Optimization of DNA shuffling for high fidelity recombination.
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.
Zhao
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Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Arnold
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FH
D005075:Biological Evolution; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D003850:Deoxyribonuclease I; D015723:Gene Library; D007202:Indicators and Reagents; D008274:Magnesium; D008345:Manganese; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D013381:Subtilisins / Q000096:biosynthesis / Q000235:genetics
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D008345:Manganese; D004247:DNA; D003850:Deoxyribonuclease I; D013381:Subtilisins; D008274:Magnesium