and thereby release multiple fluorophores for each flap, thereby providing exponential signal amplification. By using a FRET box with different dyes, QuARTS can detect multiple targets in a single reaction well. See, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation specific technology" Clin Chem 56:A199). In the embodiments described herein, the QuARTS assay can also be configured to detect multiple different targets or multiple different regions of the same target using the same FRET box, thereby generating additive fluorescent signals from a single dye.

[0330] 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, for example, by means of a Microcon TM Column (Millipore T The DNA is then purified by ultrafiltration (see, e.g., PCT / EP2004 / 011715, which is incorporated herein by reference in its entirety) according to a modified manufacturer's protocol. In some embodiments, the bisulfite-treated DNA is bound to a solid support (e.g., magnetic beads), and desulfonation and washing are performed while the DNA is bound to the support. Examples of such embodiments are provided, for example, in WO 2013 / 116375. In certain preferred embodiments, the support-bound DNA is immediately prepared for methylation assays after desulfonation and washing on the support. In some embodiments, the desulfonated DNA is eluted from the support prior to the assay.

[0331] In some embodiments, a collection of primer oligonucleotides according to the present invention is used (e.g., see Figure 1 ) and an amplification enzyme to amplify the fragments of the treated DNA. The amplification of several DNA segments can be performed simultaneously in one and the same reaction vessel. Typically, the amplification is performed using the polymerase chain reaction (PCR).

[0332] Methods for isolating DNA suitable for these assay techniques are known in the art. Specifically, some embodiments include isolating nucleic acids as described in U.S. Patent Application Serial No. 13 / 470,251 ("Isolation of Nucleic Acids", published as US2012 / 0288868), which is incorporated herein by reference in its entirety.

[0333] In some embodiments, markers as described herein can be used for the QUARTS assay performed on fecal samples. In some embodiments, a method for producing a DNA sample is provided, and in particular, a method for producing such a DNA sample, wherein the DNA sample comprises a small volume (e.g., less than 100 microliters, less than 60 microliters) of highly purified low-abundance nucleic acids and is substantially and / or effectively free of the material that inhibits the determination (e.g., PCR, INVADER, QuARTS determinations, etc.) for testing the DNA sample. Such DNA samples can be used for diagnostic assays, which qualitatively detect the presence of genes, gene variants (e.g., alleles) or genetic modifications (e.g., methylation) present in the sample taken from the patient, or quantitatively measure the activity, expression or amount of the gene, gene variant or genetic modification. For example, some cancers are associated with the presence of specific mutant alleles or specific methylation states, and therefore detection and / or quantitative detection of such mutant alleles or methylation states have predictive value in the diagnosis and treatment of cancer.

[0334] Many valuable genetic markers exist in samples with extremely low amounts, and many events that produce such markers are rare. Therefore, even sensitive detection methods such as PCR also require a large amount of DNA to provide enough low-abundance targets to meet or replace the detection threshold of the assay. Moreover, even the presence of a small amount of inhibitory substances will also damage the accuracy and precision of these assays that relate to detecting such a small amount of targets. Therefore, the method provided herein provides the necessary volume and concentration management for producing such DNA samples.

[0335] In some embodiments, the sample includes blood, serum, plasma or saliva. In some embodiments, the subject is human. Such samples can be obtained by many methods known in the art, such as those apparent to the technician. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known to those skilled in the art (including but not limited to centrifugation and filtration). Although it is generally preferred not to use invasive techniques to obtain samples, it is still preferred to obtain samples such as tissue homogenates, tissue sections and biopsy specimens. The technology is not limited to the method for preparing samples and providing nucleic acids for testing. For example, in some embodiments, using, for example, direct gene capture as described in detail in U.S. Patents 8,808,990 and 9,169,511 and WO 2012 / 155072, or by related methods, DNA is isolated from fecal samples or blood or plasma samples.

[0336] The analysis of markers can be carried out individually or simultaneously with other markers in a test sample. For example, several markers can be combined into a test to efficiently process multiple samples and may provide higher diagnosis and / or prognosis accuracy. In addition, those skilled in the art will recognize the value of testing multiple samples from the same experimenter (for example, at continuous time points). This type of test to serial samples can allow identification marker methylation state changes over time. The variation of methylation state and the lack of variation of methylation state can provide useful information about disease state, and the useful information includes but is not limited to the approximate time of identification event occurrence, the existence and amount of salvageable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and the identification of experimenter's results (including the risk of future events).

[0337] Analysis of biomarkers can be performed in a variety of physical formats. For example, microtiter plates or automation can be used to facilitate processing of large numbers of test samples. Alternatively, a single sample format can be developed to facilitate, for example, prompt immediate treatment and diagnosis in an outpatient transport or emergency room setting.

[0338] It is considered that the embodiment of the present technology is provided in the form of a kit. The kit includes embodiments of compositions, devices, equipment, etc. as described herein, and instructions for use of the kit. Such instructions describe the preparation of analytes by samples, for example, collecting samples and preparing nucleic acids by samples in appropriate methods. The 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) to facilitate storage, transportation and / or use by users of the kit. It should be understood that liquid components (e.g., buffer) can be provided in lyophilized form for user reconstruction. The kit may include controls or references for evaluating, verifying and / or ensuring the performance of the kit. For example, a kit for determining the amount of nucleic acid present in a sample may include such controls, the controls comprising the same nucleic acid or another nucleic acid of known concentration for comparison, and in some embodiments, the kit includes a detection reagent (e.g., primer) 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 components of the kit provide the functionality of a system for preparing a nucleic acid solution by a sample. In some embodiments, certain components of the system are provided by the user.

[0339] III. Application

[0340] In some embodiments, the diagnostic assay identifies the presence of a disease or condition in an individual. In some embodiments, the disease is cancer (e.g., colon cancer). In some embodiments, markers whose abnormal methylation is associated with colon cancer are used (e.g., one or more markers selected from the markers listed in Table 1, or preferably one or more of the following: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D, QKI, FER1L4). In some embodiments, the assay further comprises detecting a reference gene (e.g., β-actin, ZDHHC1, B3GALT6).

[0341] In some embodiments, the technology can be applied to treat patients (e.g., patients with colon cancer, patients with early colon cancer, or patients who may develop colon cancer), the method comprising determining the methylation state of one or more markers as provided herein, and administering treatment to the patient based on the results of determining the methylation state. The treatment can be administration of a pharmaceutical compound, a vaccine, surgery, imaging of the patient, or another test. Preferably, the use is in a clinical screening method, a prognostic assessment method, a method for monitoring therapy results, a method for identifying patients most likely to respond to a particular therapeutic treatment, a method for imaging a patient or subject, and a method for drug screening and development.

[0342] In some embodiments, the technology can be applied to methods for diagnosing colon cancer in a subject. As used herein, the terms "diagnosing" and "diagnosing" refer to methods in which a technician can estimate and even determine whether a subject suffers from a specified disease or condition or whether a specified disease or condition may develop in the future. Technicians often diagnose based on one or more diagnostic indicators (e.g., biomarkers whose methylation state indicates the presence, severity, or absence of a condition).

[0343] Along with diagnosis, clinical cancer prognosis involves determining the aggressiveness of the cancer and the likelihood of tumor recurrence to plan the most effective therapy. If a more accurate prognosis can be made or even the potential risk of developing cancer can be assessed, appropriate therapy can be selected, and in some cases a less stringent therapy can be selected for the patient. Evaluation of cancer biomarkers (e.g., determining their methylation status) can be used to separate subjects with a good prognosis and / or a low risk of developing cancer, who do not need therapy or require limited therapy, from those who are more likely to develop cancer or suffer cancer recurrence and who may benefit from more intensive treatment.

[0344] Thus, as used herein, "making a diagnosis" or "diagnosis" further includes determining the risk of developing cancer or determining a prognosis, which can provide for predicting clinical outcome (with or without medical treatment), selecting an appropriate treatment (or whether a treatment is effective), or monitoring current treatment and potentially changing that treatment based on measurement of the diagnostic biomarkers disclosed herein.

[0345] Further, in some embodiments of the present technology, multiple determinations of biomarkers can be performed over time to facilitate diagnosis and / or prognosis. Temporal changes in biomarkers can be used to predict clinical outcomes, monitor the progression of colon cancer, and / or monitor the efficacy of appropriate therapies for cancer. In such embodiments, for example, one may expect to see changes in the methylation state of one or more biomarkers disclosed herein (and possibly one or more additional biomarkers if monitored) in a biological sample over time during an effective therapy.

