title: 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.
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim/Ruhr, Germany.
authors.forename: Joaquin
authors.initials: J
authors.lastname: Sanchis
authors.forename: Layla
authors.initials: L
authors.lastname: Fernández
authors.forename: J Daniel
authors.initials: JD
authors.lastname: Carballeira
authors.forename: Jullien
authors.initials: J
authors.lastname: Drone
authors.forename: Yosephine
authors.initials: Y
authors.lastname: Gumulya
authors.forename: Horst
authors.initials: H
authors.lastname: Höbenreich
authors.forename: Daniel
authors.initials: D
authors.lastname: Kahakeaw
authors.forename: Sabrina
authors.initials: S
authors.lastname: Kille
authors.forename: Renate
authors.initials: R
authors.lastname: Lohmer
authors.forename: Jérôme J-P
authors.initials: JJ
authors.lastname: Peyralans
authors.forename: John
authors.initials: J
authors.lastname: Podtetenieff
authors.forename: Shreenath
authors.initials: S
authors.lastname: Prasad
authors.forename: Pankaj
authors.initials: P
authors.lastname: Soni
authors.forename: Andreas
authors.initials: A
authors.lastname: Taglieber
authors.forename: Sheng
authors.initials: S
authors.lastname: Wu
authors.forename: Felipe E
authors.initials: FE
authors.lastname: Zilly
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D002175:Candida / Q000201:enzymology / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004851:Epoxide Hydrolases / Q000235:genetics; D015723:Gene Library; D008049:Lipase / Q000235:genetics; D016296:Mutagenesis; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D008049:Lipase; D004851:Epoxide Hydrolases