Fujii Ryota, Kitaoka Motomitsu, Hayashi Kiyoshi
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.
Nat Protoc. 2006;1(5):2493-7. doi: 10.1038/nprot.2006.403.
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.
本文描述了一种引入随机突变的简单方法，即易错滚环扩增（RCA）。在氯化锰存在的情况下，通过RCA扩增模板质粒，并用于转化宿主菌株，从而得到一个突变文库，整个质粒中每千碱基有三到四个随机点突变。该方法的主要优点是简单。此方法既不需要设计特异性引物，也不需要摸索热循环条件。制备反应混合物只需10分钟，随后进行过夜孵育并转化宿主菌株。该方法能够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应用。
