Detection of colon neoplasia by analysis of methylated DNA
Provided herein is technology for neoplasia screening, and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of cancer, in particular, colorectal cancer.
1. 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.
2. The method of claim 1 , wherein the assay reactions are PCR-flap assay reactions, wherein the oligonucleotides are flap oligonucleotides, and wherein each of the plurality of assay reaction mixtures further comprises at least one hairpin oligonucleotide comprising a region that is complementary to a portion of a flap oligonucleotide in the assay reaction mixture.
3. The method of claim 1 , wherein the assay reaction mixtures comprise bulk fish DNA.
4. The method of claim 1 , wherein the sample is a blood sample, a plasma sample, a stool sample, or a tissue sample.
5. The method of claim 1 , wherein DNA obtained from the sample is treated with a reagent that selectively modifies unmethylated cytosine residues in the obtained DNA to produce modified residues.
6. The method of claim 5 , wherein the reagent comprises a bisulfite reagent.
7. The method of claim 1 , wherein the plurality of methylation marker primer pairs comprises at least one primer pair for amplifying a target region from a methylated marker DNA selected from the group consisting of VAV3; ZNF671; CHST2; FLII; JAM3; SFMBT2; PDGFD; DTX1; ZNF568; GRIN2D; TSPYL5; and QKI.
8. The method of claim 1 , wherein the plurality of methylation marker primer pairs comprises primer pairs for amplifying target regions from methylated marker DNAs comprising the group consisting of VAV3; ZNF671; CHST2; FLII; JAM3; SFMBT2; PDGFD; DTX1; ZNF568; GRIN2D; TSPYL5; and QKI.
9. The method of claim 1 , wherein the two to twenty-four different methylated marker DNAs comprise nucleic acid sequences selected from the group consisting of SEQ ID NOS: 1, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 121, 126, 131, 136, and their complements.
10. The method of claim 1 , wherein the two to twenty-four different methylated marker DNAs are bisulfite-converted DNAs comprising nucleic acid sequences selected from the group consisting of SEQ ID NOS: 2, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, 87, 92, 97, 102, 107, 122, 127, 132, 137, and their complements.