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Error-Prone Rolling Circle Amplification Greatly Simplifies Random Mutagenesis

  • Ryota Fujii
  • Motomitsu Kitaoka
  • Kiyoshi Hayashi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1179)

Abstract

We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl2 and is used to transform a host strain resulting in a mutant library with several random point mutations per kilobase through the entire plasmid. The primary advantage of this method is its simplicity. This protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.

Key words

Random mutagenesis Directed evolution Point mutation Rolling circle amplification Protein engineering 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Ryota Fujii
    • 1
  • Motomitsu Kitaoka
    • 2
  • Kiyoshi Hayashi
    • 3
  1. 1.Synthetic Chemicals LaboratoryMitsui Chemicals, Inc.SodegauraJapan
  2. 2.National Food Research Institute,National Agriculture and Food Research OrganizationTsukubaJapan
  3. 3.Faculty of Food and Nutritional SciencesToyo UniversityItakura-machi, Ora-gunJapan

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