[0346] The technology can be further applied to methods for determining whether to start or continue prevention or treatment of cancer in a subject. In some embodiments, the method includes providing a series of biological samples from a subject over a period of time; analyzing the series of biological samples to determine the methylation state of at least one biomarker disclosed herein in each biological sample; and comparing any measurable changes in the methylation state of one or more biomarkers in each biological sample. Any changes in the methylation state of the biomarkers during this time period can be used to predict the risk of developing cancer, predict clinical outcomes, determine whether to start or continue prevention or treatment of cancer, and whether the current therapy is effective in treating cancer. For example, a first time point can be selected before the start of treatment, and a second time point can be selected at a time after the start of treatment. The methylation state can be measured in each sample taken from different time points, and qualitative and / or quantitative differences can be recorded. Changes in the methylation state of the biomarker levels from different samples can be related to the risk of the subject developing colon cancer, the prognosis of the subject, determining the efficacy of treatment of the subject, and / or cancer progression.

[0347] In preferred embodiments, the methods and compositions of the invention are used to treat or diagnose a disease at an early stage, such as before disease symptoms appear. In some embodiments, the methods and compositions of the invention are used to treat or diagnose a disease at a clinical stage.

[0348] As described above, in some embodiments, multiple determinations can be performed to one or more diagnostic or prognostic biomarkers, and the time variation of the marker can be used to determine the diagnosis or prognosis. For example, the diagnostic marker can be determined at the initial time, and the diagnostic marker can be determined again at the second time. In such embodiments, the increase of the marker from the initial time to the second time can diagnose a specific type or severity of cancer, or a given prognosis. Similarly, the reduction of the marker from the initial time to the second time can indicate a specific type or severity of cancer, or a given prognosis. In addition, the degree of change of one or more markers can be related to the severity of cancer and future adverse events. The technician will understand that, although in certain embodiments, the same biomarker can be compared and measured at multiple time points, a given biomarker can also be measured at one time point, and a second biomarker can be measured at a second time point, and the comparison of these markers can provide diagnostic information.

[0349] As used herein, the phrase "determining a prognosis" refers to a method by which a skilled person can predict the course or outcome of a condition in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even the ability to predict that a given process or outcome is more or less likely to occur based on the methylation state of a biomarker. Instead, a skilled person will understand that the term "prognosis" refers to an increased probability that a certain process or outcome will occur; that is, the process or outcome is more likely to occur in a subject exhibiting a given condition when compared to an individual who does not exhibit the given condition. For example, in an individual who does not exhibit the condition, the likelihood of a given outcome (e.g., developing colon cancer) may be very low.

[0350] In some embodiments, statistical analysis associates prognostic indicators with a tendency toward adverse outcomes. For example, in some embodiments, as determined by a statistical significance level, a methylation state different from the methylation state in a normal control sample obtained from a patient who has never had cancer can predict that a subject is more likely to have cancer than a subject with a level more similar to the methylation state in the control sample. In addition, changes in methylation state from baseline (e.g., "normal") levels can reflect a subject's prognosis, and the degree of change in methylation state can be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and / or p value. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is incorporated herein by reference in its entirety. Exemplary confidence intervals for the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 and 0.0001.

[0351] In other embodiments, the threshold change degree of the methylation state of the prognostic or diagnostic biomarker disclosed herein can be determined, and the change degree of the methylation state of the biomarker in the biological sample is simply compared with the threshold change degree of the methylation state. The preferred threshold change of the methylation state of the biomarker provided herein is 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 "nomogram" can be established, by which the methylation state of the prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to the relevant tendency for a given result. The technician is familiar with the use of such nomograms to associate two numerical values, and understands that the uncertainty in the measurement is the same as the uncertainty of the marker concentration, because the reference is a single sample measurement value, not a population average.

[0352] In some embodiments, the control sample is analyzed in parallel with the biological sample so that the results obtained from the biological sample can be compared with the results obtained from the control sample. In addition, it is contemplated that a standard curve can be provided, using which the results of the determination of the biological sample can be compared. If a fluorescent marker is used, such a standard curve presents the methylation state of the biomarker as a function of the assay unit (e.g., fluorescence signal intensity). Using samples taken from multiple donors, a standard curve for the control methylation state of one or more of the biomarkers in normal tissues and a "risky" level of the one or more biomarkers in tissues taken from donors with colon cancer can be provided.

[0353] The analysis of markers can be carried out individually or simultaneously with other markers in a test sample. For example, several markers can be combined into a test to efficiently process multiple samples and possibly provide higher diagnosis and / or prognosis accuracy. In addition, those skilled in the art will recognize the value of testing multiple samples from the same experimenter (for example, at continuous time points). This type of test to serial samples can allow identification marker methylation state changes over time. The variation of methylation state and the lack of variation of methylation state can provide useful information about disease state, and the useful information includes but is not limited to the approximate time of identification event occurrence, the existence and amount of salvageable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and the identification of experimenter's results (including the risk of future events).

[0354] Analysis of biomarkers can be performed in a variety of physical formats. For example, microtiter plates or automation can be used to facilitate processing of large numbers of test samples. Alternatively, a single sample format can be developed to facilitate, for example, prompt immediate treatment and diagnosis in an outpatient transport or emergency room setting.

[0355] In some embodiments, if there is a measurable difference in the methylation state of at least one biomarker in the sample compared to the control methylation state, the subject is diagnosed as having colon cancer. In contrast, when no change in methylation state is identified in the biological sample, the subject can be identified as not having colon cancer, not being at risk of cancer, or having a low risk of cancer. In this regard, subjects with colon cancer or its risk can be distinguished from subjects with low to substantially no cancer or its risk. Those subjects with a risk of developing colon cancer can be included in a more intensive and / or regular screening program. On the other hand, those subjects with low to substantially no risk can avoid receiving a screening program until future screenings (e.g., screenings performed according to the present technology) indicate that a risk of colon cancer has occurred in those patients.

[0356] As described above, according to the embodiments of the methods of the present technology, detecting the change in the methylation state of the one or more biomarkers can be a qualitative determination, or can be a quantitative determination. Therefore, the step of diagnosing that the subject suffers from colon cancer or has a risk of developing colon cancer indicates that certain threshold measurements have been performed, for example, the methylation state of the one or more biomarkers in the biological sample is different from the predetermined control methylation state. In some embodiments of the method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method, wherein the control sample is tested in parallel with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of the method, the predetermined methylation state is based on a standard curve and / or is identified by a standard curve. In other embodiments of the method, the predetermined methylation state is a specific state or a range of states. Thus, the predetermined methylation state can be selected within acceptable limits that will be apparent to those skilled in the art, based in part on the embodiments of the method practiced and the desired specificity, etc.

[0357] 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. Molecular profiling of rare cells is important in biological and clinical research. Applications include characterizing circulating epithelial cells (CEpC) in the peripheral blood of cancer patients for disease prognosis and personalized treatment (see, e.g., 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). Thus, embodiments of the present disclosure provide compositions and methods for detecting the presence of metastatic cancer in a subject by identifying the presence of methylation markers in plasma or whole blood.

[0358] Experimental Examples

[0359] Example 1

[0360] Sample preparation method

[0361] Methods for DNA isolation and QUARTS assay

[0362] Provided below are exemplary methods for DNA isolation prior to analysis, and exemplary QuARTS assays, such as may be used according to embodiments of the present technology. In this example, the application of QuARTS technology to DNA from blood and various tissue samples is described, but the technology is readily applicable to other nucleic acid samples as shown in other examples.

[0363] DNA isolation from cells and plasma

[0364] For cell lines, e.g. RSC ccfDNA plasma kit (Promega Corp., Madison, WI) is used to separate genomic DNA from cell conditioned medium. According to the kit protocol, 1 mL of cell conditioned medium (CCM) is used instead of plasma, and the medium is processed according to the kit procedure. The elution volume is 100 μL, of which 70 μL is generally used for bisulfite conversion. See also U.S. Patent Application Serial No. 62 / 249,097 filed on October 30, 2015; U.S. Patent Application Serial Nos. 15 / 335,111 and 15 / 335,096 filed on October 26, 2016; and International Application Serial No. PCT / US16 / 58875 filed on October 26, 2016, each of which is incorporated herein by reference in its entirety for all purposes.

