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


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2008-11-01

AUTHORS

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

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. More... »

PAGES

387-397

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s00253-008-1678-9

DOI

http://dx.doi.org/10.1007/s00253-008-1678-9

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1046650831

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/18820909


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