Compositions and methods for performing methylation detection assays

Patent metadata

Field	Value
Publication number	US-2022042111-A1
Application number	US-202117409129-A
Country	US
Kind code	A1
Filing date	Aug 23, 2021
Priority date	Dec 12, 2014
Publication date	Feb 10, 2022
Grant date	—

Abstract

Provided herein is technology relating to performing methylation assays. In particular, the technology relates to internal controls for methylation assays.

First claim

1 - 48 . (canceled)

49 . A method of measuring amounts of methylation of marker genes in a sample from a subject, the method comprising:
a) extracting genomic 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 genomic DNA; c) measuring an amount of methylated ZDHHC1 DNA in the genomic DNA by amplifying a region of ZDHHC1 in the genomic 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 genomic DNA as a percentage of the amount of methylated ZDHHC1 DNA measured in said genomic DNA, wherein the value indicates the amount of methylation of each of the two to seven marker genes measured in the sample.

50 . The method of claim 49 , further comprising a step of treating the genomic DNA with a methylation-specific or methylation-sensitive reagent prior to step b).

51 . The method of claim 50 , wherein the methylation-specific or methylation-sensitive reagent is selected from a bisulfite reagent, a methylation-specific nuclease, and a restriction enzyme.

52 . The method of claim 49 , wherein said two to seven marker genes comprise all seven marker genes.

53 . The method of claim 49 , 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.

54 . The method of claim 53 , 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.

55 . The method of claim 54 , wherein the capture reagents comprise oligonucleotides complementary to the marker genes and the methylated ZDHHC1 DNA.

56 . The method of claim 54 , wherein the solid supports comprise magnetic beads.

57 . The method of claim 49 , 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.

58 . The method of claim 49 , further comprising a step of identifying a KRAS mutation score in said sample.

59 . The method of claim 58 , wherein identifying the KRAS mutation score comprises treating the genomic DNA with an oligonucleotide that specifically hybridizes to a KRAS nucleic acid.

60 . The method of claim 58 , wherein identifying the KRAS mutation score comprises measuring mutations in KRAS by digital melt curve analysis or quantitative allele-specific PCR.

61 . The method of claim 58 , comprising measuring an amount of methylation of ZDHHC1, BMP3, and NDRG4, and identifying a KRAS mutation score in said sample.

62 . The method of claim 49 , wherein said method further comprises a step of determining the presence of hemoglobin in said sample.

63 . The method of claim 49 , wherein said subject is a patient having or suspected of having a gastrointestinal neoplasm and/or inflammatory bowel disease.

64 . The method of claim 63 , wherein the neoplasm is a pancreas neoplasm, a colorectal neoplasm, a bile duct neoplasm, a stomach neoplasm, an esophagus neoplasm, or an adenoma.

Assignees

• Exact Sciences Dev Co Llc
• Mayo Found Medical Education & Res

Inventors

• Ahlquist David Alan
• Taylor William Russell
• Mahoney Douglas W
• Lidgard Graham P
• Allawi Hatim T
• Vaccaro Abram Michael

Classifications

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title