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Journal of Zhejiang University SCIENCE B 2009 Vol.10 No.6 P.479~482

http://doi.org/10.1631/jzus.B0820367


An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening


Author(s):  Bao-zhong ZHANG, Xin ZHANG, Xiao-ping AN, Duo-liang RAN, Yu-sen ZHOU, Jun LU, Yi-gang TONG

Affiliation(s):  State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China; more

Corresponding email(s):   tong.yigang@gmail.com, lujun98@gmail.com

Key Words:  Site-directed mutagenesis (SDM), Restriction endonuclease, Mutant screening


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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.

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publisher="Zhejiang University Press & Springer",
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%A Jun LU
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A1 - Duo-liang RAN
A1 - Yu-sen ZHOU
A1 - Jun LU
A1 - Yi-gang TONG
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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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Reference

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[7] Rabhi, I., Guedel, N., Chouk, I., Zerria, K., Barbouche, M.R., Dellagi, K., Fathallah, D.M., 2004. A novel simple and rapid PCR-based site-directed mutagenesis method. Mol. Biotechnol., 26(1):27-34.

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[9] Tseng, W.C., Lin, J.W., Wei, T.Y., Fang, T.Y., 2008. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal. Biochem., 375(2):376-378.

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