Patent ID: 8703414

Claim:
A method for quantifying the degree of methylation of a target DNA sequence, comprising: a) converting unmethylated cytosine in a target DNA sequence to uracil or to another base that can be distinguished from cytosine based on its base-pairing behavior, wherein 5-methylcytosine remains unconverted; b) amplifying the converted target DNA sequence in the presence of two real-time probes, which bind to the same amplificate in the same vessel, wherein: (i) a first probe is specific for the methylated state or the unmethylated state of the target DNA sequence, (ii) a second probe does not carry a CpG dinucleotide and no TG- or CA-dinucleotide whose cytosine base was converted by the treatment of step a), (iii) each probe contains a pair of labels that permits the independent detection of each probe, and (iv) each of the two probes hybridize to the converted target DNA sequence; c) detecting each pair of labels independent of the other pair; d) determining the extent to which the amplification has proceeded by detecting the hybridized probe signals at various time points, wherein the signal generated by the first probe is generated independently of the signal of the second probe; and e) determining the degree of methylation of the target DNA sequence by determining the ratio of signal intensities for the first and second probes.