[A] and [C] In methylation-specific TaqMan® assays, two methylation-sensitive TaqMan® probes are present together with methylation-insensitive PCR primers. Depending on the methylation status of the targeted sequence, only the TaqMan® probe specific for bisulfite converted methylated DNA or the TaqMan® probe specific for unmethylated DNA can hybridize to the target sequence. [B] and [D] Both probes are labeled with different fluorophores that are released during PCR if the probe hybridizes to the DNA. The fluorescence from the released fluorophore is proportional to the amount of accumulated PCR product.
[A] and [C] In semiquantitative MethyLight PCR, a TaqMan® probe is located between primers that bind the methylation sites of interest. Primers are specific for methylated sequences, annealing only to methylated DNA, and not to unmethylated DNA. During the PCR extension step, the fluorophore is released, and the resulting fluorescence is measured. [B] and [D] In the case of unmethylated DNA, primer annealing and extension does not occur, and no fluorescence is detected. Ideally, a separate assay is carried out with primers specific for converted, unmethylated DNA, to determine the amount of unmethylated sequences. To quantify the methylation degree, a separate assay determining the DNA quantity is required (not shown in the figure).
EpiTect MethyLight Assays enable the direct quantification of the degree of methylation in a sample by using the threshold cycle values (CT) determined by qPCR. [A] In the FAM channel, the probe detects methylated DNA, and in the [B] VIC channel, the probe detects unmethylated DNA. A 10 µg sample of bisulfite converted, methylated and unmethylated human control DNA, or defined mixtures of both DNAs containing 95% to 5% methylated DNA, as indicated in the figure, were used in a methylation quantification experiment on the ABI PRISM 7900 cycler. The EpiTect MethyLight Assay for HS_CDKN2A was used in combination with the EpiTect MethyLight PCR Kit. The degree of methylation was calculated from the CT values from the FAM (CT (CG)) and VIC (CT (TG)) channels.