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

Zhixin Shao, Huimin Zhao, Lori Giver, Frances H. Arnold

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)681-683
Number of pages3
JournalNucleic acids research
Volume26
Issue number2
DOIs
StatePublished - Jan 15 1998

ASJC Scopus subject areas

  • Genetics

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