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| A Simple and Rapid Single Primer PCR Method for Site-directed Mutagenesis |
| GAO Rui-ping, CHENG Long-bin, LI Zhen-qiu |
| College of Life Sciences, Hebei University, Baoding 071002, China |
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Abstract To solve the problem that additional primers could be inserted at the mutation site when use QuickChange site-directed mutagenesis methods to mutate some specific sites, QuickChange method was modifed as followed:A pair of complementary primers containing mutations site were synthesized, and single primer PCR amplification was done respectively. The two PCR products from single amplification were mixed and denatured and renatured followed by addition of restriction endonuclease Dpn I. The product was transformed into competent E.coli DH5α, and positive clones were screened for DNA sequencing. Several sites in amorpha-4,11-diene synthase(ADS) gene that could not be successful mutated using tranditional method had been successfully mutated with this method without any additional primers. So this novel method was practicable and could solve the problem of extra primers insertion in QuickChange site-directed mutagenesis. This also indirectly proved that additional primers insertion was caused by putting two primers in one reaction system.
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Received: 05 February 2015
Published: 25 May 2015
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