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Appl Microbiol Biotechnol. 2006 Nov;73(1):234-40. Epub 2006 Oct 5.

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

Author information

1
Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai, People's Republic of China.

Abstract

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.

PMID:
17021880
DOI:
10.1007/s00253-006-0583-3
[Indexed for MEDLINE]

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