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

Nature Protocols volume 1, pages 2493–2497 (2006)Cite this article

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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.

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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  • 22 February 2007

    changed 2007 to 2006

References

  1. Bloom, J.D. et al. Evolving strategies for enzyme engineering. Curr. Opin. Struct. Biol. 15, 447–452 (2005).

    Article  CAS  Google Scholar 

  2. Jaeger, K.E. & Eggert, T. Enantioselective biocatalysis optimized by directed evolution. Curr. Opin. Biotechnol. 15, 305–313 (2004).

    Article  CAS  Google Scholar 

  3. Arnold, F.H., Wintrode, P.L., Miyazaki, K. & Gershenson, A. How enzymes adapt: lessons from directed evolution. Trends Biochem. Sci. 26, 100–106 (2001).

    Article  CAS  Google Scholar 

  4. Johannes, T.W. & Zhao, H. Directed evolution of enzymes and biosynthetic pathways. Curr. Opin. Microbiol. 9, 261–267 (2006).

    Article  CAS  Google Scholar 

  5. Reetz, M.T. Directed evolution of enantioselective enzymes as catalysts for organic synthesis. Adv. Catal. 49, 1–69 (2006).

    CAS  Google Scholar 

  6. Aharoni, A., Griffiths, A.D. & Tawfik, D.S. High-throughput screens and selections of enzyme-encoding genes. Curr. Opin. Chem. Biol. 9, 210–216 (2005).

    Article  CAS  Google Scholar 

  7. Goddard, J.P. & Reymond, J.L. Enzyme assays for high-throughput screening. Curr. Opin. Biotechnol. 15, 314–322 (2004).

    Article  CAS  Google Scholar 

  8. Taylor, S.V., Kast, P. & Hilvert, D. Investigating and engineering enzymes by genetic selection. Angew. Chem. Int. Ed. Engl. 40, 3310–3335 (2001).

    Article  Google Scholar 

  9. Lin, H. & Cornish, V.W. Screening and selection methods for large-scale analysis of protein function. Angew. Chem. Int. Ed. Engl. 41, 4402–4425 (2002).

    Article  CAS  Google Scholar 

  10. Reetz, M.T. & Jaeger, K.E. Superior biocatalysts by directed evolution. In Biocatalysis - From Discovery to Application 31–57 (Springer-Verlag, Berlin, 1999).

    Chapter  Google Scholar 

  11. Leung, D.W., Chen, E. & Goeddel, D.W. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Techniques 1, 11–15 (1989).

    Google Scholar 

  12. Greener, A., Callahan, M. & Jerpseth, B. In in vitro Mutagenesis Protocols (ed. Trower, M.K.) (Humana press, New Jersey, 1996).

    Google Scholar 

  13. Kornberg, A. & Baker, T. In DNA Replication (W.H. Freeman & Company, NY, 1992).

    Google Scholar 

  14. Fire, A. & Xu, S.Q. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92, 4641–4645 (1995).

    Article  CAS  Google Scholar 

  15. Liu, D.Y., Daubendiek, S.L., Zillman, M.A., Ryan, K. & Kool, E.T. Rolling circle DNA synthesis: small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118, 1587–1594 (1996).

    Article  CAS  Google Scholar 

  16. Lizardi, P.M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).

    Article  CAS  Google Scholar 

  17. Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

    Article  CAS  Google Scholar 

  18. Fujii, R., Kitaoka, M. & Hayashi, K. One-step random mutagenesis by error-prone rolling circle amplification. Nucleic Acids Res. 32, e145 (2004).

    Article  Google Scholar 

  19. Ding, X., Snyder, A.K., Shaw, R., Farmerie, W.G. & Song, W.Y. Direct retransformation of yeast with plasmid DNA isolated from single yeast colonies using rolling circle amplification. BioTechniques 35, 774–779 (2003).

    Article  CAS  Google Scholar 

  20. Camps, M., Naukkarinen, J., Johnson, B.P. & Loeb, L.A. Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I. Proc. Natl. Acad. Sci. USA 100, 9727–9732 (2003).

    Article  CAS  Google Scholar 

  21. Henke, E. & Bornscheuer, U.T. Directed evolution of an esterase from Psueudomonas fluorescens. Random mutagenesis by error-prone PCR or a mutator strain and identification of mutants showing enhanced enantioselectivity by a resorufin-based fluorescence assay. Biol. Chem. 380, 1029–1033 (1999).

    Article  CAS  Google Scholar 

  22. Bornscheuer, U.T., Altenbuchner, J. & Meyer, H.H. Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones. Biotechnol. Bioeng. 58, 554–559 (1998).

    Article  CAS  Google Scholar 

  23. Reetz, M.T., Zonta, A., Schimossek, K., Liebeton, K. & Jaeger, K.E. Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew. Chem. Int. Ed. Engl. 36, 2830–2832 (1997).

    Article  CAS  Google Scholar 

  24. de Vega, M., Lazaro, J.M. & Salas, M. Phage φ29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3′-5′ exonuclease active site. J. Mol. Biol. 304, 1–9 (2000).

    Article  CAS  Google Scholar 

  25. Voss, C., Schmidt, T., Schleef, M., Friehs, K. & Flaschel, E. Production of supercoiled multimeric plasmid DNA for biopharmaceutical application. J. Biotechnol. 105, 205–213 (2003).

    Article  CAS  Google Scholar 

  26. Vakulenko, S.B. et al. Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEMpUC19 β-lactamase from Escherichia coli. J. Biol. Chem. 274, 23052–23060 (1999).

    Article  CAS  Google Scholar 

  27. Vakulenko, S.B., Toth, M., Taibi, P., Mobashery, S. & Lerner, S.A. Effects of Asp-179 mutations in TEMpUC19 β-lactamase on susceptibility to β-lactams. Antimicrob. Agents Chemother. 39, 1878–1880 (1995).

    Article  CAS  Google Scholar 

  28. Blanco, L. et al. Highly efficient DNA-synthesis by the phage φ29 DNA polymerase. J. Biol. Chem. 264, 8935–8940 (1989).

    CAS  PubMed  Google Scholar 

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Acknowledgements

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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  1. Ryota Fujii

    Present address: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, Minneapolis, 55108, USA

Authors and Affiliations

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

    Ryota Fujii, Motomitsu Kitaoka & Kiyoshi Hayashi

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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). https://doi.org/10.1038/nprot.2006.403

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