site-directed mutagenesis

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site-directed mutagenesis A technique used in genetic engineering for introducing particular changes to the base sequence of a gene at a specific site. It allows precise and specific mutations to be made and is done, for example, to modify the amino-acid sequence of the protein expressed by the gene to investigate how such a change affects the protein's structure and function. First, the gene of interest is cloned and made available as single-stranded DNA – a widely used vector for this purpose is the bacteriophage M13. Then an artificial oligonucleotide, containing perhaps 20–30 nucleotides, is constructed containing the desired change in base sequence. This is allowed to hybridize with the complementary (apart from the mutation site) single-stranded DNA and is then extended at either end by the enzyme DNA polymerase using the single-stranded DNA as template. The two strands, each with their vector, are then separated and cloned, and clones containing the mutant gene are selected.