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Using the TripleMaster PCR System for Robust Amplification of ,,, GC-Rich DNA Templates

DMSO is a co-solvent that destabilizes DNA by disrupting base pairs and thereby lowering the melting temperature of DNA. DMSO has been known to improve the amplification of some GC-rich DNA sequences, including some resistant to amplification by standard PCR techniques. While DMSO improved the yield and specificity of amplification for these targets, it may not be beneficial or necessary for other targets. In these cases, other PCR additives such as betaine, formamide, glycerol or trimethylammonium hydrochloride can be tested. However it is impossible to identify which, if any, PCR additive is best for a particular target, so it may be necessary to test several different agents separately or in combination. It should be noted that although these additives can increase the yield and specificity of PCR reactions, their effects on the melting temperature of DNA alters the optimal annealing temperature for a specific primer/template system. Therefore, optimization of the thermal cycling parameters may be required, which often requires the performance of several experiments.1, 3, 4, 5, 6

One method that eliminates the need for thermal cycling optimization experiments is the touchdown PCR method, which is used in the amplification of the MC1R target. During touchdown PCR, the annealing temperature of the first cycle is set at or a few degrees above the melting temperatures of the primers. The annealing temperature of each sequential cycle is then programmed for incrementally lower temperatures so that the annealing temperature range of the entire program spans approximately 15C. This favors amplification of the target amplicon by ensuring that the first annealing event involves the template sequence
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