chapter  42
6 Pages

Site-Directed Mutagenesis

Site-directed mutagenesis is a technique that allows the in vitro modification of the sequence of amino acids of a protein in order to evaluate the importance of the modified amino acids in the functioning of the protein. The principle is to mutate the gene in vitro and to produce the protein in vitro. In order to do this, it is necessary to have the cDNA or the coding sequence of this protein. The nucleotide sequence, modified in vitro, is reintroduced in a microorganism (generally a bacterium, see Profiles 10 and 39). The transformed microorganism synthesizes the protein from a mutant gene to produce a polypeptide harbouring the mutation. The biochemical characteristics of this mutated protein are then analysed in order to study the importance of mutated amino acids in the functioning of the protein. Three types of mutations can occur: deletion, insertion, or substitution of bases.