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

  • Wei Zhang
  • Bengt MannervikEmail author
Original Paper

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.

Keywords

Protein engineering Saturation mutagenesis Megaprimer approach QuikChange method 

Abbreviations

QCM

QuikChange site-directed mutagenesis method

GST

Glutathione transferase

PCR

Polymerase chain reaction

Notes

Acknowledgments

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

References

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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  1. 1.Department of Chemistry, BMCUppsala UniversityUppsalaSweden
  2. 2.Department of NeurochemistryStockholm UniversityStockholmSweden

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