Factors affecting PCR-mediated recombination.
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 alpha. 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 in vivo repair of heteroduplex DNA molecules in Escherichia coli by a RecA-independent mechanism, perhaps the mismatch repair, results in the formation of chimeric molecules. Using Bacillus subtilis as the host, however, can significantly diminish non-PCR RecA-independent in vivo recombination, owing to the fact that transforming DNA molecules enter B. subtilis as single strands. Combined, these results suggest that using Pfu DNA polymerase for amplification and B. subtilis as the host for transformation may significantly reduce chimera formation.
Shafikhani
Sasha
S
Genencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA. sshafikh@uclink.berkeley.edu
D001412:Bacillus subtilis / Q000235:genetics; D002678:Chimera; D004274:DNA, Recombinant / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011938:Rec A Recombinases; D011995:Recombination, Genetic; D019914:Taq Polymerase
D004274:DNA, Recombinant; D015345:Oligonucleotide Probes; C072146:Pfu DNA polymerase; D011938:Rec A Recombinases; D019914:Taq Polymerase; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase