title: Novel Random Mutagenesis Method for Directed Evolution.
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.
authors.affiliation: 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. hfeng@scu.edu.cn.
authors.forename: Hong
authors.initials: H
authors.lastname: Feng
authors.affiliation: 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.
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Hong-Yan
authors.initials: HY
authors.lastname: Zhao
mesh_terms: D001709:Biotechnology / Q000379:methods; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods; D017930:Genes, Reporter / Q000235:genetics; D007613:Kanamycin Resistance / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods
chemical_list: D043224:Deoxyribonuclease (Pyrimidine Dimer)
keywords: DNA shuffling; Endonuclease V; Molecular evolution; Mutagenesis; PCR; dITP