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 .- 48 . (canceled)
49 . A composition comprising a reaction mixture, comprising:
a) nucleic acid from a human sample;
b) for each of one or more methylation marker genes selected from the group consisting of vimentin, septin 9, NDRG4, BMP3, VAV3, SFMBT2, and TFPI2a, a pair of primers that specifically hybridize to and are extendible by a DNA polymerase from a region within the methylation marker gene in a target DNA when the target DNA is present in the nucleic acid from the human sample, wherein the target DNA comprises a differentially methylated region of the methylation marker gene that is differently methylated between DNA from a human subject having a neoplasm and DNA from a human subject who does not have a neoplasm;
c) amplicon DNA amplified from at least one target DNA by extension of a pair of primers of b) and comprising a differentially methylated region of the methylation marker gene;
d) a pair of ZDHHC1 primers that specifically hybridize to and are extendible by a DNA polymerase from a region within methylated ZDHHC1 DNA;
e) ZDHHC1 amplicon DNA amplified from methylated ZDHHC1 DNA by extension of a pair of primers of d); and
f) a thermostable DNA polymerase.
50 . The composition of claim 49 , wherein the nucleic acid from the human sample comprises DNA treated with a methylation-sensitive restriction enzyme or with a reagent that selectively modifies unmethylated cytosine nucleotides in DNA.
51 . The composition of claim 49 , further comprising one or more oligonucleotides that each specifically hybridize to a differentially methylated region of a methylation marker gene in one or more amplicon DNAs amplified from the at least one target DNA.
52 . The composition of claim 51 , wherein the one or more oligonucleotides comprise reporter molecules.
53 . The composition of claim 52 , where said reporter molecules comprise fluorophores.
54 . The composition of claim 51 , wherein the one or more oligonucleotides comprise oligonucleotides comprising a flap sequence.
55 . The composition of claim 49 , further comprising one or more of a FRET cassette and a FEN-1 endonuclease.
56 . The composition of claim 49 , wherein the reaction mixture further comprises:
ii) dNTPs; and
iii) a buffer comprising Mg ++ .
57 . The composition of claim 49 , wherein nucleic acid from a human sample comprises DNA prepared from a blood or plasma sample.
58 . The composition of claim 49 , wherein nucleic acid from a human sample comprises DNA prepared from a fecal sample.
59 . The composition of claim 49 , wherein nucleic acid from a human sample comprises DNA prepared from a tissue sample.
60 . A method of measuring amounts of 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) treating the extracted DNA with a reagent that modifies DNA in a methylation-specific manner to produce treated DNA;
c) amplifying the treated DNA using a pair of primers specific for each of one or more methylated markers selected from the group consisting of vimentin, septin 9, NDRG4, BMP3, VAV3, SFMBT2_897, and TFPI2a in the treated DNA;
d) amplifying the treated DNA using a pair of primers specific for methylated reference marker ZDHHC1 in the treated DNA;
e) measuring an amount of amplified methylated marker DNA for each of the one or more methylated markers and an amount of amplified methylated reference marker ZDHHC1 DNA; and
e) calculating a value for the amount of amplified methylated marker DNA for each of the one or more methylated markers relative to the amount of amplified methylated reference marker ZDHHC1 DNA, wherein the value indicates the amount of methylation of each of the one or more methylated markers in the biological sample.
61 . The method of claim 60 , wherein the biological sample comprises a blood or plasma sample.
62 . The method of claim 60 , wherein the biological sample comprises a fecal sample.
63 . The method of claim 60 , wherein the biological sample comprises a tissue sample.
64 . The method of 60 , wherein step c) and step d) take place within a single reaction mixture.
65 . The method of claim 60 , wherein the reagent is selected from a bisulfite reagent, a methylation-specific nuclease, and a restriction enzyme.
66 . The method of claim 60 , wherein the measuring of step e) comprises using one or more of polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nuclease, mass-based separation, and target capture.
67 . The method of claim 60 , wherein said method further comprises a step of assaying the biological sample to detect hemoglobin.
68 . The method of claim 60 , wherein said human subject is a patient having or suspected of having a gastrointestinal neoplasm and/or inflammatory bowel disease.