Cassette mutagenesis

It uses complementary restriction enzyme digest ends on the target DNA and gene cassette to achieve specificity.

[1][2][3][4][5] First, restriction enzymes are used to cleave near the target sequence on DNA contained in a suitable vector.

The length of the sequence flanked by the restriction sites is also a limiting factor due to the use of synthetic gene cassettes.

[2][3] Since one gene cassette can contain multiple mutations, less total oligonucleotide synthesis and purification is needed.

[2] Once the vector is set up with flanking restriction sites, all manipulations (i.e., mutagenesis, sequencing, expression) can be performed in the same plasmid.