<P> 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 N - 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 . </P> <P> 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 . </P> <P> The basic procedure requires the synthesis of a short DNA primer . This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest . The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion . The single - strand primer is then extended using a DNA polymerase, which copies the rest of the gene . The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned . Finally, mutants are selected by DNA sequencing to check that they contain the desired mutation . </P> <P> The original method using single - primer extension was inefficient due to a low yield of mutants . This resulting mixture contains both the original unmutated template as well as the mutant strand, producing a mixed population of mutant and non-mutant progenies . Furthermore, the template used is methylated while the mutant strand is unmethylated, and the mutants may be counter-selected due to presence of mismatch repair system that favors the methylated template DNA, resulting in fewer mutants . Many approaches have since been developed to improve the efficiency of mutagenesis . </P>

Site-directed mutagenesis relies on mut proteins to cause base replacements