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

  • Aurelio Hidalgo
  • Anna Schließmann
  • Uwe T. Bornscheuer
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Focused directed evolution Megaprimer PCR Cassette mutagenesis Gene randomization Gene recombination 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Aurelio Hidalgo
    • 1
  • Anna Schließmann
    • 2
  • Uwe T. Bornscheuer
    • 2
  1. 1.Center for Molecular Biology, UAM-CSICMadridSpain
  2. 2.Department of Biotechnology and Enzyme Catalysis, Institute of BiochemistryGreifswald UniversityGreifswaldGermany

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