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 |
|---|---|
| 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 |
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Keywords
- Enterokinase gene
- Hexapeptide gene
- Shift primer
- Site-directed multibase mutagenesis
ASJC Scopus subject areas
- Biotechnology
- Applied Microbiology and Biotechnology
- Genetics
- Molecular Biology
- Agronomy and Crop Science
Cite this
Construction of a high-efficiency multi-site-directed mutagenesis. / Tan, Haidong; Li, Yueguang; Chen, Ling; Kasai, Tomonari; Seno, Masaharu.
In: African Journal of Biotechnology, Vol. 10, No. 3, 17.01.2011, p. 449-452.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Construction of a high-efficiency multi-site-directed mutagenesis
AU - Tan, Haidong
AU - Li, Yueguang
AU - Chen, Ling
AU - Kasai, Tomonari
AU - Seno, Masaharu
PY - 2011/1/17
Y1 - 2011/1/17
N2 - 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.
AB - 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.
KW - Enterokinase gene
KW - Hexapeptide gene
KW - Shift primer
KW - Site-directed multibase mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=79251508689&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79251508689&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:79251508689
VL - 10
SP - 449
EP - 452
JO - African Journal of Biotechnology
JF - African Journal of Biotechnology
SN - 1684-5315
IS - 3
ER -