MethyLight is a quantitative and sensitive assay for the detection of low frequencies of hypermethylated alleles of regions of the genes. It is a fluorescent-based, real-time PCR Method. TaqMan fluorescent probes are used in the amplification of bisulfite-converted DNA. This method can be used with clinical samples like FFPE and urine sediments.
The figure below displays a MethyLight real-time PCR process. For methylated sites, a TaqMan probe is located between primers that bind the methylation sites of interest. During the PCR extension, the fluorophore is released, and the fluorescence is measured. In unmethylated DNA, primer annealing and extension do not occur, and no fluorescence is detected. A separate assay can be carried out with specific primers for unmethylated DNA to determine the amount of sequences.16
