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.

Autor: Bichet A; Department of Biochemistry, Building No. 9.2, Saarland University, D-66041 Saarbruecken, Germany., Bureik M, Lenz N, Bernhardt R
Jazyk: angličtina
Zdroj: Applied biochemistry and biotechnology [Appl Biochem Biotechnol] 2004 May; Vol. 117 (2), pp. 115-22.
DOI: 10.1385/abab:117:2:115
Abstrakt: 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.
Databáze: MEDLINE
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