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.
Original language | English |
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Pages (from-to) | 449-452 |
Number of pages | 4 |
Journal | African Journal of Biotechnology |
Volume | 10 |
Issue number | 3 |
Publication status | Published - Jan 17 2011 |
Keywords
- 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