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 β-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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Acknowledgments
This research was supported by the Shanghai Project for International Scientific and Technological Cooperation (055407068), Fund of National Natural Science Foundation of China (30471258; 30370987), Shanghai Rising-Star Program (05QMX1445), Shanghai Natural Science Foundation (04ZR14116), Shanghai Key Basic Research Project (05DJ14008), Project of key laboratory of Shanghai (05dz223266), Shanghai Prospering Agriculture through Science and Technology Foundation (2001 3–12), and Development Foundation of SAAS (2006-10).
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Peng, RH., Xiong, AS. & Yao, QH. A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis. Appl Microbiol Biotechnol 73, 234–240 (2006). https://doi.org/10.1007/s00253-006-0583-3
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DOI: https://doi.org/10.1007/s00253-006-0583-3