title: Development and Use of a Novel Random Mutagenesis Method: In Situ Error-Prone PCR (is-epPCR).
abstract: Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.
authors.affiliation: Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang, Jiangsu, 212013, China. weilanshao@ujs.edu.cn.
authors.forename: Weilan
authors.initials: W
authors.lastname: Shao
authors.affiliation: Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
authors.forename: Kesen
authors.initials: K
authors.lastname: Ma
authors.affiliation: Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Le
authors.affiliation: Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Wang
authors.affiliation: Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
authors.forename: Chong
authors.initials: C
authors.lastname: Sha
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004247:DNA
keywords: Amplification of circular plasmids; Directed evolution; In situ error-prone PCR; Random mutagenesis; Selection marker swapping; Thermostable DNA ligase