The Site-directed mutagenesis reference article from the English Wikipedia on 24-Jul-2004
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Site-directed mutagenesis

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(in vitro mutagenesis)

Site-directed mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, site-directed mutagenesis requires that the wild-type gene sequence be known. An oligonucleotide with its sequence containing a desired mutation is chemically synthesized. The oligonucleotide is attached by base pair hydrogen bonding to the complementary wild-type gene sequence. The synthetic oligonucleotide is used as a primer for the in vitro synthesis of a new DNA strand that is complementary to the original (template) strand. The DNA synthesis is performed by adding the enzyme DNA polymerase to the DNA template. The newly synthesized strand of DNA has the primer and the desired mutation incorporated into it. By using a pair of primers and the polymerase chain reaction it is possible to amplify the newly created DNA molecule and produce enough copies to make further manipulation of the new DNA possible. Typically, the mutated DNA is then inserted into an expression vector by means of restriction enzymes and DNA ligase. The expression vector is then typically inserted into a cell where it can be used as a genetic template for the synthesis of a mutated protein. The biological activity of the mutated protein can then be compared to that of the wild-type protein.