[0365] An example of a complete process for isolating DNA from a blood sample for use in, for example, a detection assay is provided in this example. Optional bisulfite conversion and detection methods are also described.

[0366] I. Blood Processing

[0367] Collect whole blood in anticoagulant EDTA or Streck cell-free DNA BCT tubes. An exemplary procedure is as follows:

[0368] 1. Draw 10 mL of whole blood into a vacutainer (anticoagulant EDTA or Streck BCT), collecting the entire volume to ensure the correct ratio of blood to anticoagulant.

[0369] 2. After collection, gently mix the blood by inverting the tube 8 to 10 times to mix the blood and anticoagulant and keep at room temperature until centrifugation, which should be done within 4 hours of blood collection.

[0370] 3. Centrifuge the blood sample at 1500g (±100g) for 10 minutes at room temperature in a swing-out rotor (swing-out head). Do not use the brake to stop the centrifuge.

[0371] 4. Carefully aspirate the supernatant (plasma) at room temperature and pool in a centrifuge tube. Make sure not to disrupt the cell layer or transfer any cells.

[0372] 5. Carefully transfer 4 mL aliquots of supernatant to cryovials.

[0373] 6. As each aliquot is made, tightly cap and place on ice. This process should be completed within 1 hour of centrifugation.

[0374] 7. Ensure that cryovials are properly labeled with relevant information, including details of additives present in the blood.

[0375] 8. Samples can be kept frozen at -20°C for up to 48 hours before being transferred to a -80°C freezer.

[0376] II. Preparation of control DNA during synthesis

[0377] Complementary strands of methylated zebrafish DNA having the sequence shown below were synthesized using standard DNA synthesis methods such as addition of phosphoramidites to incorporate 5-methyl C bases at the indicated positions. The synthesized strands were annealed to generate double-stranded DNA fragments used as process controls.

[0378]

[0379] A. Annealing and preparation of synthetic control for concentrated zebrafish (ZF-RASS F1 180mer)

[0380] 1. Reconstitute lyophilized single-stranded oligonucleotides at a concentration of 1 μM in 10 mM Tris (pH 8.0), 0.1 mM EDTA.

[0381] 2. Prepare 10X annealing buffer with 500 mM NaCl, 200 mM Tris-HCl pH 8.0, and 20 mM MgCl2.

[0382] 3. Annealing the synthetic strands:

[0383] In a total volume of 100 μL, combine equimolar amounts of each single-stranded oligonucleotide in 1X annealing buffer, e.g., as shown in the following table:

[0384]

[0385] 4. Heat the annealing mixture to 98°C for 11-15 minutes.

[0386] 5. Remove the tube from the heat source and centrifuge briefly to allow the aggregate to collect at the bottom of the tube.

[0387] 6. Incubate the reaction tube at room temperature for 10 to 25 minutes.

[0388] 7. Add 0.9 mL of fish DNA diluent (20 ng / mL of large fish DNA in Te (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)) to adjust the concentration of zebrafish RASSF1 DNA fragment in the genomic fish DNA vector to 1.0 x 10 10 10 copies / μl of annealed double-stranded synthetic zebrafish RASSF1 DNA.

[0389] 8. Dilute process controls to desired concentrations with 10 mM Tris (pH 8.0), 0.1 mM EDTA, e.g., as described in the table below, and store at -20°C or -80°C.

[0390]

[0391] B. Preparation of 100x Stock Process Control (12,000 copies / μL of zebrafish RASSF1 DNA in 200 ng / μL of large fish DNA)

[0392] 1. Thaw the reagents

[0393] 2. Vortex and centrifuge the thawed reagents

[0394] 3. Add the following reagents to a 50 mL conical tube

[0395]

[0396] 4. Aliquot into labeled 0.5 mL tubes and store at -20 °C

[0397] C. Preparation of 1x process control stock solution (120 copies / μL of zebrafish RASSF1 DNA in 2 ng / μL fish DNA)

[0398] 1. Thaw the reagents

[0399] 2. Vortex and centrifuge the thawed reagents

[0400] 3. Add the following reagents to a 50 mL conical tube:

[0401] Reagents 1mL 5mL 10mL 100x Zebrafish Process Control 10μL 50μL 100μL 10mM Tris (pH 8.0), 0.1mM EDTA 990μL 4950μL 9900μL

[0402] 4. Aliquot 0.3 mL into labeled 0.5 mL tubes and store at -20 °C

[0403] III. DNA Extraction from Plasma

[0404] 1. Thaw plasma, prepare reagents, label tubes, and clean and set up the biosafety cabinet for extraction

[0405] 2. Add 300 μL of Proteinase K (20 mg / mL) to a 50 mL conical tube for each sample.

[0406] 3. Add 2-4 mL of plasma sample to each 50 mL conical tube (do not vortex).

[0407] 4. Swirl or pipette to mix and let stand at room temperature for 5 minutes.

[0408] 5. Add 4-6 mL of Lysis Buffer 1 (LB1) solution to bring the volume to approximately 8 mL.

[0409] LB1 preparation:

[0410] 0.1 mL of 120 copies / μL zebrafish RASSF1 DNA process control as described above;

[0411] 0.9-2.9 mL of 10 mM Tris (pH 8.0), 0.1 mM EDTA (e.g., use 2.9 mL for a 2 mL plasma sample)

[0412] 3 mL of 4.3 M guanidine thiocyanate containing 10% IGEPAL (from a stock solution of 5.3 g IGEPAL CA-630 combined with 45 mL 4.8 M guanidine thiocyanate)

[0413] 6. Invert the tube 3 times.

[0414] 7. Place the tube on a benchtop shaker (room temperature) and shake at 500 rpm for 30 minutes at room temperature.

[0415] 8. Add 200 μL of silica bound beads (16 μg particles / μL) and mix by vortexing.

[0416] 9. Add 7 mL of Lysis Buffer 2 (LB2) solution and mix by vortexing.

[0417] LB2 preparation:

[0418] 4 mL 4.3 M guanidine thiocyanate mixed with 10% IGEPAL

[0419] 3 mL 100% isopropyl alcohol

[0420] (Lysis Buffer 2 can be added before, after or in parallel with the silica-bound beads)

[0421] 10. Invert the tube 3 times.

[0422] 11. Place the tube on a benchtop shaker and shake at 500 rpm for 30 minutes at room temperature.

[0423] 12. Place the tube on the capture aspirator and run the program with magnetic bead aggregation for 10 minutes, then aspirate. This will allow the beads to aggregate for 10 minutes, then remove all liquid from the tube.

[0424] 13. Add 0.9 mL of Wash Solution 1 (3 M guanidine HCl or guanidine thiocyanate, 56.8% EtOH) to resuspend the bound beads and mix by vortexing.

[0425] 14. Place the tube on a benchtop shaker and shake at 400 rpm for 2 minutes at room temperature.

[0426] (All subsequent steps can be completed on the STARlet automation platform.)

[0427] 15. Mix by repeated pipetting and transfer the contained beads to a 96 deep well plate.

[0428] 16. Place the plate on the magnetic stand for 10 minutes.

[0429] 17. Aspirate the supernatant to waste.

[0430] 18. Add 1 mL of Wash Solution 2 (80% ethanol, 10 mM Tris pH 8.0).

[0431] 19. Mix for 3 minutes.

[0432] 20. Place the tubes on the magnetic rack for 10 minutes.

[0433] 21. Aspirate the supernatant to waste.

[0434] 22. Add 0.5 mL of Wash Solution 2.

[0435] 23. Mix for 3 minutes.

[0436] 24. Place the tubes on the magnetic rack for 5 minutes.

[0437] 25. Aspirate the supernatant to waste.

[0438] 26. Add 0.25 mL of Wash Solution 2.

[0439] 27. Mix for 3 minutes.

[0440] 28. Place the tubes on the magnetic rack for 5 minutes.

[0441] 29. Aspirate the supernatant to waste.

[0442] 30. Add 0.25 mL of Wash Solution 2.

[0443] 31. Mix for 3 minutes.

[0444] 32. Place the tubes on the magnetic rack for 5 minutes.

