( 9.3 Amplification of GC-rich targets ...)TripleMaster PCR System

9.3 Amplification of GC-rich targets

With the TripleMaster PCR System, DNA sequences with a biased nucleotide composition (GC-content >70 %; homopolymeric stretches), repeats and inverted repeats can be amplified.

General hints

The reaction parameters, especially for GC-rich targets, may require some modification:
- Test which reaction buffer performs best with the appropriate target (Tuning Buffer versus HighFidelity Buffer) - Increase the temperature for template denaturation to 98C (not feasible for long targets >5 kb). - Use the upper limit of the time range given in Table 7 (processivity and extension rate of polymerases on difficult templates is slower). - Work at higher dNTP concentrations (300-500 M) than given in the protocol for high fidelity PCR. - For extremely GC-rich targets (>70 %) use GC-destabilizing co-solvents in combination with the Tuning or HighFidelity Buffer (DMSO: 2-8 %; glycerol: 2-5 %; betaine: 0.5- 2.5 M; trimethylammonium hydrochloride: 50-100 M; acetamide: 1-5 %). Reduce annealing temperature by 2C decrements with co-solvents. - Increase the denaturation temperature gradually up to 98C using the gradient function of the Mastercycler gradient, but restrict the denaturation time in each cycle to maximal 10 seconds. - Introduce a special pre-amplification denaturation step at 98C with the buffer/template/
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