[A] Both primers used for methylated LDOC1 PCR were modified such that a mismatch was introduced at the 3' end of either the forward or the reverse primer. This modification mimics the situation of unchanged primers annealing to bisulfite converted DNA, which differs due to a missing methylation at one CpG site. [B] Using HotStarTaq d-Tect Polymerase, PCR was only successful in the absence of a mismatch at the 3' end of the primer used. A single mismatch is sufficient for correct MSP results.
PCR for methylated [A] p16, [B] NEFH, and [C] NPTX2 were performed using different amounts of methylated DNA (0 pg, 100 pg, and 1 ng) against a background of 10 ng unmethylated DNA. The reactions were run at 3 different temperatures (48°C, 53°C, and 58°C) using 4 different DNA polymerases. HotStarTaq d-Tect shows highly specific PCR results over the whole temperature range, whereas other polymerases either failed or gave highly unspecific bands.
[B] During annealing,methylation-specific primers also often bind to the unmethylated, converted DNA with a mismatch or several mismatches at the 3' end of the primer. [A] and [B]Taq DNA Polymerase can efficiently elongate these primers, regardless of the primer mismatch. Due to its increased ability to discriminate between mismatches, genetically engineered HotStarTaq d-Tect Polymerase recognizes a mismatch at the 3' end of the methylation-specific primer, and prevents primer extension. [C] HotStarTaq d-Tect Polymerase only elongates primers without mismatch. [D] Thus, HotStarTaq d-Tect Polymerase increases reliability in MSP PCR, and prevents false-positive amplification reactions, facilitating primer design.