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Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Abstract

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

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Acknowledgement

This research was supported by the German–Israeli Project Cooperation (DIP), the Deutsche Forschungsgemeinschaft (Schwerpunkt 1170; “Directed Evolution to Optimize and Understand Molecular Biocatalysis”; Project RE 359/13-1) and the Fonds der Chemischen Industrie.

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This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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Correspondence to Manfred T. Reetz.

Additional information

Joaquin Sanchis, Layla Fernández, and J. Daniel Carballeira contributed equally.

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Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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Sanchis, J., Fernández, L., Carballeira, J.D. et al. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Appl Microbiol Biotechnol 81, 387–397 (2008). https://doi.org/10.1007/s00253-008-1678-9

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Keywords

  • Directed evolution
  • Saturation mutagenesis
  • PCR
  • Megaprimer
  • Antiprimer
  • Difficult-to-amplify templates