High-fidelity in vitro recombination using a proofreading polymerase

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