Site-directed mutagenesis

Abstract

The development of genetic engineering, whereby a specific gene or cDNA (c is copy or complementary) can be isolated as part of a minichromosome that can replicate and be expressed (by transcription and translation) in a living cell, has made possible in vitro techniques for micromanipulation (i.e. site-directed mutagenesis) of a cloned gene, to make defined changes in the portion of the gene that encodes its protein product. The methods by which this micromanipulation of a structural gene are effected fall under three broad headings: (i) the production of random single base-pair substitutions by chemical or enzymatic means; (ii) the construction of heteroduplex DNA by annealing single-strand target DNA with a chemically synthesized mutagenic oligonucleotide and (iii) the total or partial synthesis of mutant duplex DNA from chemically synthesized oligonucleotides. As a consequence it is now possible to modify a gene so that any amino acid in its product protein can be replaced by any other amino acid.