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


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2006-12

AUTHORS

Ryota Fujii, Motomitsu Kitaoka, Kiyoshi Hayashi

ABSTRACT

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. More... »

PAGES

2493-2497

References to SciGraph publications

  • 1998-07. Mutation detection and single-molecule counting using isothermal rolling-circle amplification in NATURE GENETICS
  • 1999-02-26. Superior Biocatalysts by Directed Evolution in BIOCATALYSIS - FROM DISCOVERY TO APPLICATION
  • Journal

    TITLE

    Nature Protocols

    ISSUE

    5

    VOLUME

    1

    Author Affiliations

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nprot.2006.403

    DOI

    http://dx.doi.org/10.1038/nprot.2006.403

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1035794549

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/17406496


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    38 schema:description 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.
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