Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates
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 restrictio...
Published in: | Applied Microbiology and Biotechnology |
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Online Access: | https://hal.archives-ouvertes.fr/hal-00354234 https://doi.org/10.1007/s00253-008-1678-9 |
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ftccsdartic:oai:HAL:hal-00354234v1 2023-05-15T13:36:04+02:00 Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates Sanchis, Joaquin Fernandez, Layla Daniel Carballeira, J. Drone, Jullien Gumulya, Yosephine Höbenreich, Horst Kille, Sabrina Lohmer, Renate Jp Peyralans, Jérôme Podtetenieff, John Prasad, Shreenath Soni, Pankaj Wu, Sheng E Zilly, Felipe Reetz, Manfred 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) 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) 2008-11-01 https://hal.archives-ouvertes.fr/hal-00354234 https://doi.org/10.1007/s00253-008-1678-9 en eng HAL CCSD Springer Verlag info:eu-repo/semantics/altIdentifier/doi/10.1007/s00253-008-1678-9 info:eu-repo/semantics/altIdentifier/pmid/18820909 hal-00354234 https://hal.archives-ouvertes.fr/hal-00354234 doi:10.1007/s00253-008-1678-9 PUBMED: 18820909 ISSN: 0175-7598 EISSN: 1432-0614 Applied Microbiology and Biotechnology https://hal.archives-ouvertes.fr/hal-00354234 Applied Microbiology and Biotechnology, Springer Verlag, 2008, 81, pp.387-397. ⟨10.1007/s00253-008-1678-9⟩ PCR Megaprimer Antiprimer Difficult-to-amplify templates Directed evolution Saturation mutagenesis [SDV.BIO]Life Sciences [q-bio]/Biotechnology [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology info:eu-repo/semantics/article Journal articles 2008 ftccsdartic https://doi.org/10.1007/s00253-008-1678-9 2021-12-12T06:15:21Z 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. Article in Journal/Newspaper Antarc* Antarctica Archive ouverte HAL (Hyper Article en Ligne, CCSD - Centre pour la Communication Scientifique Directe) Applied Microbiology and Biotechnology 81 2 387 397 |
institution |
Open Polar |
collection |
Archive ouverte HAL (Hyper Article en Ligne, CCSD - Centre pour la Communication Scientifique Directe) |
op_collection_id |
ftccsdartic |
language |
English |
topic |
PCR Megaprimer Antiprimer Difficult-to-amplify templates Directed evolution Saturation mutagenesis [SDV.BIO]Life Sciences [q-bio]/Biotechnology [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology |
spellingShingle |
PCR Megaprimer Antiprimer Difficult-to-amplify templates Directed evolution Saturation mutagenesis [SDV.BIO]Life Sciences [q-bio]/Biotechnology [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Sanchis, Joaquin Fernandez, Layla Daniel Carballeira, J. Drone, Jullien Gumulya, Yosephine Höbenreich, Horst Kille, Sabrina Lohmer, Renate Jp Peyralans, Jérôme Podtetenieff, John Prasad, Shreenath Soni, Pankaj Wu, Sheng E Zilly, Felipe Reetz, Manfred Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates |
topic_facet |
PCR Megaprimer Antiprimer Difficult-to-amplify templates Directed evolution Saturation mutagenesis [SDV.BIO]Life Sciences [q-bio]/Biotechnology [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology |
description |
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. |
author2 |
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) 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) |
format |
Article in Journal/Newspaper |
author |
Sanchis, Joaquin Fernandez, Layla Daniel Carballeira, J. Drone, Jullien Gumulya, Yosephine Höbenreich, Horst Kille, Sabrina Lohmer, Renate Jp Peyralans, Jérôme Podtetenieff, John Prasad, Shreenath Soni, Pankaj Wu, Sheng E Zilly, Felipe Reetz, Manfred |
author_facet |
Sanchis, Joaquin Fernandez, Layla Daniel Carballeira, J. Drone, Jullien Gumulya, Yosephine Höbenreich, Horst Kille, Sabrina Lohmer, Renate Jp Peyralans, Jérôme Podtetenieff, John Prasad, Shreenath Soni, Pankaj Wu, Sheng E Zilly, Felipe Reetz, Manfred |
author_sort |
Sanchis, Joaquin |
title |
Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates |
title_short |
Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates |
title_full |
Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates |
title_fullStr |
Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates |
title_full_unstemmed |
Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates |
title_sort |
improved pcr method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates |
publisher |
HAL CCSD |
publishDate |
2008 |
url |
https://hal.archives-ouvertes.fr/hal-00354234 https://doi.org/10.1007/s00253-008-1678-9 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
ISSN: 0175-7598 EISSN: 1432-0614 Applied Microbiology and Biotechnology https://hal.archives-ouvertes.fr/hal-00354234 Applied Microbiology and Biotechnology, Springer Verlag, 2008, 81, pp.387-397. ⟨10.1007/s00253-008-1678-9⟩ |
op_relation |
info:eu-repo/semantics/altIdentifier/doi/10.1007/s00253-008-1678-9 info:eu-repo/semantics/altIdentifier/pmid/18820909 hal-00354234 https://hal.archives-ouvertes.fr/hal-00354234 doi:10.1007/s00253-008-1678-9 PUBMED: 18820909 |
op_doi |
https://doi.org/10.1007/s00253-008-1678-9 |
container_title |
Applied Microbiology and Biotechnology |
container_volume |
81 |
container_issue |
2 |
container_start_page |
387 |
op_container_end_page |
397 |
_version_ |
1766074048698122240 |