Optimization of DNA shuffling for high fidelity recombination

Abstract

A convenient 'DNA shuffling' protocol for random recombination of homologous genes in vitro with a very low rate of associated point mutagenesis (0.05%) is described. In addition, the mutagenesis rate can be controlled over a wide range by the inclusion of Mn2+or Mg2+during DNase I digestion, by choice of DNA polymerase used during gene reassembly as well as how the genes are prepared for shuffling (PCR amplification versus restriction enzyme digestion of plasmid DNA). These protocols should be useful for in vitro protein evolution, for DNA based computing and for structure-function studies of evolutionarily related genes.