Nucleic Acids Research Advance Access published online on August 15, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm583
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Methods Online |
One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning
Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, California 92521, USA
*To whom correspondence should be addressed. Tel: +1 951 827 6473; Fax: +1 951 827 3087; Email: jeffbach{at}citrus.ucr.edu
Received April 16, 2007. Revised July 16, 2007. Accepted July 17, 2007.
Recombination cloning encompasses a set of technologies that transfer gene sequences between vectors through site-specific recombination. Due in part to the instability of linear DNA in bacteria, both the initial capture and subsequent transfer of gene sequences is often performed using purified recombination enzymes. However, we find linear DNAs flanked by loxP sites recombine efficiently in bacteria expressing Cre recombinase and the lambda Gam protein, suggesting Cre/lox recombination of linear substrates can be performed in vivo. As one approach towards exploiting this capability, we describe a method for constructing large (>1 x 106 recombinants) libraries of gene mutations in a format compatible with recombination cloning. In this method, gene sequences are cloned into recombination entry plasmids and whole-plasmid PCR is used to produce mutagenized plasmid amplicons flanked by loxP. The PCR products are converted back into circular plasmids by transforming Cre/Gam-expressing bacteria, after which the mutant libraries are transferred to expression vectors and screened for phenotypes of interest. We further show that linear DNA fragments flanked by loxP repeats can be efficiently recombined into loxP-containing vectors through this same one-step transformation procedure. Thus, the approach reported here could be adapted as general cloning method.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.