Methods of detecting MLH1 methylation

A method for detecting methylation of a promoter in a DNA sample comprising:

• (a) incubating a DNA sample with a methylation-sensitive restriction enzyme, wherein a first target nucleic acid sequence in a promoter in the sample comprises a recognition site for the methylation-sensitive restriction enzyme, and a second target nucleic acid sequence in a control gene in the sample comprises a recognition site for the methylation-sensitive restriction enzyme;
• (b) incubating the sample with a plurality of probes for querying a plurality of target nucleic acids in the sample, wherein the plurality of probes comprises
• (i) a first locus specific probe comprising a first target specific region complementary to the first target nucleic acid sequence in the promoter, wherein the sequence of the first locus specific probe comprises a methylation sensitive restriction site;
• and
• (ii) a second locus specific probe comprising a second target specific region complementary to the second target nucleic acid sequence in the control gene, wherein the sequence of the second locus specific probe does not comprise a methylation sensitive restriction site, and wherein the first locus specific probe and second locus specific probe are detectably labelled;
• (c) hybridizing the plurality of probes to the plurality of target nucleic acids in the sample to form a plurality of hybridization complexes;
• (d) amplifying the plurality of hybridization complexes to produce a plurality of amplicons, wherein amplification is carried out with a plurality of primer sets comprising:
• (i) a first forward primer comprising a region that is complementary to a nucleic acid sequence located 5′ from the first target nucleic acid sequence in the promoter;
• (ii) a first reverse primer comprising a region that is complementary to a nucleic acid sequence located 3′ from the first target nucleic acid sequence in the promoter;
• (iii) a second forward primer comprising a region that is complementary to a nucleic acid sequence located 5′ from the second target nucleic acid sequence in the control gene;
• and
• (iv) a second reverse primer comprising a region that is complementary to a nucleic acid sequence located 3′ from the second target nucleic acid sequence in the control gene;
• and
• (e) detecting the plurality of amplicons, wherein detecting an amplicon comprising the first target nucleic acid sequence in the promoter indicates methylation of the first target nucleic acid sequence in the promoter in the sample.