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Nucleic Acids Research 2004 32(19):e145; doi:10.1093/nar/gnh147
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Published online 26 October 2004

Nucleic Acids Research, Vol. 32 No. 19 © Oxford University Press 2004; all rights reserved

One-step random mutagenesis by error-prone rolling circle amplification

Ryota Fujii, Motomitsu Kitaoka* and Kiyoshi Hayashi

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan

* To whom correspondence should be addressed. Tel: +81 29 838 8071; Fax: +81 29 838 7321; Email: mkitaoka{at}nfri.affrc.go.jp

Received August 18, 2004; Revised and Accepted October 10, 2004

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3–4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique.


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