Detection of colon neoplasia by analysis of methylated DNA

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

a) amplifying DNA from the sample with PCR amplification reagents in one or more reaction mixtures, wherein the PCR amplification reagents comprise:

i) methylation marker primer pairs for amplifying target regions from two to thirteen different methylated marker DNAs selected from the group consisting of ankyrin repeat domain 13B (ANKRD13B); carbohydrate sulfotransferase 2 (CHST2); cyclin and CBS domain divalent metal cation transport mediator 1 (CNNM1); deltex E3 ubiquitin ligase 1 (DTX1); dysferlin (FER1L4); glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D); junctional adhesion molecule 3 (JAM3); platelet derived growth factor D (PDGFD); QKI, KH domain containing RNA binding (QKI); Scm like with four mbt domains 2 (SFMBT2); vav guanine nucleotide exchange factor 3 (VAV3); zinc finger protein 568 (ZNF568); and zinc finger protein 671 (ZNF671); and

ii) at least one control primer pair for amplifying a target region from at least one control DNA;

and

b) measuring amplified amounts of the target regions of the two to thirteen different methylated marker DNAs, and an amplified amount of the target region from the at least one control DNA, wherein the at least one control DNA comprises a region of a B3GALT6 gene.

2. The method of claim 1 , wherein said amplifying DNA from the sample is in one reaction mixture.

3. The method of claim 1 , wherein the sample comprises a blood sample, a plasma sample, a stool sample, or a tissue sample.

4. The method of claim 1 , wherein the assaying comprises treating DNA obtained from the sample with a methylation-specific reagent.

5. The method of claim 4 , wherein the methylation-specific reagent selectively modifies cytosine bases in the obtained DNA to produce modified bases.

6. The method of claim 5 , wherein the methylation-specific reagent comprises a bisulfite reagent.

7. The method of claim 1 , wherein the one or more reaction mixtures comprise methylation marker primer pairs for amplifying target regions from each methylated marker DNA in the group consisting of ANKRD13B, CHST2, CNNM1, DTX1, FER1L4, GRIN2D, JAM3, PDGFD, QKI, SFMBT2, VAV3, ZNF568, and ZNF671.

8. The method of claim 1 , wherein the one or more reaction mixtures comprise bulk fish DNA.

9. The method of claim 1 , wherein the one or more reaction mixtures comprise PCR-flap assay reagents, wherein the PCR-flap assay reagents comprise:

i) flap oligonucleotides that specifically hybridize to target regions amplified from the two to thirteen methylated marker DNAs selected from the group consisting of ANKRD13B, CHST2, CNNM1, DTX1, FER1L4, GRIN2D, JAM3, PDGFD, QKI, SFMBT2, VAV3, ZNF568, and ZNF671; and

ii) a flap oligonucleotide that specifically hybridizes to a target region from the at least one control DNA.

10. The method of claim 9 , wherein the PCR-flap assay reagents further comprise at least one hairpin oligonucleotide comprising a region that is complementary to a 5′ flap portion of at least one flap oligonucleotide.

11. The method of claim 10 , wherein the at least one hairpin oligonucleotide is a FRET cassette.

12. The method of claim 9 , wherein the PCR-flap assay reagents further comprise one or more of dNTPs, a buffer comprising Mg ++ , a FRET cassette, and a FEN-1 endonuclease.

13. The method of 1 , further comprising assaying the sample for carcinoembryonic antigen (CEA) protein.