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

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...

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Bibliographic Details
Published in:Journal of Zhejiang University. B. Science 2009-06, Vol.10 (6), p.479-482
Main Authors: Zhang, Bao-zhong, Zhang, Xin, An, Xiao-ping, Ran, Duo-liang, Zhou, Yu-sen, Lu, Jun, Tong, Yi-gang
Format: Article
Language:English
Subjects:
Bacteria
Biomedical and Life Sciences
Biomedicine
Biotechnology
Deoxyribonucleic acid
DNA
DNA序列
Escherichia coli
Escherichia coli - genetics
Escherichia coli Proteins - genetics
Genetic Testing - methods
Mutagenesis
Mutagenesis, Site-Directed - methods
Mutation - genetics
Polymerase chain reaction
Polymerase Chain Reaction - methods
Proteins
反向PCR
定点突变
序列设计
聚合酶链反应
质粒DNA
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Summary: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.
ISSN:1673-1581
1862-1783
DOI:10.1631/jzus.B0820367