Volkov, Alexander A. and Shao, Zhixin and Arnold, Frances H. (1999) Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair. Nucleic Acids Research, 27 (18). e18. ISSN 0305-1048. http://resolver.caltech.edu/CaltechAUTHORS:VOLnar99
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We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).
|Additional Information:||Copyright© Oxford University Press, 1999. Received January 28, 1999; Revised May 30, 1999; Accepted June 14, 1999 This research was supported in part by the Department of Energy. The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.|
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|Deposited On:||08 Oct 2005|
|Last Modified:||26 Dec 2012 08:41|
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