Error-prone rolling circle amplification: the simplest random mutagenesis protocol
Error-prone rolling circle amplification: the simplest random mutagenesis protocol
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DOI：
10.1038/nprot.2006.403
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Hayashi, Kiyoshi
中科院分区：
文献类型：
--
作者：
Fujii, Ryota;Kitaoka, Motomitsu;Hayashi, Kiyoshi
描述了一种引入随机突变的简单方案，称为易错滚环扩增（RCA）。在 MnCl2 存在下，通过 RCA 扩增模板质粒，并用于转化宿主菌株，得到整个质粒中每千碱基具有 3 至 4 个随机点突变的突变体文库。这种方法的主要优点是它的简单性。该方案既不需要设计特定的引物，也不需要探索热循环条件。只需 10 分钟即可制备反应混合物，然后过夜孵育并转化宿主菌株。该方法可以快速制备随机突变的质粒文库，并将使随机诱变得到更广泛的采用。
A simple protocol to introduce random mutations, named error-prone rolling circle amplification (RCA), is described. A template plasmid is amplified by RCA in the presence of MnCl2 and used for transformation of a host strain to give a mutant library with three to four random point mutations per kilobase throughout the entire plasmid. The prime advantage of this method is its simplicity. This protocol requires neither the design of specific primers nor the exploration of thermal cycling conditions. It takes just 10 min to prepare the reaction mixture, followed by overnight incubation and transformation of a host strain. This method permits rapid preparation of randomly mutated plasmid libraries, and will enable the wider adoption of random mutagenesis.