1. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates
- Author
-
Horst Höbenreich, Sheng Wu, Sabrina Kille, Renate Lohmer, Felipe E. Zilly, Shreenath Prasad, Jérôme J.-P. Peyralans, Andreas Taglieber, Yosephine Gumulya, Daniel Kahakeaw, Jullien Drone, John Podtetenieff, J. Daniel Carballeira, Joaquin Sanchis, Manfred T. Reetz, Layla Fernández, Pankaj Soni, Max-Planck-Institut für Kohlenforschung (Coal Research), Max-Planck-Gesellschaft, Institut Charles Gerhardt Montpellier - Institut de Chimie Moléculaire et des Matériaux de Montpellier (ICGM ICMMM), and Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut de Chimie du CNRS (INC)
- Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Antiprimer ,Mutagenesis (molecular biology technique) ,Computational biology ,Biology ,Polymerase Chain Reaction ,01 natural sciences ,Applied Microbiology and Biotechnology ,law.invention ,Megaprimer ,03 medical and health sciences ,Plasmid ,Bacterial Proteins ,Cytochrome P-450 Enzyme System ,law ,Methods ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Genomic library ,Saturated mutagenesis ,Polymerase chain reaction ,Candida ,Gene Library ,NADPH-Ferrihemoprotein Reductase ,030304 developmental biology ,Bacillus megaterium ,Epoxide Hydrolases ,Genetics ,Saturation mutagenesis ,0303 health sciences ,Difficult-to-amplify templates ,010405 organic chemistry ,fungi ,Lipase ,General Medicine ,Directed evolution ,biology.organism_classification ,0104 chemical sciences ,PCR ,Mutagenesis ,Pseudomonas aeruginosa ,Aspergillus niger ,Directed Molecular Evolution ,Primer (molecular biology) ,Plasmids ,Biotechnology - Abstract
International audience; 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.
- Published
- 2008