Oligonucleotide cassette mutagenesis

In another valuable approach, cassette mutagenesis, plasmid DNA is cut with a pair of restriction enzymes to remove a short segment (Figure 6.37). A synthetic double-stranded oligonucleotide (the cassette) with cohesive ends that are complementary to the ends of the cut plasmid is then added and ligated. Each plasmid now contains the desired mutation. It is convenient to introduce into the plasmid unique restriction sites spaced about 40 nucleotides apart so that mutations can be readily made anywhere in the sequence. 

Figure 6.37. Cassette Mutagenesis. DNA is cleaved at a pair of unique restriction sites by two different restriction endonuclease. A synthetic oligonucleotide with ends that are complementary to these sites (the cassette) is then ligated to the cleaved DNA. The method is highly versatile because the inserted DNA can have any desired sequence.

Outline the procedure of oligonucleotide-directed mutagenesis, including cassette mutagenesis. 

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