title: The "Bringer" strategy: a very fast and highly efficient method for construction of mutant libraries by error-prone polymerase chain reaction of ring-closed plasmids.
abstract: Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR). Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property. However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure. Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures. Primers binding to the beta-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible. The functionality of this approach was demonstrated by mutating the alpha-peptide coding region of the lacZ gene.
authors.affiliation: Department of Biochemistry, Building No. 9.2, Saarland University, D-66041 Saarbruecken, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bichet
authors.forename: Matthias
authors.initials: M
authors.lastname: Bureik
authors.forename: Natalie
authors.initials: N
authors.lastname: Lenz
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*