ABSTRACT: Site-directed methods for the generation of genetic

diversity are essential tools in the field of directed enzyme evolution.
The Golden Gate cloning technique has been proven to be an
efficient tool for a variety of cloning setups. The utilization of restriction
enzymes which cut outside of their recognition domain allows
the assembly of multiple gene fragments obtained by PCR
amplification without altering the open reading frame of the reconstituted
gene. In this technical note, we developed a protocol
termed Golden Mutagenesis for the rapid, easy, reliable and inexpensive
construction of mutagenesis libraries. One to five positions
within a coding sequence could be altered simultaneously using a
protocol which can be performed within two days. To facilitate the
implementation of this technique, a software library for automated
primer design and for the graphical evaluation of the sequencing
results has been developed, allowing an easy determination of the
library quality. 

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