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One-Pot Simple Methodology for Cassette Randomization and Recombination for Focused Directed Evolution (OSCARR)

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Directed Evolution Library Creation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1179))

Abstract

The OSCARR methodology (One-pot Simple methodology for CAssette Randomization and Recombination) bridges the gap between site-directed mutagenesis and full randomization by making use of carefully designed mutagenic cassettes and an optimized one-pot megaprimer PCR. The method is especially suited to construct libraries of up to ten randomized codons for focused directed evolution, exhibits up to 97 % efficiency in the amplification of mutated over wild-type products, and is sufficiently versatile to allow mutagenesis and recombination of several cassettes within the same gene.

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Author information

Authors and Affiliations

  1. Center for Molecular Biology, UAM-CSIC, Madrid, Spain

    Aurelio Hidalgo

  2. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany

    Anna Schließmann & Uwe T. Bornscheuer

Authors
  1. Aurelio Hidalgo
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  2. Anna Schließmann
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  3. Uwe T. Bornscheuer
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Corresponding author

Correspondence to Uwe T. Bornscheuer .

Editor information

Editors and Affiliations

  1. Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia

    Elizabeth M.J. Gillam

  2. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

    Janine N. Copp

  3. School of Biological Sciences, Victoria University, Wellington, New Zealand

    David Ackerley

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© 2014 Springer Science+Business Media New York

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Hidalgo, A., Schließmann, A., Bornscheuer, U.T. (2014). One-Pot Simple Methodology for Cassette Randomization and Recombination for Focused Directed Evolution (OSCARR). In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_14

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  • DOI: https://doi.org/10.1007/978-1-4939-1053-3_14

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-1052-6

  • Online ISBN: 978-1-4939-1053-3

  • eBook Packages: Springer Protocols

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