A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis.
Deep Mutational Scanning
Directed Evolution
Nicking Mutagenesis
Oligo pools
Protein Design
Protein Engineering
Site-saturation mutagenesis
Journal
Bio-protocol
ISSN: 2331-8325
Titre abrégé: Bio Protoc
Pays: United States
ID NLM: 101635102
Informations de publication
Date de publication:
05 Aug 2020
05 Aug 2020
Historique:
received:
18
03
2020
revised:
20
05
2020
accepted:
26
05
2020
entrez:
4
3
2021
pubmed:
5
3
2021
medline:
5
3
2021
Statut:
epublish
Résumé
Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking restriction endonuclease and exonuclease treatment. Mutagenic primers encoding the desired mutations are annealed to the resulting circular single-stranded DNA, extended with high-fidelity polymerase, and ligated into covalently closed circular DNA by
Identifiants
pubmed: 33659364
doi: 10.21769/BioProtoc.3697
pii: e3697
pmc: PMC7842585
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e3697Subventions
Organisme : NIAID NIH HHS
ID : R01 AI141452
Pays : United States
Informations de copyright
Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.
Déclaration de conflit d'intérêts
Competing interestsT.A.W. is on a U.S. patent application 16/115.029 covering the nicking mutagenesis method. P.J.S and Z.T.B. do not have competing interests.
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