IMPROVED POLYMERASE FIDELITY IN PCR-SSCPA

Polymerase fidelity is important in any application of the polymerase chain reaction (PCR). In single-strand conformation polymorphism analy sis (SSCPA) where one may be looking for a small number of altered DNA strands in the presence of thousands of unchanged strands it is criti cal. We have examined the effect of PCR conditions, product purificati on and SSCP analysis on the measured error rates with 4 thermostable p olymerases (Taq, Vent, Pyrostase and Pfu). Error rates have been calcu lated by densitometric scanning of SSCPA gel images. Using PCR conditi ons which maximize fidelity and eliminating products which include lar ge additions or deletions we have achieved error rates of 10(-5) to 10 (-6). Such low rates heighten the probability that relatively infreque nt mutations will be identified. Further, the densitometric scanning o f gel images provides a useful modification of conventional SSCPA whic h facilitates such identification.