| Autor: |
Bichet, Andreas, Bureik, Matthias, Lenz, Natalie, Bernhardt, Rita |
| Zdroj: |
Applied Biochemistry and Biotechnology; May 2004, Vol. 117 Issue: 2 p115-122, 8p |
| 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 β-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 α-peptide coding region of the lacZ gene. |
| Databáze: |
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