Full Length Research Paper

Construction of a high-efficiency multi-site-directed mutagenesis

Haidong Tan¹, Yueguang Li², Ling Chen³, Tomonari Kasai³ and Masaharu Seno³*

¹Biotechnology Department, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, People’s Republic of China.

²Department of General Surgery, Tianjin 4th Centre Hospital, Zhongshanlu, Hebeiqu, Tianjin 300140, People’s Republic of China.

³Department of Medical and Bioengineering Science, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.

*Corresponding author. E-mail: mseno@cc.okayama-u.ac.jp. Fax: +81-86-251-8216.

Abbreviations: PCR, Polymerase chain reaction; MSD, multi-site-directed mutagenesis; EK, enterokinase; LB, lysogenic broth; ccc, circular covalently closed.

Accepted 15 November, 2010

   Abstract

Although site-directed mutagenesis has been used in many fields, it still has low rate of success and high cost because of low-yield target products. A modified method for multi-site-directed mutagenesis was developed with shifted primer design and cold-start polymerase chain reaction (PCR). The developed method was successfully applied to hexapeptide gene synthesis and recombinant enterokinase gene modification in the plasmids pET41a and pET24b-EK. The efficiency was pronounced at a 1:10 molar ratio of 7-base mutant products to 705-bp fragment products as control. Even in a 10-base substitution mutagenic PCR, a 1:50 molar ratio of mutant products to 705-bp fragment products was reached. Meanwhile, the quality of mutants was proved through the transformation efficiency and sequencing. This method was beneficial to prepare high-quality multibase mutagenesis and also implied that large-scale multibase mutagenesis was feasible, efficient, economical, and productive.

Key words: Site-directed multibase mutagenesis, shift primer, hexapeptide gene, enterokinase gene.