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

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About: Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : wasabi.inria.fr associated with source document(s)

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type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
title	Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates
Creator	Daniel, J Drone, Jullien Fernández, Layla Gumulya, Yosephine Höbenreich, Horst Jérôme, J.-P Kahakeaw, Daniel Kille, Sabrina Lohmer, Renate Peyralans, John Podtetenieff, Shreenath Prasad, Pankaj Reetz, Sanchis, Joaquin Soni, Andreas Taglieber, Sheng Wu, Felipe Zilly, Manfred
source	PMC
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.
has issue date	2008-11-01(xsd:dateTime)
bibo:doi	10.1007/s00253-008-1678-9
bibo:pmid	18820909
has license	cc-by-nc
sha1sum (hex)	fac3c7b30a3795146510e99b46c147b0dd575d75
schema:url	https://doi.org/10.1007/s00253-008-1678-9
resource representing a document's title	Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates
has PubMed Central identifier	PMC7419347
has PubMed identifier	18820909
schema:publication	Appl Microbiol Biotechnol
resource representing a document's body	covid:fac3c7b30a3795146510e99b46c147b0dd575d75#body_text
is schema:about of	named entity 'PCR' named entity 'application' named entity 'METHOD' named entity 'TEMPLATE' named entity 'AMPLIFY' named entity 'APPLIED' named entity 'SINGLE' named entity 'REACTION' named entity '27S' named entity 'USED' named entity 'MUTAGENIC' named entity 'enzymes' named entity 'protocols' named entity 'fail' named entity 'devised' named entity 'protocols' named entity 'improvement' named entity 'mutagenic' named entity 'PCR' named entity 'plasmid' named entity 'Stratagene' named entity 'Pseudomonas aeruginosa' named entity 'gene' named entity 'PCR' named entity 'saturation mutagenesis' named entity 'overlap extension PCR' named entity 'DpnI' named entity 'plasmid' named entity 'PCR-amplified' named entity 'saturation mutagenesis' named entity 'plasmid' named entity 'gene' named entity 'linear stage' named entity 'Dam methylation' named entity 'molecular biological' named entity 'plasmid' named entity 'mutagenesis' named entity 'bacteria' named entity 'saturation mutagenesis' named entity 'primers' named entity 'primers' named entity 'HPLC' named entity 'plasmid' named entity 'gene' named entity 'directed evolution' named entity 'gene' named entity 'anneal' named entity 'plasmids' named entity 'codon' named entity 'genomic DNA' named entity 'enantioselectivity' named entity 'GC content' named entity 'PCR' named entity 'nucleotide' named entity 'nucleotides' named entity 'plasmid' named entity 'DpnI' named entity 'TTT' named entity 'PCRs' named entity 'mutagenic' named entity 'site-directed mutagenesis' named entity 'A. niger' named entity 'oligonucleotide' named entity 'plasmid'

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