Jan, 2011
Construction of a high-efficiency multi-site-directed mutagenesis
AFRICAN JOURNAL OF BIOTECHNOLOGY
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- Volume
- 10
- Number
- 3
- First page
- 449
- Last page
- 452
- Language
- English
- Publishing type
- Research paper (scientific journal)
- Publisher
- ACADEMIC JOURNALS
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.
- Link information
- ID information
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- ISSN : 1684-5315
- Web of Science ID : WOS:000286397500022