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An efficient one-step site-directed and site-saturation mutagenesis protocol

Zheng, Lei ; Baumann, Ulrich ; Reymond, Jean-Louis

In: Nucleic Acids Research, 2004, vol. 32, no. 14, p. e115-e115

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    Titre
    • An efficient one-step site-directed and site-saturation mutagenesis protocol
    Auteur
    • Zheng, Lei. Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
    • Baumann, Ulrich. Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
    • Reymond, Jean-Louis. Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Nucleic Acids Research, 2004, vol. 32, no. 14, p. e115-e115. Oxford University Press
    Autre version électronique
    Classification
    • Santé
    Mots clés
    Identifiant OAI-PMH
    • oai:doc.rero.ch:292617
    Summary
    We have developed a new primer design method based on the QuickChange™ site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange™ protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries