Mutational approaches insertional mutagenesis

There are two strategies to use insertional mutagenesis for cloning purposes. One is the direct tagging approach. In this case a mutant, for example a spontaneous or chemically induced mutant, has already been identified, but no information on the nature of the mutated gene is available. 

Insertions Cassette Mutagenesis. In another valuable approach, cassette mutagenesis, plasmid DNA is cut with a pair of restriction enzymes to remove a short segment (Figure 5.21). 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. 

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