Patent ID: 7045319

Claim:
A method for detecting methylated cytosine residues at a CpG site in a target gene, said method comprising: (a) deaminating a single-stranded target DNA polynucleotide under conditions which convert unmethylated cytosine residues to uracil residues while not converting methylated cytosine residues to uracil; (b) hybridizing a target site probe with said deaminated single-stranded target DNA polynucleotide wherein said target site probe and said deaminated target DNA polynucleotide form a bubble complex comprising a first double-stranded region upstream of said CpG site, a single-stranded region comprising said CpG site, and a second double-stranded region downstream of said CpG site; (c) incubating said deaminated target DNA polynucleotide and said target site probe with an initiator, a terminator, and an RNA-polymerase, wherein at least one of said initiator and said terminator are complementary to the CpG site; (d) reiteratively synthesizing an oligonucleotide transcript that is complementary to said CpG site from said deaminated target DNA polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple reiterative oligonucleotide transcripts; and (e) detecting or quantifying said reiterative oligonucleotide transcripts, thereby detecting methylated cytosine residues.