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

  • H Tan
  • Y Li
  • L Chen
  • T Kasai
  • M Seno
Keywords: Site-directed multibase mutagenesis, shift primer, hexapeptide gene, enterokinase gene.

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.

Published
2013-08-13
Section
Articles

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eISSN: 1684-5315