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

Joaquin Sanchis, Layla Fernandez, J. Daniel Carballeira, Jullien Drone, Yosephine Gumulya, Horst Hoebenreich, Daniel Kahakeaw, Sabrina Kille, Renate Lohmer, Jerome J. -P. Peyralans, John Podtetenieff, Shreenath Prasad, Pankaj Soni, Andreas Taglieber, Shuang sheng Wu, Felipe E. Zilly, Manfred T Reetz

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Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange (TM) 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.

Original languageEnglish
Pages (from-to)387-397
Number of pages11
JournalApplied Microbiology and Biotechnology
Issue number2
Publication statusPublished - Nov 2008
Externally publishedYes


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

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