EpiTect MethyLight PCR Kits can be used in combination with EpiTect MethyLight Assays or other dual-labeled assays for sensitive quantification of DNA methylation in bisulfite converted DNA. Depending on the level used for sequence discrimination, MethyLight analyses can be performed in a quantitative or semiquantitative format.
Dual-labeled probes, including TaqMan® probes, are sequence-specific oligonucleotides with a fluorophore and a quencher moiety attached. The fluorophore is at the 5' end of the probe, and the quencher moiety is usually located internally or at the 3' end. During the elongation phase of PCR, the probe is cleaved by the 5' → 3' exonuclease activity the polymerase, separating the fluorophore from the quencher moiety. This results in detectable fluorescence that is proportional to the amount of accumulated PCR product.
Quantitative MethyLight real-time PCR with methylation-specific probes
For quantitative methylation analysis using MethyLight PCR, two dual-labeled probes with different fluorescent labels are used in combination with one set of PCR primers. Bisulfite converted DNA requires a different probe sequence for methylated and unmethylated CpG sites. The primers are specific for unmethylated sites. One of the probes binds specifically to methylated DNA at the site of interest, and the other binds specifically to unmethylated DNA at the same site. Since the probes contain different fluorophores, the amounts of methylated and unmethylated sequence can be determined (see figure " Quantitative MethyLight real-time PCR").
Semiquantitative MethyLight real-time PCR with methylation-specific primers
For semiquantitative MethyLight analysis, a dual-labeled probe is used together with a set of PCR primers that specifically binds to the methylation sites of interest. Bisulfite converted DNA requires a different primer sequence for methylated and unmethylated CpG sites, so the primers need to be specific to methylated, bisulfite converted DNA. If the methylation-specific primer binds to the DNA, it will be elongated, and the 5' → 3' exonuclease activity of Taq DNA Polymerase will lead to the degradation of the primer and the release of the fluorophore. As the fluorophore is now separated from the quencher moiety, its fluorescence is detectable (see figure " Semiquantitative MethyLight real-time PCR").
The EpiTect MethyLight Master Mix has been specifically developed for highly sensitive detection of methylated and unmethylated DNA using sequence-specific probes. In addition to various salts and additives, the buffer also contains a specially optimized combination of KCl and (NH4)2SO4, which promotes a high ratio of specific-to-nonspecific primer and probe binding during the annealing step of each PCR cycle. This allows discriminative hybridization of the probes, and the detection of unmethylated and/or methylated target sequences. The stringent primer annealing conditions lead to increased PCR specificity when amplifying bisulfite converted DNA, enabling reliable probe-based methylation detection.
Control reactions in MethyLight PCR
Control reactions must be carried out to ensure that MethyLight PCR probes and primers specifically bind methylated or unmethylated DNA. Such reactions require bisulfite converted control DNA (fully methylated and fully unmethylated) in various concentrations. In addition, mixtures of these control DNAs can serve as quantification standards when determining methylation degrees. With the EpiTect Control DNA Set, QIAGEN provides the quality-controlled DNAs needed — methylated bisulfite converted, unmethylated bisulfite converted, and normal human genomic DNA — in a ready-to-use kit format for standardized and reliable methylation-specific control reactions.