用于定向进化的新型随机诱变方法

Novel Random Mutagenesis Method for Directed Evolution.

作者信息

Feng Hong, Wang Hai-Yan, Zhao Hong-Yan

机构信息

The Key Laboratory for Bio-resources and Eco-Environment of Ministry of Education, The Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, 29, Wangjiang Rd, Chengdu, 610064, Sichuan, China.

出版信息

Methods Mol Biol. 2017;1498:483-490. doi: 10.1007/978-1-4939-6472-7_32.

DOI:10.1007/978-1-4939-6472-7_32

PMID:27709596

Abstract

Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

摘要

定向进化是一种强大的基因诱变策略，已在科学研究和生物技术产业中用于蛋白质工程。1994年，Stemmer开发了定向进化的常规方法（Stemmer，《美国国家科学院院刊》91，10747 - 10751，1994；Stemmer，《自然》370，389 - 391，1994）。从那时起，人们引入了各种方法，每种方法根据目标基因和程序都有其优点和局限性。在本章中，将描述一种结合了使用dITP的诱变PCR和核酸内切酶V切割的新型替代定向进化方法。卡那霉素抗性基因用作报告基因以验证这种新型定向进化方法。这种定向进化方法已被证明在实践中是高效、可重复且易于操作的。