Compositions and methods for performing methylation detection assays

Patent metadata

Field	Value
Publication number	US-2016168643-A1
Application number	US-201514966938-A
Country	US
Kind code	A1
Filing date	Dec 11, 2015
Priority date	Dec 12, 2014
Publication date	Jun 16, 2016
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 . A method of characterizing a sample obtained from a subject, the method comprising:
a) assaying a sample from a subject for an amount of at least one methylated marker gene selected from the group consisting of vimentin, septin 9, NDRG4, BMP3, VAV3, SFMBT2 897, and TFPI2a in a sample obtained from a subject; b) assaying said sample for an amount of methylated ZDHHC1 DNA in said sample; c) comparing the amount of said at least one methylated marker gene to the amount of methylated ZDHHC1 DNA in said sample to determine a methylation state for said at least one marker gene in said sample; and d) generating a record reporting the methylation state for said at least one marker gene in said sample.

2 . The method of claim 1 , wherein said at least one marker is at least two of said markers.

3 . The method of claim 1 , wherein said at least one marker is all of said markers.

4 . The method of claim 1 wherein the assaying comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, or target capture.

5 - 14 . (canceled)

15 . 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.

16 . The method of claim 1 , wherein said assay further comprises the step of identifying a KRAS mutation score in said sample, preferably wherein said measuring of said K-ras mutation score is measured by digital melt curve analysis or quantitative allele-specific PCR.

17 . The method of claim 16 , wherein said assay comprises detecting methylation state of ZDHHC1, BMP3, NDRG4, and identifying a KRAS mutation score in said sample.

18 . The method of claim 1 , wherein said method further comprises the step of determining the presence of hemoglobin in said sample.

19 . The method of claim 1 , wherein said subject is a patient having inflammatory bowel disease.

20 . A kit, comprising:
a) at least one oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to ZDHHC1; and b) at least one additional oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to marker selected from the group consisting of vimentin, NDRG4, BMP3, VAV3, SFMBT2_897, and TFPI2a.

21 . (canceled)

22 . The kit of claim 20 , wherein said kit further comprises bisulfate.

23 . The kit of claim 20 , wherein said kit further comprises at least one oligonucleotide, wherein at least a portion of said oligonucleotide specifically hybridizes to KRAS.

24 . The kit of claim 20 , wherein said kit further comprises reagents for detecting the presence of hemoglobin in a stool sample.

25 . The kit of claim 20 , wherein said oligonucleotide is selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a nucleic acid probe, and an INVADER oligonucleotide.

26 - 28 . (canceled)

29 . The kit of claim 20 , wherein said kit comprises capture reagents comprising oligonucleotides complementary to ZDHHC1 and said one or more markers genes.

30 . A composition, comprising:
a) a complex of a ZDHHC1 nucleic acid and at least one oligonucleotide, wherein at least a portion of said oligonucleotide is hybridized to said ZDHHC1 nucleic acid; and b) one or more reaction mixtures comprising a complex of a target nucleic selected from the group consisting of vimentin, NDRG4, BMP3, VAV3, SFMBT2 897, and TFPI2a and one or more oligonucleotides that specifically hybridize to one or more target genes.

31 . (canceled)

32 . A method of characterizing a sample from a subject having inflammatory bowel disease, comprising:
a) assaying a sample of stool from a subject to determine the presence of methylated ZDHHC1 DNA and β-actin DNA, b) calculating a ratio of β-actin DNA to methylated ZDHHC1 DNA in said sample of stool; c) comparing the ratio of β-actin DNA to methylated ZDHHC1 DNA to a ratio of β-actin DNA to methylated ZDHHC1 DNA correlated to a known severity of inflammation in IBD, wherein the ratio correlated to the known severity of inflammation was previously determined by assaying a sample of stool from the same subject, or wherein the ratio correlated to the known severity of inflammation was determined by assaying a sample of stool from a normal subject; and d) generating a record reporting whether the ratio of β-actin DNA to methylated ZDHHC1 DNA in said subject is lower or higher than the ratio correlated to the known severity of inflammation, or whether the ratio of β-actin DNA to methylated ZDHHC1 DNA in said subject is the same as the ratio correlated to the known severity of inflammation.

33 - 36 . (canceled)

37 . The method of claim 32 , wherein the assaying comprises bisulfate converting said ZDHHC1 DNA and said β-actin DNA.

38 . A composition comprising a strand of DNA comprising the nucleotide sequence of SEQ ID NO.27, in a reaction mixture with one or more nucleic acids selected from the group consisting of: oligonucleotides of SEQ ID NOS:3-10, 33, 59, 60, 56-58, and 63-65, a strand of DNA comprising the nucleotide sequence of SEQ ID NO:54, a strand of DNA comprising the nucleotide sequence of SEQ ID NO:61, and a strand of DNA comprising the nucleotide sequence of SEQ ID NO: 68.

39 . The composition of claim 38 , further comprising a detection probe oligonucleotide, wherein the detection probe oligonucleotide comprises a region that is complementary to a portion of said strand of DNA comprising the nucleotide sequence of SEQ ID NO:27.

40 . The composition of claim 39 , wherein said detection probe oligonucleotide comprises a region that is complementary to a portion of SEQ ID NO 27.

41 . The composition of claim 39 , wherein said detection probe oligonucleotide comprises a reporter molecule.

42 . The composition of claim 41 , where said reporter molecule comprises a fluorophore.

43 . The composition of claim 39 , wherein said detection probe comprises a flap sequence.

44 . The composition of claim 39 , further comprising one or more of a FRET cassette; a FEN-1 endonuclease and a thermostable DNA polymerase.

45 . The composition of claim 44 , wherein said thermostable DNA polymerase is a bacterial DNA polymerase.

46 . A reaction mixture comprising the composition of claim 39 .

47 . A set of reaction mixtures comprising a strand of DNA comprising the nucleotide sequence of SEQ ID NO.27 and one or more nucleic acids selected from the group consisting of: oligonucleotides of SEQ ID NOS:3-10, 33, 59, 60, 56-58, and 63-65, a strand of DNA comprising the nucleotide sequence of SEQ ID NO:54, a strand of DNA comprising the nucleotide sequence of SEQ ID NO:61, and a strand of DNA comprising the nucleotide sequence of SEQ ID NO: 68.

48 . The set of reaction mixtures of claim 47 , wherein said strand of DNA comprising the nucleotide sequence of SEQ ID NO.27 is not in a reaction mixture with any of said one or more nucleic acids.

Assignees

• Exact Sciences Corp
• Mayo Foundation

Inventors

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

Classifications

• C12Q1/6886 Primary

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

• C12Q2600/154

  Methylation markers · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/166

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

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title