An improved dual-tube megaprimer approach for multi-site saturation mutagenesis
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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.
KeywordsProtein engineering Saturation mutagenesis Megaprimer approach QuikChange method
QuikChange site-directed mutagenesis method
Polymerase chain reaction
This work was supported by grants from the Swedish Research Council and the Swedish Cancer Society.
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