[0445] 33. Aspirate the supernatant to waste.

[0446] 34. Place the plate on a heat block at 70°C for 15 minutes while shaking.

[0447] 35. Add 125 μL of elution buffer (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA).

[0448] 36. Incubate at 65°C for 25 minutes with shaking.

[0449] 37. Place the plate on a magnet and allow the beads to aggregate and cool for 8 minutes.

[0450] 38. Transfer the eluate to a 96-well plate and store at -80° C. The recoverable / transferable volume is approximately 100 μL.

[0451] IV. DNA Quantification before Bisulfite Treatment

[0452] To measure DNA in samples with the ACTB gene and assess zebrafish process control recovery, DNA can be measured prior to further processing. Set up the QuARTS PCR-flap assay using 10 μL of extracted DNA using the following protocol:

[0453] 1. Prepare a 10X oligonucleotide mix containing 2 μM each of the forward and reverse primers, 5 μM of the probe and FRET box, and 250 μM each of deoxynucleoside triphosphates (dNTPs). (Primers, probes, and FRET sequences, see below)

[0454]

[0455]

[0456] 2. Prepare the master mix as follows:

[0457] Components Volume per reaction (μL) water 15.50 10X Oligonucleotide Mix 3.00 20X QuARTS Enzyme Mix* 1.50 Total volume 20.0

[0458] *20X enzyme mix contains 1 unit / μL GoTaq Hot Start Polymerase (Promega), 292ng / μL Creavase 2.0 Flap Endonuclease (Hologic).

[0459] 3. Use a pipette to transfer 10 μL of each sample into a well of a 96-well plate.

[0460] 4. Add 20 μL of the master mix to each well of the plate.

[0461] 5. Seal the plate and centrifuge at 3000 rpm for 1 minute.

[0462] 6. Run the plate on an ABI 7500 or Light Cycler 480 real-time thermal cycler using the following reaction conditions:

[0463]

[0464]

[0465] V. Bisulfite Conversion and Purification of DNA

[0466] 1. Thaw all extracted DNA samples from the DNA extraction in the plasma step and spin down the DNA.

[0467] 2. Reagent Preparation:

[0468]

[0469] 3. Add 5 μL of 100 ng / μL BSA DNA carrier solution to each well of a deep well plate (DWP).

[0470] 4. Add 80 μL of each sample to DWP.

[0471] 5. Add 5 μL of freshly prepared 1.6 N NaOH to each well in the DWP.

[0472] 6. Mix carefully by pipetting with a pipette set to 30-40 μL to avoid bubbles.

[0473] 7. Incubate at 42°C for 20 minutes.

[0474] 8. Add 120 μL of BIS SLN to each well.

[0475] 9. Incubate at 66°C for 75 minutes with mixing during the first 3 minutes.

[0476] 10. Add 750 μL of BND SLN

[0477] 11. Pre-mix silica beads (BND BDS) and add 50 μL of silica beads (BND BDS) to the wells of the DWP.

[0478] 12. Mix on a heated shaker at 1,200 rpm at 30°C for 30 minutes.

[0479] 13. Allow the beads to aggregate on the magnetic plate for 5 minutes and then aspirate the solution to waste.

[0480] 14. Add 1 mL of wash buffer (CNV WSH) and move the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0481] 15. Allow the beads to aggregate on the magnetic plate for 5 minutes and then aspirate the solution to waste.

[0482] 16. Add 0.25 mL of wash buffer (CNV WSH) and move the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0483] 17. Allow the beads to collect on the magnetic plate and then aspirate the solution to waste.

[0484] 18. Add 0.2 mL of desulfonation buffer (DES SLN) and mix at 1,200 rpm for 7 minutes at 30°C.

[0485] 19. Allow the beads to aggregate on the magnetic plate for 2 minutes and then aspirate the solution to waste.

[0486] 20. Add 0.25 mL of wash buffer (CNV WSH) and move the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0487] 21. Allow the beads to aggregate on the magnetic plate for 2 minutes and then aspirate the solution to waste.

[0488] 22. Add 0.25 mL of wash buffer (CNV WSH) and move the plate to a heated shaker and mix at 1,200 rpm for 3 minutes.

[0489] 23. Allow the beads to aggregate on the magnetic plate for 2 minutes and then aspirate the solution to waste.

[0490] 24. Dry the plate by moving to a heated shaker and incubating at 70°C for 15 minutes while mixing at 1,200 rpm.

[0491] 25. Add 80 μL of Elution Buffer (ELU BFR) to all samples in DWP.

[0492] 26. Incubate at 65°C for 25 minutes while mixing at 1,200 rpm.

[0493] 27. Manually transfer the eluate to a 96-well plate and store at -80°C

[0494] 28. The recoverable / transferable volume is approximately 65 μL.

[0495] VI. QuARTS-X Multiplex Flap Assay for Methylated DNA Detection and Quantification

[0496] A. Multiplex PCR (mPCR) setup:

[0497] 1. Prepare a 10X primer mix containing forward and reverse primers for each target methylation marker, each to a final concentration of 750 nM. As described in the above example, use 10 mM Tris-HCl (pH 8), 0.1 mM EDTA as the diluent.

[0498] 2. Prepare 10X multiplex PCR buffer containing 100 mM MOPS (pH 7.5), 75 mM MgCl2, 0.08% Tween 20, 0.08% IGEPALCA-630, and 2.5 mM dNTP.

[0499] 3. Prepare multiplex PCR master mix as follows:

[0500]

[0501] 4. Thaw the DNA and centrifuge the plate.

[0502] 5. Add 25 μL of the master mix to the 96-well plate.

[0503] 6. Add 50 μL of each sample to each well.

[0504] 7. Seal the plate with an aluminum foil seal (do not use a strip cap).

[0505] 8. Place in a heated lid thermal cycler and cycle using the following profile for about 5 to 20 cycles, preferably about 10 to 13 cycles:

[0506]

[0507] 9. After thermal cycling is complete, perform a 1:10 dilution of the amplicon as follows:

[0508] a) Transfer 180 μL of 10 mM Tris-HCl (pH 8), 0.1 mM EDTA to each well of a deep well plate.

[0509] b) Add 20 μL of amplification sample to each pre-filled well.

[0510] c) Mix the diluted sample by repeated pipetting using a new tip and a 200 μL pipette (be careful not to create aerosols).

[0511] d) Seal the diluted plate with a plastic seal.

[0512] e) Centrifuge the diluted plate at 1000 rpm for 1 minute.

[0513] f) Seal any remaining undiluted multiplex PCR products with a new aluminum foil seal and place at -80°C.

[0514] B. QuARTS assay for multiplexed amplified DNA:

[0515] 1. Thaw Fish DNA Diluent (20 ng / μL) and use to dilute the plasmid calibrators required in the assay (see, e.g., U.S. Patent Application Serial No. 15 / 033,803, which is incorporated herein by reference). Use the following table as a guide for dilutions:

[0516]

[0517] 2. Prepare a 10X triple QuARTS oligonucleotide mix for markers A, B, and C (e.g., markers of interest, plus running controls and internal controls such as β-actin or B3GALT6) using the table below (see, e.g., U.S. patent application Ser. No. 62 / 364,082, which is incorporated herein by reference).

[0518]

[0519] For example, the following might be used to detect bisulfite-treated β-actin, B3GALT6, and zebrafish RASSF1 markers:

[0520]

[0521]

[0522] 3. Prepare the QuARTS flap assay master mix using the following table:

[0523] Components Volume per reaction (μL) water 15.5 10X Triple Oligonucleotide Mix 3.0 20X QuARTS Enzyme Mix 1.5 Total volume 20.0

[0524] *20X enzyme mix contains 1 unit / μL of GoTaq Hot Start Polymerase (Promega), 292 ng / μL of Cleavase 2.0 Flap Endonuclease (Hologic).

[0525] 4. Using a 96-well ABI plate, pipette 20 μL of the QuARTS master mix into each well.

[0526] 5. Add 10 μL of appropriate calibrators or diluted mPCR samples.

[0527] 6. Seal the plate with ABI clear plastic seal.

[0528] 7. Centrifuge the plate at 3000 rpm for 1 minute.

