CLC number: Q3
On-line Access:
Received: 2008-11-17
Revision Accepted: 2009-03-03
Crosschecked: 2009-04-24
Cited: 7
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Bao-zhong ZHANG, Xin ZHANG, Xiao-ping AN, Duo-liang RAN, Yu-sen ZHOU, Jun LU, Yi-gang TONG. An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening[J]. Journal of Zhejiang University Science B, 2009, 10(6): 479-482.
@article{title="An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening",
author="Bao-zhong ZHANG, Xin ZHANG, Xiao-ping AN, Duo-liang RAN, Yu-sen ZHOU, Jun LU, Yi-gang TONG",
journal="Journal of Zhejiang University Science B",
volume="10",
number="6",
pages="479-482",
year="2009",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B0820367"
}
%0 Journal Article
%T An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening
%A Bao-zhong ZHANG
%A Xin ZHANG
%A Xiao-ping AN
%A Duo-liang RAN
%A Yu-sen ZHOU
%A Jun LU
%A Yi-gang TONG
%J Journal of Zhejiang University SCIENCE B
%V 10
%N 6
%P 479-482
%@ 1673-1581
%D 2009
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B0820367
TY - JOUR
T1 - An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening
A1 - Bao-zhong ZHANG
A1 - Xin ZHANG
A1 - Xiao-ping AN
A1 - Duo-liang RAN
A1 - Yu-sen ZHOU
A1 - Jun LU
A1 - Yi-gang TONG
J0 - Journal of Zhejiang University Science B
VL - 10
IS - 6
SP - 479
EP - 482
%@ 1673-1581
Y1 - 2009
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B0820367
Abstract: site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.
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