State Key Laboratory of Cellular Stress Biology and Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.
Anal Biochem. 2011 Nov 15;418(2):304-6. doi: 10.1016/j.ab.2011.07.008. Epub 2011 Jul 18.
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.
QuikChange 是一种常用于蛋白质和核酸结构与功能研究的定点突变方法。然而，标准的 protocol 通常在产生所需突变时效率不高。在这里，我们提出了一种新的引物设计策略，即中央重叠引物（COP），它使用一对不同长度的双份引物，其中短引物与长引物的中间区域互补。COP 方法在不使用超感受态大肠杆菌细胞或费力筛选阳性克隆的情况下，高效且稳健地产生了大约 90%的突变率。
