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Anal Biochem. 2011 Nov 15;418(2):304-6. doi: 10.1016/j.ab.2011.07.008. Epub 2011 Jul 18.

An efficient approach for site-directed mutagenesis using central overlapping primers.

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State Key Laboratory of Cellular Stress Biology and Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.


QuikChange is a popular method for site-directed mutagenesis in structural and functional studies of proteins and nucleic acids. However, the standard protocol is often inefficient in producing the desired mutations. Here we present a novel strategy for primer design, central overlapping primers (COP), which employs a pair of bipartite primers of different lengths, with the short primer complementary to the middle region of the long primer. The COP method is efficient and robust in generating approximately 90% mutation rate without supercompetent Escherichia coli cells or laborious screening for positive clones.

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