[0529] 8. Place the plate in an ABI thermal cycler programmed to run the following thermal protocol and start the instrument

[0530]

[0531] For example, an aliquot (e.g., 10 μL) of diluted pre-amplified DNA is used in a QuARTS PCR-flap assay as described above. See also U.S. patent application Ser. No. 62 / 249,097, filed Oct. 30, 2015; U.S. patent application Ser. No. 15 / 335,096, filed Oct. 26, 2016, and U.S. patent application Ser. No. PCT / US16 / 58875, filed Oct. 26, 2016, each of which is incorporated herein by reference in its entirety for all purposes.

[0532] Example 2

[0533] Selection and testing of methylation markers for colorectal cancer detection in plasma

[0534] Reduced representation bisulfite sequencing (RRBS) data were obtained on tissues from 19 patients with colon cancer, 19 patients with polyps, 19 healthy patients, and DNA extracted from buffy coats of 19 healthy patients.

[0535] After alignment to an in silico bisulfite-converted version of the human genome sequence, average methylation at each CpG island was calculated for each sample type (i.e., tissue or buffy coat), and marker regions were selected based on the following criteria:

[0536] Select regions that are 50 base pairs or longer.

[0537] For the QuARTS flap assay design, the selected regions have a minimum of 1 methylated CpG in each of the following regions: a) the probe region, b) the forward primer binding region, and c) the reverse primer binding region. For both the forward and reverse primers, it is preferred that the methylated CpG is close to the 3' end of the primer, but not at the 3' terminal nucleotide. Exemplary flap endonuclease assay oligonucleotides are shown in Figure 1 middle.

[0538] • Preferably, the buffy coat methylation at any CpG in the target region does not exceed >0.5%.

[0539] • Preferably, the cancer tissue in the target region is methylated > 10%.

[0540] • For assays designed for tissue analysis, normal tissue methylation in the region of interest is preferably <0.5%.

[0541] Based on the above criteria, the markers ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; and ZNF671 were selected and the QuARTS flap assay was designed for them, as shown in Figure 5 . Figure 1 shown.

[0542] 27 markers selected from the tissue screening results were tripled for the determination of β-actin converted by bisulfite and used to test the DNA isolated from the plasma samples as described above. According to the manufacturer's protocol (LuminexCorp.), the CEA protein in plasma was measured using the Luminex Magplex assay. DNA from 2mL plasma samples (89 cancers and 95 normals) was extracted and eluted in 125 μL. 10 μL aliquots of the extracted DNA were used in the QuARTS assay to detect β-actin and zebrafish synthetic targets. 80 μL aliquots of the DNA were bisulfite converted as described in Example 1 and eluted in 70 μL.

[0543] As described in Example 1, Figure 1 Multiplex PCR reactions were performed on 50 μL aliquots of bisulfite-converted DNA samples using forward and reverse primers for the targets indicated in , and the markers were detected using the QuARTS flap assay.

[0544] Based on the sensitivity of various markers, the following 12 methylation markers were selected for further analysis: VAV3, ZNF671, CHST2, FLI1, JAM3, SFMBT2, PDGFD, DTX1, TSPYL5, ZNF568, GRIN2D, QKI

[0545] All 12 markers are used together as Figure 1 Preamplification was performed using the primers indicated for these markers. Figure 1 The pre-amplified material was analyzed in a multiplex QuARTS assay as described in Example 1 using the primers and probes shown in . The multiplex assay was grouped as follows:

[0546]

[0547] In addition to the above, CEA protein was measured for the same samples as described above. The data and results are shown in Figure 3 and 4 The sensitivity of various markers at 90% specificity is as follows:

[0548] Markers Sensitivity at 90% specificity ZNF671 49％ TSPYL5 46％ QKI 41％ JAM3 40％ DTX1 40％ GRIN2D 38％ ZNF568 37％ CEA protein 36％ FLI1 36％ SFMBT2 35％ PDGFD 35％ CHST2 33％ VAV3 31％

[0549] At 95% individual cutoff values ​​for various markers, the following final sensitivities were obtained using the combined data set.

[0550] Cancer Stage Negative Positive Total number of samples Sensitivity I 14 7 21 33％ II 7 18 25 72％ III 7 17 24 71％ IV 1 18 19 95％ total 60 89 67％

[0551] The combined specificity of the assay was (88 / 95 = 92.6%).

[0552] Thus, the combination of these 12 markers plus the CEA protein had a sensitivity of 67% for all cancer tissues tested (88 of 95 cancers) and a specificity of 92.6%. This panel of methylated DNA markers measured on tissue achieves extremely high discrimination for all types of colon cancer while remaining negative in normal colon tissue. The determination of this panel of markers can also be applied to blood- or body fluid-based tests and can be applied, for example, to colon cancer screening.

[0553] Multiple target sequences reported to one dye

[0554] The following experiments involve an amplified flap cleavage assay configured so that multiple target-specific primary cleavage reactions report to a single FRET box, thereby generating a fluorescent signal in a single dye channel. The different targets to be detected can be, for example, different markers or genes, different mutations, or different regions of a single marker or gene. Example 3 involves the use of a single FRET box and dye channel to detect methylation of multiple different markers associated with cancer, such as colorectal cancer, and Example 4 involves the use of a single FRET box and dye channel to detect multiple regions within a single marker.

[0555] Reagents used in the following experiments:

[0556]

[0557]

[0558]

[0559] Example 3

[0560] Multiple markers reporting to one dye

[0561] As discussed above, in some embodiments, it is desirable to use a single FRET box and a single dye channel in a single reaction with a greater number of markers. In tests developed for detecting multiple markers reporting to a single FRET box and a single dye, markers with similar reaction efficiencies (i.e., each target copy produces the same amount of detectable signal) are selected to be combined in multiple reactions reporting to a single dye channel. The advantage of combining detection assays with identical or similar reaction efficiencies is that any single calibrator used for one of the assays can be used as a calibration standard for any and all efficiency-matched detection assays.

[0562] Three markers were selected for testing in a multiple marker / one dye system (SFMBT2, VAV3, and CHST2). These target DNAs were mixed in an oligonucleotide mix where the assay oligonucleotides for all three markers were configured to report to the same FRET box and thus to the same dye (FAM). The three disease-related markers reporting to the FAM dye were combined in the same reaction along with bisulfite-converted β-actin DNA (using a QUASAR 670 FRET box) for detection as a control.

[0563] When tested against plasmid calibrators, the data showed that using multiple markers reporting to a single dye is an efficient approach that overcomes the need to run markers in separate wells.

[0564] Example 3.1

[0565] For the QuARTS Flap Endonuclease Assay with multiple different labels run in a multiplex reaction reporting to a single FRET box, the reaction efficiency of each individual label was first analyzed so that the reactions could be balanced when combined in a multiplex configuration. The assay was run to determine the assay performance of three selected labels (VAV3, SFMBT2_897, and CHST2_7890) that reported to one dye (FAM), were dually labeled with bisulfite converted β-actin (BTACT), and were configured to generate signals reported to a Quasar 670 channel.

[0566] The assay was also configured to determine whether each marker would exhibit similar QuARTS assay performance (slope / intercept / Cp) when the three markers reported to the same channel (FAM).

[0567] An oligonucleotide mixture was prepared containing reagents for detecting all three methylation markers reporting to the FAM FRET box. The oligonucleotide mixture contained the BTACT reagent for detecting the report to the Quasar 670 as a control. The oligonucleotide mixture was tested against plasmid targets containing various plasmids containing marker target DNA and BTACT DNA. Calculations were performed to understand whether the calibration curve of one marker could be used to accurately quantify the other markers. All reactions were repeated 4 times.

