通过基于聚合酶链式反应的交错延伸过程（StEP）进行体外“有性”进化。

In vitro 'sexual' evolution through the PCR-based staggered extension process (StEP).

作者信息

Zhao Huimin, Zha Wenjuan

机构信息

Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

出版信息

Nat Protoc. 2006;1(4):1865-71. doi: 10.1038/nprot.2006.309.

DOI:10.1038/nprot.2006.309

PMID:17487170

Abstract

This protocol describes a directed evolution method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) is essentially a modified PCR that uses highly abbreviated annealing and extension steps to generate staggered DNA fragments and promote crossover events along the full length of the template sequence(s). The resulting library of chimeric polynucleotide sequence(s) is subjected to subsequent high-throughput functional analysis. The recombination efficiency of the StEP method is comparable to that of the most widely used in vitro DNA recombination method, DNA shuffling. However, the StEP method does not require DNA fragmentation and can be carried out in a single tube. This protocol can be completed in 4-6 h.

摘要

本方案描述了一种用于多核苷酸序列体外诱变和重组的定向进化方法。交错延伸过程（StEP）本质上是一种改良的聚合酶链式反应（PCR），它使用高度简化的退火和延伸步骤来生成交错的DNA片段，并促进沿模板序列全长的交叉事件。所得的嵌合多核苷酸序列文库随后进行高通量功能分析。StEP方法的重组效率与最广泛使用的体外DNA重组方法——DNA改组相当。然而，StEP方法不需要DNA片段化，并且可以在单个试管中进行。本方案可在4至6小时内完成。