DETECTION OF DNA METHYLATION
In a first aspect, the invention concerns a method for detecting or quantifying DNA methylation at a locus. In one embodiment, a methylation-sensitive endonuclease is formulated together with a polymerase enzyme in an appropriate reaction mixture such that amplification of DNA occurs in a methylation specific manor. Quantitative DNA amplification at selected loci can be used to determine the level of methylation. Kits and reagents for performing such methods are also provided.
1 . A method for detecting the prevalence of methylation of a DNA sequence in a DNA sample comprising:
(i) contacting the DNA sample with a reaction mixture comprising a methylation-sensitive endonuclease (MSE), a DNA polymerase, oligonucleotide primers flanking the DNA sequence and a buffer formulated to facilitate activity of the MSE and the DNA polymerase;
(ii) incubating the sample under conditions permissive for DNA cleavage by the MSE;
(iii) incubating the sample under conditions permissive for quantitative DNA polymerization; and
(iv) quantitating the DNA amplification to thereby quantitate the proportion of methylation in the DNA sequence of the DNA sample,
wherein the reaction mixture volume is not adjusted during steps (ii)-(iii).
2 . The method of claim 1 , wherein the MSE is an enzyme that does not cleave DNA at methylated positions.
3 . The method of claim 1 , wherein the MSE is AciI, HpaII, Hinp1I, HpyCH41V, AatII, AccII, AclI, AfeI, AgeI, AscI, AsiSI, AvaI, BmgBI, BsaAI, BsaHI, BsiEI, BsiWI, BsmBI, BspDI, BstBI, ClaI, EagI, FauI, FseI, FspI, HaeII, HgaI, HhaI, Hpy99I, KasI, MluI, NaeI, NarI, NgoMIV, NotI, NruI, PaeR7I, PmlI, PvuI, RsrII, SacII, SalI, SfoI, SgrAI, SmaI, SnaBI or ZraI.
4 . The method of claim 1 , wherein the MSE is BisI, GlaI or McrBC.
5 . The method of claim 1 , wherein the reaction mixture comprises at least two MSE with specificity to different DNA sequences.
6 . The method of claim 1 , wherein the DNA polymerase is thermophilic polymerase.
7 . The method of claim 1 , wherein the reaction mixture further comprises a hot-start DNA polymerase system.
8 . The method of claim 1 , wherein quantitating the methylation in the DNA sample comprises comparing the amount of DNA amplification to an amplification standard.
9 . The method of claim 8 , wherein the amplification standard is produced by subjecting a portion of the DNA sample to steps (i)-(iv) wherein the MSE is inactivated or substantially absent from the reaction mixture.
10 . The method of claim 1 , wherein the oligonucleotide primers comprise a label.
11 . The method of claim 1 , wherein the reaction mixture further comprises an oligonucleotide probe that binds to the DNA sequence and comprises a label.
12 . The method of claim 11 , wherein the label is a fluorescent label, a radioactive label, a sequence label, an enzymatic label or an affinity label.
13 . The method of claim 1 , wherein the reaction mixture comprises free nucleotides that comprise a label or a label that binds to double-stranded DNA.
14 . (canceled)
15 . The method of claim 1 , wherein step (iv) detecting DNA amplification comprises detecting a label.
16 . The method of claim 1 , wherein the DNA sample is a mammalian genomic DNA sample.
17 . The method of claim 16 , wherein the mammalian genomic DNA is from a human subject a cell line or tissue bank.
18 . (canceled)
19 . The method of claim 17 , wherein the mammalian genomic DNA is from a blood sample, a tissue biopsy sample, a urine sample or a saliva sample.
20 . A reaction mixture comprising a methylation-sensitive endonuclease (MSE), a DNA polymerase and a buffer formulated to facilitate activity of the methylation-sensitive DNA endonuclease and quantitative activity of the DNA polymerase.
21 - 30 . (canceled)
31 . A kit comprising a vial comprising a reaction mixture according to claim 20 .
32 - 33 . (canceled)
34 . A method for quantifying site-specific DNA methylation prevalence in a genomic DNA sample comprising the steps of:
(a) digesting half of the DNA sample with methylation-sensitive endonuclease (MSE) to cleave non-methylated DNA;
(b) incubating the second half of the DNA sample with inactivated MSE;
(c) amplifying the MSE-treated DNA using a DNA polymerase and oligonucleotide primers in the presence of an oligonucleotide probe or dye to produce amplified samples; and
(d) determining the methylation status be measuring Ct values for the amplified samples and comparing the values to established Ct values correlated to percent DNA methylation,
wherein the reaction mixture volume of the two samples is not adjusted during steps (a)-(c).