A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis.

ABSTRACT: 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 Taq DNA ligase. The heteroduplex DNA is resolved by selective degradation of the template strand. The complementary strand is synthesized and ligated, resulting in a library of mutated covalentl

SUBMITTER: Steiner PJ 

PROVIDER: S-EPMC7842585 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature