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

Haidong Tan, Yueguang Li, Ling Chen, Tomonari Kasai, Masaharu Seno

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)449-452
Number of pages4
JournalAfrican Journal of Biotechnology
Issue number3
Publication statusPublished - Jan 17 2011


  • Enterokinase gene
  • Hexapeptide gene
  • Shift primer
  • Site-directed multibase mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Genetics
  • Agronomy and Crop Science


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