A convenient and robust method for construction of combinatorial and random mutant libraries

被引：8

作者：

An, Yingfeng ^{[1
]}

Wu, Wenfang ^{[1
]}

Lv, Anguo ^{[1
]}

机构：

[1] Chinese Acad Sci, Inst Appl Ecol, Shenyang 110016, Peoples R China

来源：

BIOCHIMIE | 2010年 / 92卷 / 08期

关键词：

Mutant library; DNA shuffling; Error-prone PCR; Ligase-independent cloning; IN-VITRO; DNA; EVOLUTION; FAMILY; FRAGMENTATION; RECOMBINATION;

D O I：

10.1016/j.biochi.2010.03.016

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Here we describe a convenient and robust ligase-independent method for construction of combinatorial and random mutant libraries. The homologous genes flanked by plasmid-derived DNA sequences are fragmented, and the random fragments are reassembled in a self-priming polymerase reaction to obtain chimeric genes. The product is then mixed with linearized vector and two pairs of flanking primers, followed by assembly of the chimeric genes and linearized vector by PCR to introduce recombinant plasmids of a combinatorial library. Commonly, it is difficult to find proper restriction sites during the construction of recombinant plasmids after DNA shuffling with multiple homologous genes. However, this disadvantage can be overcome by using the ligase-independent method because the steps of DNA digestion and ligation can be avoided during library construction. Similarly, DNA sequences with random mutations introduced by error-prone PCR can be used to construct recombinant plasmids of a random mutant library with this method. Additionally, this method can meet the needs of large and comprehensive DNA library construction. (C) 2010 Elsevier Masson SAS. All rights reserved.

引用

页码：1081 / 1084

页数：4

共 10 条

[1] Revealing biases inherent in recombination protocols [J].

Chaparro-Riggers, Javier F. ;

Loo, Bernard L. W. ;

Polizzi, Karen M. ;

Gibbs, Phillip R. ;

Tang, Xiao-Song ;

Nelson, Mark J. ;

Bommarius, Andreas S. .

BMC BIOTECHNOLOGY, 2007, 7 (1)

[2] DNA shuffling of a family of genes from diverse species accelerates directed evolution [J].

Crameri, A ;

Raillard, SA ;

Bermudez, E ;

Stemmer, WPC .

NATURE, 1998, 391 (6664) :288-291

[3] Novel family shuffling methods for the in vitro evolution of enzymes [J].

Kikuchi, M ;

Ohnishi, K ;

Harayama, S .

GENE, 1999, 236 (01) :159-167

[4] An effective family shuffling method using single-stranded DNA [J].

Kikuchi, M ;

Ohnishi, K ;

Harayama, S .

GENE, 2000, 243 (1-2) :133-137

[5] Random DNA fragmentation with endonuclease V: application to DNA shuffling [J].

Miyazaki, K .

NUCLEIC ACIDS RESEARCH, 2002, 30 (24) :e139

[6] Regulation of average length of complex PCR product [J].

Shagin, Dmitry A. ;

Lukyanov, Konstantin A. ;

Vagner, Laura L. ;

Matz, Mikhail, V .

NUCLEIC ACIDS RESEARCH, 1999, 27 (18)

[7] RAPID EVOLUTION OF A PROTEIN IN-VITRO BY DNA SHUFFLING [J].

STEMMER, WPC .

NATURE, 1994, 370 (6488) :389-391

[8] DNA SHUFFLING BY RANDOM FRAGMENTATION AND REASSEMBLY - IN-VITRO RECOMBINATION FOR MOLECULAR EVOLUTION [J].

STEMMER, WPC .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (22) :10747-10751

[9] Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis [J].

Stutzman-Engwall, K ;

Conlon, S ;

Fedechko, R ;

McArthur, H ;

Pekrun, K ;

Chen, Y ;

Jenne, S ;

La, C ;

Trinh, N ;

Kim, S ;

Zhang, YX ;

Fox, R ;

Gustafsson, C ;

Krebber, A .

METABOLIC ENGINEERING, 2005, 7 (01) :27-37

[10] Amplification of complex gene libraries by emulsion PCR [J].

Williams, Richard ;

Peisajovich, Sergio G. ;

Miller, Oliver J. ;

Magdassi, Shlomo ;

Tawfik, Dan S. ;

Griffiths, Andrew D. .

NATURE METHODS, 2006, 3 (07) :545-550

← 1 →