Improved polymerase fidelity in PCR-SSCPA.

ABSTRACT

Polymerase fidelity is important in any application of the polymerase chain reaction (PCR). In single-strand conformation polymorphism analysis (SSCPA) where one may be looking for a small number of altered DNA strands in the presence of thousands of unchanged strands it is critical. We have examined the effect of PCR conditions, product purification and SSCP analysis on the measured error rates with 4 thermostable polymerases (Taq, Vent, Pyrostase and Pfu). Error rates have been calculated by densitometric scanning of SSCPA gel images. Using PCR conditions which maximize fidelity and eliminating products which include large additions or deletions we have achieved error rates of 10(-5) to 10(-6). Such low rates heighten the probability that relatively infrequent mutations will be identified. Further, the densitometric scanning of gel images provides a useful modification of conventional SSCPA which facilitates such identification.