Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.
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.
Sanchis
Joaquin
J
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim/Ruhr, Germany.
Fernández
Layla
L
Carballeira
J Daniel
JD
Drone
Jullien
J
Gumulya
Yosephine
Y
Höbenreich
Horst
H
Kahakeaw
Daniel
D
Kille
Sabrina
S
Lohmer
Renate
R
Peyralans
Jérôme J-P
JJ
Podtetenieff
John
J
Prasad
Shreenath
S
Soni
Pankaj
P
Taglieber
Andreas
A
Wu
Sheng
S
Zilly
Felipe E
FE
Reetz
Manfred T
MT
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
D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D008049:Lipase; D004851:Epoxide Hydrolases