Molecular evolution by staggered extension process (StEP) in vitro recombination | Nature Biotechnology Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Abstract We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynu-cleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65°C is 50 times that of wild-type subtilisin E. This is a preview of subscription content, access via your institution Access options Access through your institution Subscription info for Chinese customers We have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal. Go to naturechina.com Buy this article Purchase on SpringerLink Instant access to the full article PDF. 39,95 € Prices may be subject to local taxes which are calculated during checkout Similar content being viewed by others Human DNA polymerase ε is a source of C>T mutations at CpG dinucleotides Article Open access 10 October 2024 Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell Article Open access 22 October 2024 Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries Article Open access 10 July 2024 References Stahl, F.W. 1987. Genetic recombination. Sci. Am. 256: 1–101. Article Google Scholar Kucherlapati, R. and Smith, G.R. 1988. Genetic recombination. American Society of Microbiology, Washington, DC. Google Scholar Holland, J.H. 1992. Genetic algorithms. Sci. Am. 267: 66–72. Article Google Scholar Holland, J.H. 1992. 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Arnold View author publications Search author on: PubMed Google Scholar Corresponding author Correspondence to Frances H. Arnold . Rights and permissions Reprints and permissions About this article Cite this article Zhao, H., Giver, L., Shao, Z. et al. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16 , 258–261 (1998). https://doi.org/10.1038/nbt0398-258 Download citation Received : 05 November 1997 Accepted : 23 January 1998 Issue date : 01 March 1998 DOI : https://doi.org/10.1038/nbt0398-258 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. 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