Compositions and methods for performing methylation detection assays
Provided herein is technology relating to performing methylation assays. In particular, the technology relates to internal controls for methylation assays.
1. A method of measuring methylation of marker genes in a sample from a subject, the method comprising:
a) extracting DNA from a biological sample from a human subject;
b) measuring amounts of methylated marker DNA for two to seven marker genes selected from the group consisting of vimentin, septin 9, NDRG4, BMP3, VAV3, SFMBT2_897, and TFPI2a in the DNA;
c) measuring an amount of methylated ZDHHC1 DNA in the DNA by amplifying a region of ZDHHC1 in the DNA using a pair of primers that hybridize within a sequence corresponding to SEQ ID NO:26 and its complement, SEQ ID NO:27 and its complement, or SEQ ID NO:33 and its complement, to produce an amplified product; and
d calculating a value for the amount of each of said two to seven methylated marker DNAs measured in said DNA as a percentage of the amount of methylated ZDHHC1 DNA measured in said DNA, wherein the value indicates the amount of methylation of each of the two to seven marker genes measured in the sample.
2. The method of claim 1 , further comprising a step of treating the DNA with a methylation-specific or methylation-sensitive reagent prior to step b).
3. The method of claim 2 , wherein the methylation-specific or methylation-sensitive reagent is selected from a bisulfite reagent, a methylation-specific nuclease, and a restriction enzyme.
4. The method of claim 1 , wherein said two to seven marker genes comprise all seven marker genes.
5. The method of claim 1 , wherein the measuring comprises using one or more of polymerase chain reaction, flap endonuclease assay, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, and target capture.
6. The method of claim 5 , wherein the measuring using target capture comprises capturing the marker genes and the methylated ZDHHC1 DNA with one or more solid supports comprising capture reagents.
7. The method of claim 6 , wherein the capture reagents comprise oligonucleotides complementary to the marker genes and the methylated ZDHHC1 DNA.
8. The method of claim 6 , wherein the solid supports comprise magnetic beads.
9. The method of claim 1 , wherein the sample is a stool sample, a tissue sample, a pancreatic juice sample, a pancreatic cyst fluid sample, a blood sample, or a urine sample.
10. The method of claim 1 , wherein said method further comprises a step of determining the presence of hemoglobin in said sample.
11. The method of claim 1 , wherein said subject is a patient having or suspected of having a gastrointestinal neoplasm and/or inflammatory bowel disease.
12. The method of claim 11 , wherein the neoplasm is a pancreas neoplasm, a colorectal neoplasm, a bile duct neoplasm, a stomach neoplasm, an esophagus neoplasm, or an adenoma.