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One of the most powerful techniques developed to study the structure/function relationships of proteins has been to mutate a gene to verify the effect of the mutation, both in vitro and in vivo. In general, any DNA fragment, showing regulatory functions or not, can be studied by mutagenesis approach. Early attempts of DNA mutagenesis were non-site-specific and made using radiation or chemical mutagens. Analogs of nucleotides and other chemicals were later used to generate localized point mutations, but they were not specific point mutations. Site-directed mutagenesis is the method that allows to make specific and intentional changes to the DNA sequence, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules. In 1978 Prof. Michael Smith described the first technique of site-directed mutagenesis, which was developed in collaboration with Prof. Clyde Hutchinson. They used as primer a 12-nucleotide oligomer with single nucleotide mismatch, as template φX174 DNA, and the E. coli DNA polymerase I to synthesize DNA, to construct a closed circular double-stranded DNA with the mutagenic oligonucleotide in one strand.
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