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Random-priming in vitro recombination: an effective tool for directed evolution

Shao, Zhixin and Zhao, Huimin and Giver, Lori and Arnold, Frances H. (1998) Random-priming in vitro recombination: an effective tool for directed evolution. Nucleic Acids Research, 26 (2). pp. 681-683. ISSN 0305-1048.

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A simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences is reported. The method involves priming template polynucleotide(s) with random-sequence primers and extending to generate a pool of short DNA fragments which contain a controllable level of point mutations. The fragments are reassembled during cycles of denaturation, annealing and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. Screening or selecting the expressed gene products leads to new variants with improved functions, as demonstrated by the recombination of genes encoding different thermostable subtilisins in order to obtain enzymes more stable than either parent.

Item Type:Article
Additional Information:Copyright © 1998 by Oxford University Received September 15, 1997; Revised and Accepted November 21, 1997 This research is supported by Eli Lilly and Co., the US Office of Naval Research (N0014-96-1-340 and N00014-97-1-0433), the US Department of Energy’s program in Biological and Chemical Technologies Research within the Office of Industrial Technologies, Energy Efficiency and Renewables. Z.S. is grateful to Drs Peggy Arps and Kentaro Miyazaki for helpful discussions and to Michaeleen Callahan for excellent technical assistance.
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Deposited On:08 Oct 2005
Last Modified:26 Dec 2012 08:41

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