3-6.2.2 Cassette mutagenesis

In cassette mutagenesis, a fragment of DNA containing the mutation in the gene of interest is synthesized. Then it has been inserted into a plasmid after the cleavage by a restriction enzyme at a particular site in the plasmid and subsequent ligation. Usually the restriction enzymes that cleave the plasmid and candidate oligonucleotide are same to allow generation of sticky ends of the plasmid and insert to ligate to one another. This method can generate mutants at close to 100% efficiency, but in practice limited by the availability of suitable restriction sites.