[0568] plan:

[0569] Prepare a stock plasmid dilution containing one marker plasmid and one BTACT control plasmid at 20 ng / μL of fish DNA in 10 mM Tris, 0.1 mM EDTA as follows (see reagent table above):

[0570]

[0571]

[0572] VAV3 / BTACT Plasmid Mix Ci, cp / μL Cf,cp / μL Volume added μL VAV3 Plasmid 1.00E+05 1.00E+04 50 BTACT Plasmid 1.00E+05 1.00E+04 50 fDNA Diluent NA NA 400 Total volume NA NA 500

[0573] From the 3 plasmid mixtures prepared above, prepare the following dilutions:

[0574] Cf,cp / μL Ci, cp / μL df Added Ci, μL Diluent, μL Total volume 1.00E+05 1.00E+04 10 50 450 500 1.00E+04 1.00E+03 10 50 450 500 1.00E+03 1.00E+02 10 50 450 500 1.00E+02 1.00E+01 10 50 450 500 1.00E+01 1.00E+00 10 50 450 500

[0575] Prepare a 10X oligonucleotide mix containing assay oligonucleotides (primers, probe, FRET cassette) and dNTPs as follows:

[0576]

[0577] QuARTS Flap Endonuclease Assay Reaction Setup:

[0578] Prepare the master mix for the QuARTS amplification reaction as follows:

[0579] Master mix preparation: 96-well plate -

[0580]

[0581] The reaction setup is as follows:

[0582] Use a multichannel pipette to pipette 20 μl of the master mix into a 96-well QuARTS plate and add 10 μl of sample.

[0583] The plate was sealed and centrifuged at 3000 rpm for 1 minute.

[0584] The plate was run on a LightCycler 480 using the following conditions, detecting on FAM, HEX and Quasar 670 channels: 465-510, 533-580 and 618-660 nM

[0585]

[0586] result:

[0587] Strand number counting using VAV3 / BTACT plasmid calibrator standard curve:

[0588]

[0589] Strand counting using the SFMBT2_897 / BTACT plasmid calibrator standard curve:

[0590]

[0591]

[0592] Strand number counting using CHST2_7890 / / BTACT plasmid calibrator standard curve:

[0593] These data show that:

[0594] No cross-reactive or background signals are generated when the marker and control are amplified and detected together;

[0595] The Cp values ​​of CHST2_7890 and VAV3 are similar;

[0596] The Cp value of SFMBT2_897 occurs at an earlier cycle than CHST2_7890 and VAV3, indicating that this is a faster QuARTS assay reaction;

[0597] • The SFMBT2_897 calibrator and oligo mix combination underestimated the strand counts of VAV3 and CHST2_7890 due to the faster reaction of SFMBT2_897.

[0598] The CHST2_7890 calibrator provided a VAV3 calculation that indicated the assay performance was comparable to the CHST2_7890 assay response, but overestimated the amount of SFMBT2_897;

[0599] The VAV3 calibrator provided a calculated result for CHST2_7890 that indicated the assay performance was comparable to the VAV3 assay response, but produced an overestimation of the amount of SFMBT2_897; and

[0600] • To balance the reaction, the performance of the QuARTS assay detecting SFMBT2_897 needs to be reduced to match the performance of the SFMBT2_897 and CHST2_7890 targets.

[0601] Experiment 3.2

[0602] The above data show that the SFMBT2_897 assay reaction produces a higher signal, indicating a faster reaction. In order to multiplex these markers, the SFMBT2_897 assay should be improved to match the efficiency of the slower assay (i.e., to match the signal output of the VAV3 and CHST2_7890 assays). The following experiment tested whether changing the concentration of the forward primer of SFMBT2_897 would achieve this.

[0603] plan:

[0604] Assays were performed as described in Experiment 3.1 above. A 10X oligonucleotide mixture was assembled containing the components listed above, but with a reduced amount of SFMBT2_897 forward primer for generating a final assay concentration of 200 nM (as in Experiment 3.1), 100 nM, or 50 nM. The concentration of all other assay primers in the final reaction mixture was 200 nM, and the Light Cycler protocol was as described in Experiment 3.1.

[0605] The results showed that reducing the SFMBT2_897 forward primer concentration did not seem to affect the slope or intercept of the signal curve reflecting PCR efficiency (data not shown). In addition, the Cp value did not change, so the chain number calculated for SFMBT2_897 did not match the chain number calculated for other marker targets.

[0606] Experiment 3.3 :

[0607] The following experiments tested whether varying the concentration of the SFMBT2_897 probe would reduce the efficiency of the SFMBT2_897 assay to match the signal output of the CHST2_7890 and VAV3 amplification reactions.

[0608] The assay was performed as described in Experiment 3.1 above. A 10X oligonucleotide mix was assembled containing the components listed above, but with an amount of SFMBT2_897 probe oligonucleotide to produce a final assay concentration of 250 nM or 100 nM, while CHST2_7890 and VAV3 probes were present at 500 nM (as described in Experiment 3.1). The Light Cycler protocol was as described in Experiment 3.1.

[0609] result:

[0610] Strand number counting using VAV3 / BTACT plasmid calibrator standard curve:

[0611]

[0612]

[0613] Strand counting using the SFMBT2_897 / BTACT plasmid calibrator standard curve:

[0614]

[0615] Strand counting using the CHST2_7890 / BTACT plasmid calibrator standard curve:

[0616]

[0617]

[0618] result:

[0619] These data show that adjusting the probe concentration down resulted in a slight increase in intercept and a slight increase in PCR % efficiency. The Cp value also increased, so the calculation of the strand count gave a value similar to that calculated using the other markers as calibration standards.

[0620] The 250 nM SFMBT2_897 probe concentration resulted in similar chain count calculations for the three markers, with the SFMBT2_897 chain counts slightly higher than the other markers. The 50 nM concentration of the probe produced a slightly underestimated chain count, but this probe provided some improvement. Therefore, the 200 nM SFMBT2_897 probe concentration was selected for further testing.

[0621] Experiment 3.4 :

[0622] This experiment tested the standard conditions described in Experiment 3.1 for a 10X oligonucleotide mixture providing 200 nM SFMBT2-897 probe and 500 nM other probes (all marker probes were used at 500 nM). This experiment will also determine if there is an additive effect with multiple targets in a single reaction, all using the same FRET box and dye reporter signal. Single, double and triple combinations of plasmid targets were used, all target combinations included the BTACT target as a control.

[0623] Plasmid dilutions of one marker plus control:

[0624] For reactions using a single marker plasmid plus a BTACT control plasmid, a mixture was prepared containing 1.00E+04 copies / μL of each plasmid in a diluent of 20 ng / μL fish DNA in 10 mM Tris, 0.1 mM EDTA. The marker plasmids are described in the reagent table in Experiment 3.1. The targets in the plasmid mixture were as follows:

[0625] -SFMBT2_897 / BTACT

[0626] -CHST2_7890 / BTACT

[0627] -VAV3 / BTACT

[0628] Plasmid dilutions for both markers plus control:

[0629] For reactions using two marker plasmids plus a BTACT control plasmid, a mixture was prepared containing 1.00E+04 copies / μL of each plasmid in 20 ng / μL of fish DNA diluent in 10 mM Tris, 0.1 mM EDTA. The targets in the plasmid mixture were as follows:

[0630] -SFMBT2_897 / VAV3 / BTACT

[0631] -CHST2_7890 / VAV3 / BTACT

[0632] -CHST2_7890 / SFMBT2_897 / BTACT

[0633] Plasmid dilutions of the three markers plus control:

[0634] For reactions using the three marker plasmids plus the BTACT control plasmid, a mixture was prepared containing 1.00E+04 copies / μL of each plasmid in 20 ng / μL of fish DNA diluent in 10 mM Tris, 0.1 mM EDTA. The plasmid mixture was as follows:

[0635] -VAV3 / CHST2_7890 / SFMBT2_897 / BTACT

[0636] Each plasmid mixture was used to prepare solutions having 1.00E+03 copies / μL and 1.00E+02 copies / μL of each plasmid in fish DNA diluent.

[0637] 10X oligonucleotide mix containing primers and probes for all 3 markers and the BTACT control plasmid and having probe concentrations to produce 500 nM probe in each QuARTS assay reaction, except for the SFMBT2_897 probe, which was provided in an amount to provide a 200 nM SFMBT2_897 probe concentration in each reaction. The QuARTS assay components were mixed and assayed on a light cycler as described in Experiment 3.1.

[0638] result:

[0639] Strand number counting using VAV3 / BTACT plasmid calibrator standard curve:

[0640]

[0641] Strand number counting for single markers plus control plasmid:

[0642]

[0643] Strand number counts for both markers plus control plasmid:

[0644]

[0645] Add up the expected number of chains

[0646]

[0647]

[0648] Add up the expected number of chains

[0649]

[0650]

[0651] Add up the expected number of chains

[0652]

[0653] Strand number counts for three markers plus a control plasmid:

[0654]

[0655] Add up the expected number of chains

[0656]

[0657] Strand counting using the SFMBT2_897 / BTACT plasmid calibrator standard curve:

[0658]

[0659] Strand number counting for single markers plus control plasmid:

[0660]

[0661] Strand number counts for both markers plus control plasmid:

[0662]

[0663] Add up the expected number of chains

[0664]

[0665]

[0666] Add up the expected number of chains

[0667]

[0668]

[0669] Add up the expected number of chains

[0670]

[0671] Strand number counts for three markers plus a control plasmid:

[0672]

[0673] Add up the expected number of chains

[0674]

[0675] Strand counting using the CHST2_7890 / BTACT plasmid calibrator standard curve:

[0676]

[0677]

[0678] Strand number counting for single markers plus control plasmid:

[0679]

[0680] Strand number counts for both markers plus control plasmid:

[0681]

[0682] Add up the expected number of chains

[0683]

[0684]

[0685]

[0686] Add up the expected number of chains

[0687]

[0688]

[0689] Add up the expected number of chains

[0690]

[0691] Count the strands of the three markers plus the control plasmid:

[0692]

[0693] Add up the expected number of chains

[0694]

[0695] These data confirm the results shown in Experiment 3.2, indicating that tuning down the SFMBT2_897 probe concentration to 200 nM makes the efficiency of this assay reaction consistent with the efficiency of the reaction detecting VAV3 and CHST2_7890. They also show that when multiple targets in a reaction report signals to the same FRET box and dye channel, the results show an additive effect in the amount of fluorescent signal generated in the reaction. Surprisingly, no increase in background or cross-reactivity was observed.

[0696] The data further show that when the VAV3 dilution series is used as a calibration standard, the strand counts for SFMBT2_897 and CHST2_7890 DNA calculated from the data at the low end of the curve are an overestimation of the actual amount added to these reactions. The VAV3 amplification curve is more variable at the low end of the standard curve, leading to an overestimation of the strand counts for the other markers.

[0697] Experiment 3.5 :

[0698] In this experiment, when the VAV3 calibration curve was used as a reference curve for calculating DNA concentrations, the probe and primer concentrations for the VAV3 marker were adjusted to reduce overestimation of low-level targets.

[0699] For the VAV3 calibration curve, a dilution series with a combination of VAV3 plasmid and BTACT plasmid was as described in Experiment 3.4. Plasmid dilutions with all three markers plus a BTACT control were used.

[0700] Prepare a 10X oligonucleotide mix containing primers and probes for all 3 markers and the BTACT control plasmid, providing primers and probes that produce the concentrations shown below:

[0701] 1. VAV3 (400 nM primer) / SFMBT2_897 (200 nM probe) / CHST2_7890 / BTACT

[0702] 2. VAV3 (750 nM probe) / SFMBT2_897 (200 nM probe) / CHST2_7890 / BTACT

[0703] 3. VAV3 / SFMBT2_897 (200 nM probe) / CHST2_7890 / BTACT

[0704] In addition to the changes in primer and probe concentrations indicated above, all other primers had a final reaction concentration of 200 nM each, and all other probes had a final reaction concentration of 500 nM each. QuARTS assay reactions were mixed and assayed on a light cycler as described in Experiment 3.1.

[0705] Both condition modifications improved the slope of the low calibrator in the VAV3 assay, but these conditions did produce the same signal as the single marker oligonucleotide mixture. The data showed that the single marker mixture did not have the problem of overestimation of strand counts at the lower end of the standard curve. Based on these data, 400nM of each VAV3 primer and 500nM probe were selected for the investigation described in the testing of clinical samples.

[0706] Experiment 3.6

[0707] This experiment tested a multiple marker / 1 dye sample configuration on human clinical plasma samples. The plasma samples were previously tested using the standard one marker: one dye approach, as described in Example 2. The same samples were retested using an oligonucleotide mix with VAV3, SFMBT2897, and CHST27890 reporting to one fluorescent channel (FAM).

[0708] In Example 2, DNA was prepared from a series of plasma samples and the target DNA was amplified for the QuART assay. Figure 3 ) generated amplicon material was diluted 1:10 and tested using the 3-target / 1 control oligonucleotide mix described above in Experiment 3.5.

[0709] Single marker / BTACT plasmid calibrator dilutions were prepared as described in Experiment 3.1. A 10X oligonucleotide mix containing primers and probes for all three markers and BTACT control DNA was used and configured to generate reactions with 400 nM of each VAV3 primer and 200 nM SFMBT2897 probe, and with 200 nM of all other primers and 500 nM of all other probes, as described in Experiment 3.5. The QuARTS assay was mixed and assayed on a light cycler as described in Experiment 3.1. Each reaction was performed in duplicate.

[0711] Each of the three different marker calibration curves was used to calculate the target chain count counts for each sample separately. Regardless of which standard curve was used, the resulting chain count values ​​were similar. In addition, the chain counts for each sample using a single dye configuration were close to the combined chain counts of the marker set measured using a separate FRET box and dye channel in Example 2. Further, when using multiple markers that report to a dye configuration, the chain counts were detected to be zero, i.e., samples that did not generate signals in the Example 2 experiment, remained zero, indicating that the background signal did not increase when the multiple reactions were reported to the FRET box / single dye channel.

[0712] These results show that using a plurality of different target sites reported to a FRET box and the same dye, for example, a plurality of different marker genes, can increase detection sensitivity, and also show that multiple combinations do not need to be subject to the number limit of the available dye channels for signal detection. In addition, the use of this approach is not limited to having a single dye in each reaction well. For example, determination can be configured to have three (or more) markers reported to the first dye (for example, FAM) and three (or more) markers reported to the second dye (for example, HEX), so that the quantity of the marker that can be tested in the single reaction to a single nucleic acid sample preparation is doubled. Can be for other marker set and / or for one or more internal control targets use other dye channels.

[0713] Example 4

[0714] Multiple regions of a marker reporting to one dye

[0715] For three methylation markers, VAV3 (877), SFMBT2 (897), and CHST2 (7890), that showed low to zero chain counts in normal plasma using the methods described above, additional QuARTS assay oligonucleotide sets targeting additional regions within each marker were designed and tested to see whether detecting additional regions of the marker in the same reaction and reporting to the same dye channel would increase the signal-to-noise ratio for each marker, thereby increasing the sensitivity of the assay (e.g., in cancer detection).

[0716] For each of these markers, two distinct regions were identified that were differentially methylated between cancer and normal tissues by RRBS. These regions were:

[0717] -VAV3 zone 877: chr1:108507618-108507675

[0718] -VAV3 region 11878: chr1: 108507406-108507499

[0719] -SFMBT2 region 895:chr10:7452337-7452406

[0720] -SFMBT2 region 897:chr10:7452865-7452922

[0721] -CHST2 region 7890: chr3:142838847-142839000

[0722] -CHST2 region 7889:chr3:142838300-142838388

[0723] Experiment 4.1

[0724] The CHST2 regions (7889 and 7890) that report on the HEX dye were tested individually and in combined reactions to evaluate any synergy between the two regions when combined. Calibrator plasmids containing the CHST2 insert were diluted as described in Experiment 3.1 to produce a dilution series of 1E4 to 1E0 copies per μL. For individual detection of region 7889, the assay reaction contained the forward and reverse primers and arm 1 probe of CHST2_7889, the arm 1 HEX FRET box, and the primers and arm 3 probe of the BTACT control, together with the arm 3 Quasar 670 FRET box. For individual detection of region 7890, the assay reaction contained the forward and reverse primers and arm 1 probe of CHST2_7890, the arm 1 HEX FRET box, and the primers and arm 3 probe of the BTACT control, together with the arm 3 Quasar 670 FRET box. The combined reaction contained the complete set of Arm 1 probes and primers for CHST2_7889 and 7890, along with an oligonucleotide for detection of BTACT and two identical FRET cassettes.

[0725] The 10X oligonucleotide mix contained primers and probes to produce concentrations of 500 nM of each probe and 200 nM of each primer in each QuARTS assay reaction. The QuARTS assay components were mixed and assayed on a light cycler as described in Experiment 3.1.

[0726] It was found that using a single FRET box to have both regions report to the same dye in the combined reaction did not result in any increase in signal. CHST2_7889 amplification was significantly more efficient and appeared to dominate the signal produced, suggesting that the different reactions should be modified to have more similar efficiencies, as discussed in Example 3 above.

