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

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• 作者：Huiping Wang¹ ; Nan Zhou¹ ; Feng Ding ; Zhaofang Li ; Ruichuan Chen ; Aidong Han  ; ;   ; ^{ahan@xmu.edu.cn} ; Runzhong Liu  ; ;   ; ^{liurz@xmu.edu.cn}
• 关键词：Site-directed mutagenesis ; QuikChange ; Central overlapping primers (COP)
• 刊名：Analytical Biochemistry
• 出版年：2011
• 出版时间：15 November 2011
• 年：2011
• 卷：418
• 期：2
• 页码：304-306
• 全文大小：250 K

文摘

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.