High-fidelity in vitro recombination using a proofreading polymerase

Abstract

We describe a convenient PCR-based protocol for in vitro recombination of homologous genes, thereby minimizing the rate of associated point mutations. High-fidelity recombination conditions were obtained using Vent DNA polymerase, which, in contrast to Taq DNA polymerase, shows significant proofreading activity and ranges among the slowest thermostable DNA polymerases, allowing tight control of the polymerase-catalyzed DNA extension. To determine the mutagenesis rate and to analyze the efficiency of recombination. 89 clones from a standard experiment were randomly selected for further analysis. Sequence comparison revealed that 21% (19/89) of the clones result from different recombination events in the marker-containing region (260 bp). The overall mutation rate is only 0.02%, which is the lowest rate thus far reported for in vitro recombination experiments.