[0727] Experiment 4.2

[0728] Experiments were performed to determine what probe concentration should be used to balance the reaction kinetics between the different regions for each pair of regions in each marker {CHST2 (7889 and 7890), SFMBT2 (895 and 897), and VAV3 (877 and 11878)}. 10X oligonucleotide mixes were prepared to provide the following assay oligonucleotide mixes at the indicated final concentrations:

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735] The QuARTS assay components were mixed and assayed on a light cycler as described in Experiment 3.1. The average Cp values ​​obtained under different reaction conditions were as follows:

[0736]

[0737] These data indicate that by varying the probe concentration, the Cp values ​​for individual assays can be adjusted to <1 Cp for each of the five points of the calibration curve for each of the two regions for each marker. For the markers tested, the following probe concentrations were used in the QuARTS assay reactions, yielding ensemble-balanced reaction efficiencies for the target regions:

[0738] Markers [Probe] -A5-FAM [Probe]-A1-HEX SFMBT2_895 - 0.5uM SFMBT2_897 - 0.125uM CHST2_7889 0.25uM - CHST2_7890 1uM - VAV3_877 1uM - VAV3_11878 0.25uM -

[0739] Experiment 4.3

[0740] New triplex reactions (see Example 2 for original triplex reaction configuration) were designed to use multiple regions / one dye assay configurations in multiplex reactions. "Library 17" below lists a collection of 6 markers that were co-amplified with a β-actin control and then analyzed in a triplex QuARTS assay in the groups shown below. Library 17+MR-OD is suitable for multiple regions / one dye assay configurations including those for SFMBT2, VAV3, and CHST2 markers. JAM3, ZNF671, and ZNF568 assay designs were Figure 1 and Figure 2 The 3 or 4 letter abbreviation for each group in the library is the first letter of each gene name, with A representing the β-actin control.

[0741]

[0742] The new triplicate formulation was tested on a plasmid calibration dilution series containing a library 17 multiplex that included all target regions in the groups listed above in a series of dilutions that provided 2e5 to 2e1 strands of each target for each assay reaction. The final concentrations of probes for SFMBT2, VAV3, and CHST2 MR-OD were as described in the results of Experiment 4.2. Probes for JAM3, ZNF671, and ZNF568 markers and BTACT control were 1 μM. All FRET boxes were 500 nM in the final reaction mixture. The QuARTS assay components were mixed and assayed on a light cycler as described in Experiment 3.1

[0743] The triplicates containing VAV3-877 plus VAV-11878 performed as expected, giving an increase of approximately 2-3 times the chain count compared to the target count added to the reaction, while the target had only one region targeted. However, the triplicates containing CHST2-7889_CHST-7890 and SFMBT2-895_SFMBT2-897 did not show the expected summed signal. Further experiments were performed using probes of CHST2-7889_CHST2-7890 and SFMBT2-895_SFMBT2-897 at different concentrations to test them in a multiple QuARTS assay grouped as shown above. In the triple format, the probe concentrations of CHST2_7889 and CHST2_7890 can be varied to achieve the expected MR_OD results (i.e., results with expected summed values ​​for each reaction) based on the plasmid calibration curve. However, the SFMBT2_895 and SFMBT2_897 assays, although improved using altered probe concentrations, still produced signals below the expected 200% level expected for detection of both regions when used in a triplex format. Nonetheless, the following modified probe concentrations were selected for testing the triplex assay on plasma samples.

[0744]

[0745] Experiment 4.4

[0746] This experiment examined the effect of combining multiplex preamplification and triplex QuARTS assay detection using a multiple regions-one dye assay design to test human plasma samples from normal and cancer patients. The experiment compared detection of 13 methylation markers of library 17 (plus a process control, ZF_RASSF1) to detection of 63 normal plasma samples and 12 colon cancer plasma samples using the library 17+MR_OD configuration. The markers of library 17 were co-amplified together in preamplification, and the preamplified DNA was then detected in the grouped reactions listed above and as described in detail in Example 1.

[0747]

[0748] The triplicate names contain the first letter of each marker included, plus 'A' for the β-actin control. Double letters in the triplicate names in the right column (eg, "JSSA") indicate a single marker tested in two different areas.

[0749] DNA was isolated from plasma samples as described in Example 1. Bisulfite conversion, multiplex preamplification, and QuARTS assays on multiplex amplified DNA were performed as described in Example 1. Prior to bisulfite conversion, an aliquot of the isolated DNA was saved for testing for KRAS 38A and 35C mutations on unconverted DNA. The amplification primers and detection probes used for each marker were as described in Example 1. Figure 1 and 2 shown.

[0750] Logistic linear regression fits using the number of chains per reaction for VAV3, SFMBT2, CHST2, and ZNF671 showed considerable advantages when QuARTs was used in combination with MR_OD (multiple regions_one dye) compared to the standard QuARTs assay configuration, as shown below. In these analyses, marker ZNF671 was the main contributor to the assay results and was included in the logistic fits for QuARTs alone and QuARTs+MR_OD. As described above, KRAS38A and 35C mutations were also tested on unconverted DNA.

[0751] Using a standard triplex assay, the following sensitivity and specificity were obtained using multiplex preamplification:

[0752] Multiplexing with standard QuARTs assays

[0753]

[0754] When using a multiple regions / one dye configuration, sensitivity and specificity are as follows:

[0755] Multiplexing analysis using the QuARTs assay using multiple regions_one dye (MR_OD)

[0756]

[0757] Although the sample amount was small, the use of this multiple regions-one dye (FRET cassette) configuration showed a significant improvement in sensitivity, but the solution may lead to some loss of specificity.

[0758] It should be noted that while this example detects DNA isolated from plasma samples, the use of this panel of markers and modified multiplexed QuARTS assay as described above can be applied to stool or other blood or body fluid based testing and can be applied to, for example, colon cancer and other cancer screening.

[0759] All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the various embodiments described herein belong. When the definition of a term in an incorporated reference appears to be different from the definition provided in this teaching, the definition provided in this teaching shall prevail.

[0760] Various modifications and variations of the uses of the described compositions, methods, and techniques will be apparent to those skilled in the art without departing from the scope and spirit of the described techniques. Although the present 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. In fact, various modifications of the described modes for carrying out the present invention that are apparent to those skilled in the art of pharmacology, biochemistry, medical science, or related fields are intended to fall within the scope of the appended claims.

Claims

1. A method of characterizing a sample from a subject, comprising: a) providing a sample from a subject; b) determining the amount of at least one methylation marker DNA in the sample, wherein the methylation marker DNA is selected from the group of methylation marker genes consisting of: ANKRD13B; CHST2; CNNM1; GRIN2D; JAM3; LRRC4; OPLAH; SEP9; SFMBT2; SLC12A8; TBX15; ZDHHC1; ZNF304; ZNF568; ZNF671; DOCK2; DTX1; FERMT3; OPLAH; PDGFD; PKIA; PPP2R5C; TBX15; TSPYL5; VAV3; FER1L4; and ZNF671; b) assaying the sample to obtain the amount of a reference nucleic acid in the sample.

2. The method of claim 1, wherein the sample from the subject comprises a blood or plasma sample.

3. A method according to claim 1 or claim 2, wherein the assay comprises obtaining a sample comprising DNA from a subject and treating the DNA obtained from the sample with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues.

4. The method according to claim 1, wherein the at least one methylation marker DNA is selected from the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI. The method according to claim 1 , wherein the at least one methylation marker DNA comprises at least two methylation marker DNAs.

6. The method of claim 1, wherein the at least one methylation marker DNA comprises the group consisting of: VAV3; ZNF671; CHST2; FLI1; JAM3; SFMBT2; PDGFD; DTX1; TSPYL5; ZNF568; GRIN2D and QKI.

7. The method of claim 1, wherein the method further comprises measuring carcinoembryonic antigen (CEA) protein in the sample.

8. The method of claim 3, wherein the reagent comprises a bisulfite reagent.

9. The method of claim 1, wherein determining the amount of methylation marker DNA in the sample comprises determining the methylation status of a base in the methylation marker gene.

10. The method of claim 1, wherein determining the amount of the methylation marker in the sample comprises determining the degree of methylation at a plurality of bases in a methylation marker gene.

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