Compositions and methods for performing methylation detection assays
View Patent ↗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 the amount of methylated marker DNAs in a sample from a subject, the method comprising:
a) extracting genomic DNA from a biological sample from a human subject;
b) treating the extracted genomic DNA with bisulfite;
c) measuring amounts of methylated marker DNA for two genes to seven genes selected from the group consisting of vimentin, septin 9, NDRG4, BMP3, VA V3, SFMBT2 897, and TFPI2a in the bisulfite-treated genomic DNA;
d) measuring the amount of methylated ZDHHC1 DNA in the bisulfite-treated genomic DNA by amplifying a region of ZDHHC1 in the bisulfite treated genomic DNA using a pair of primers, wherein the amplifying produces an amplified product comprising a region of SEQ ID NO: 27, and contacting the amplified product with one or more oligonucleotides that comprise a sequence selected from the group consisting of SEQ ID NO: 28-32 and 34-36;
e) calculating a value for the amount of each of said two to seven methylated marker genes measured in said bisulfite-treated genomic DNA as a percentage of the amount of methylated ZDHHC1 DNA measured in said bisulfite-treated genomic DNA, wherein the value indicates the amount of each of the two to seven methylated marker DNAs measured in the sample.
2. The method of claim 1 , wherein said two to seven marker genes comprise all seven marker genes.
3. The method of claim 1 wherein the measuring comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, or target capture.
4. 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.
5. The method of claim 1 , further comprising a step of identifying a KRAS mutation score in said sample.
6. The method of claim 5 , comprising detecting methylation state of ZDHHC1, BMP3, NDRG4, and identifying a KRAS mutation score in said sample.
7. The method of claim 1 , wherein said method further comprises the step of determining the presence of hemoglobin in said sample.
8. The method of claim 1 , wherein said subject is a patient having inflammatory bowel disease.