Site-directed mutagenesis
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.
Site-directed mutagenesis is one of the most important techniques in laboratory for introducing a mutation into a DNA sequence. However, with decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis.
Early attempts at mutagenesis using radiation or chemical mutagens were non-site-specific, generating random mutations. Analogs of nucleotides and other chemicals were later used to generate localized point mutations, examples of such chemicals are aminopurine,nitrosoguanidine, and bisulfite. Site-directed mutagenesis was achieved in 1974 in the laboratory of Charles Weissmann using a nucleotide analogue N4-hydroxycytidine, which induces transition of GC to AT. These methods of mutagenesis, however, are limited by the kind of mutation they can achieve, and they are not as specific as later site-directed mutagenesis methods.
In 1971, Clyde Hutchison and Marshall Edgell showed that it is possible to produce mutants with small fragments of phage ϕX174 and restriction nucleases. Hutchison later produced with his collaborator Michael Smith in 1978 a more flexible approach to site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA polymerase. For his part in the development of this process, Michael Smith later shared the Nobel Prize in Chemistry in October 1993 with Kary B. Mullis, who invented polymerase chain reaction.
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