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A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis

文献类型： 外文期刊

作者： Peng, Ri-He 1 ; Xiong, Ai-Sheng 1 ; Yao, Quan-Hong 1 ;

作者机构： 1.Shanghai Acad Agr Sci, Shanghai Key Lab Agr Genet & Breeding, AgroBiotechnol Res Ctr, Shanghai, Peoples R China

关键词： POLYMERASE-CHAIN-REACTION;ENZYME CATALYSIS;DNA;MUTATIONS;GENERATION;PLASMID;PRIMER;ACID

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

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收录情况： SCI

摘要： A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli beta-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.

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