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

Sanchis, Joaquin, Fernandez, Layla, Carballeira, J. Daniel, Drone, Jullien, Gumulya, Yosephine, Hoebenreich, Horst, Kahakeaw, Daniel, Kille, Sabrina, Lohmer, Renate, Peyralans, Jerome J. -P., Podtetenieff, John, Prasad, Shreenath, Soni, Pankaj, Taglieber, Andreas, Wu, Sheng, Zilly, Felipe E. and Reetz, Manfred T. (2008) Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: Application to difficult-to-amplify templates. Applied Microbiology and Biotechnology, 81 2: 387-397. doi:10.1007/s00253-008-1678-9


Author Sanchis, Joaquin
Fernandez, Layla
Carballeira, J. Daniel
Drone, Jullien
Gumulya, Yosephine
Hoebenreich, Horst
Kahakeaw, Daniel
Kille, Sabrina
Lohmer, Renate
Peyralans, Jerome J. -P.
Podtetenieff, John
Prasad, Shreenath
Soni, Pankaj
Taglieber, Andreas
Wu, Sheng
Zilly, Felipe E.
Reetz, Manfred T.
Title Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: Application to difficult-to-amplify templates
Journal name Applied Microbiology and Biotechnology   Check publisher's open access policy
ISSN 0175-7598
Publication date 2008
Sub-type Article (original research)
DOI 10.1007/s00253-008-1678-9
Volume 81
Issue 2
Start page 387
End page 397
Total pages 11
Language eng
Subject 1305 Biotechnology
2402 Applied Microbiology and Biotechnology
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.
Keyword Antiprimer
Difficult-to-amplify templates
Directed evolution
Megaprimer
PCR
Saturation mutagenesis
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
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