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Error-prone rolling circle amplification: the simplest random mutagenesis protocol

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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.

NOTE: In the PDF version of this article initially published online, the publication date was shown as 29 December 2007 instead of 29 December 2006. The error has been corrected in the PDF version of the article.

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Figure 1: Mechanism of rolling circle amplification17.
Figure 2: Re-circularization of RCA product in vivo.
Figure 3: Error-prone RCA.
Figure 4: Schematic diagram comparing error-prone RCA with conventional random mutagenesis methods.

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    changed 2007 to 2006


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This study was supported in part by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

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Correspondence to Motomitsu Kitaoka.

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Fujii, R., Kitaoka, M. & Hayashi, K. Error-prone rolling circle amplification: the simplest random mutagenesis protocol. Nat Protoc 1, 2493–2497 (2006).

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