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An improved dual-tube megaprimer approach for multi-site saturation mutagenesis

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Abstract

Saturation mutagenesis is a powerful tool in protein engineering. Even though QuikChange site-directed mutagenesis method is dominantly used in laboratories, it could not be successfully applied to the generation of a focused mutant library of human glutathione transferase A2-2. In the present study, we further developed an improved versatile dual-tube approach of randomizing difficult-to-amplify targets, exhibiting significant improvement towards equal distribution of nucleotides at randomized sites compared to other published methods.

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Abbreviations

QCM:

QuikChange site-directed mutagenesis method

GST:

Glutathione transferase

PCR:

Polymerase chain reaction

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Acknowledgments

This work was supported by grants from the Swedish Research Council and the Swedish Cancer Society.

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Authors and Affiliations

  1. Department of Chemistry, BMC, Uppsala University, Box 576, 75123, Uppsala, Sweden

    Wei Zhang & Bengt Mannervik

  2. Department of Neurochemistry, Stockholm University, 10691, Stockholm, Sweden

    Bengt Mannervik

Authors
  1. Wei Zhang
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  2. Bengt Mannervik
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Correspondence to Bengt Mannervik.

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Zhang, W., Mannervik, B. An improved dual-tube megaprimer approach for multi-site saturation mutagenesis. World J Microbiol Biotechnol 29, 667–672 (2013). https://doi.org/10.1007/s11274-012-1222-z

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  • DOI: https://doi.org/10.1007/s11274-012-1222-z

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