Factors affecting PCR‐mediated recombination

Abstract In the past decade, polymerase chain reaction (PCR) has become an important tool for the identification of previously unknown microorganisms and the analysis of environmental microbial diversity. Several studies published during recent years, however, have demonstrated that products obtained after PCR using Taq or Vent DNA polymerases will contain hybrid molecules when several homologous target sequences such as multigene families, alleles, or RNA viruses are co‐amplified. In this report, we examined the recombination frequency and the extent of template switching during PCR using Taq, Pfu and RTth/Vent DNA polymerases. As a test system we constructed a series of plasmids carrying between one and three frame shift mutations in the gene coding for the protease subtilisin or deletions of approximately 100 bp in the lacZ α . Highest recombination frequencies were observed when these mutants were co‐amplified with Taq followed by RTth/Vent DNA polymerases. Pfu DNA polymerase displayed no discernable recombination activity under normal PCR conditions. Data also suggest that