Base specific cleavage of methylation-specific amplification products in combination with mass analysis

摘要

The present invention relates to a method of distinguishing between a false positive methylation specific amplification and a true methylation specific amplification, comprising the steps of: (a) treating a target nucleic acid molecule with a reagent that modifies an unmethylated selected nucleotide to produce a different nucleotide; (b) contacting the treated target nucleic acid molecule with a methylation state specific primer complementary to a first target nucleic acid region containing one or more of the selected nucleotides; (c) treating the contacted target nucleic acid molecule under nucleic acid synthesis conditions; (d) treating the synthesized products under base specific cleavage conditions; and (e) detecting the mass of the cleaved products, wherein: a change in mass of one or more cleaved products compared to a reference mass indicates that a nucleotide locus in a second region in a target is methylated, wherein the second region does not overlap with the first region, whereby presence of one or more methylated loci in the second region confirms true methylation specific amplification.