Detection of colon neoplasia by analysis of methylated DNA

A method of characterizing a sample from a human subject, the method comprising assaying the sample for amounts of a plurality of different methylated marker DNAs and amounts of at least one control DNA, the method comprising:

• a) combining DNA from the sample in a pre-amplification reaction mixture comprising PCR amplification reagents, wherein the PCR amplification reagents comprise:
• i) a plurality of methylation marker primer pairs for amplifying target regions from two to twenty-four different methylated marker DNAs selected from the group of ankyrin repeat domain 13B (ANKRD13B);
• carbohydrate sulfotransferase 2 (CHST2);
• cyclin and CBS domain divalent metal cation transport mediator 1 (CNNM1);
• dedicator of cytokinesis 2 (DOCK2);
• deltex E3 ubiquitin ligase 1 (DTX1);
• fermitin family member 3 (FERMT3);
• Fli-1 proto-oncogene, ETS transcription factor (FLII);
• glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D);
• junctional adhesion molecule 3 (JAM3);
• leucine rich repeat containing 4 (LRRC4);
• 5-oxoprolinase, ATP-hydrolysing (OPLAH);
• platelet derived growth factor D (PDGFD);
• cAMP-dependent protein kinase inhibitor alpha (PKIA);
• protein phosphatase 2 regulatory subunit B′gamma (PPP2R5C);
• QKI, KH domain containing RNA binding (QKI);
• Septin-9 (SEP9);
• Scm like with four mbt domains 2 (SFMBT2);
• solute carrier family 12 member 8 (SLC12A8);
• T-box transcription factor 15 (TBX15);
• TSPY like 5 (TSPYL5);
• vav guanine nucleotide exchange factor 3 (VAV3);
• zinc finger protein 304 (ZNF304);
• zinc finger protein 568 (ZNF568);
• and zinc finger protein 671 (ZNF671);
• and
• ii) a control primer pair for amplifying a target region from beta-1,3- galactosyltransferase 6 (B3GALT6) control DNA;
• b) exposing the pre-amplification reaction mixture to thermal cycling conditions wherein two to twenty-four different methylation marker target regions and a B3GALT6 control DNA target region are amplified to produce a pre-amplified mixture;
• c) partitioning the pre-amplified mixture into a plurality of assay reaction mixtures, wherein each assay reaction mixture comprises:
• i) additional amounts of at least one methylation marker primer pair and of the control primer pair for amplifying a target region from B3GALT6 control DNA used in step a);
• and
• ii) oligonucleotides hybridizable to the methylation marker target region(s) and B3GALT6 control DNA target region amplifiable with the primer pairs of c) i);
• d) measuring amounts of two to twenty-four different methylation marker target regions and the target region from B3GALT6 control DNA in the plurality of assay reaction mixtures in a process comprising exposing the plurality of assay reaction mixtures to thermal cycling conditions wherein amplification of two to twenty-four different methylated marker target regions and the B3GALT6 control DNA target region occurs;
• and
• e) calculating values for the amounts of the two to twenty-four different methylated marker target regions amplified in each of the assay reaction mixtures in step d) as a percentage of the amount of the B3GALT6 control DNA target region amplified in each of the assay reaction mixtures, wherein the calculated values indicate the amount of each of the two to twenty-four different methylated marker DNAs in the sample.