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Mutagenesis Protocols in Saccharomyces cerevisiae by In Vivo Overlap Extension

  • Miguel Alcalde
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 634)

Abstract

A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant libraries for directed evolution experiments. In this context, in vivo overlap extension (IVOE) is a particularly attractive protocol that takes advantage of the eukaryotic apparatus to carry out combinatorial saturation mutagenesis, site-directed recombination or site-directed mutagenesis, avoiding ligation steps and additional PCR reactions that are common to standard in vitro protocols.

Key words

IVOE Saccharomyces cerevisiae Combinatorial saturation mutagenesis In vivo recombination Directed evolution 

Notes

Acknowledgments

This work was supported by the Spanish Ministry of Science and Innovation (projects CCG08-CSIC/PPQ-3706; PIE 200880I033) and EU project FP7-NMP4-SL-2009-229255.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Instituto de Catálisis y Petroleoquímica (CSIC)MadridSpain

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