| 摘要 |
<p>The method for in vitro mutagenesis of a nucleic acid to produce single stranded DNA containing one or more base alterations includes the steps of: a) preparing an insoluble support carrying a nucleic acid to be mutagenised; b) adding a DNA oligonucleotide primer, which has the desired base alteration(s) but sufficient homology to hybridise with the appropriate site on the said first nucleic acid and allowing the primer to bind thereto; c) hybridising where necessary a further primer corresponding to the end of the nucleic acid at which synthesis is to start and adding a polymerase or reverse transcriptase, and nucleotides to form a DNA strand having the desired alteration(s) and including a ligase to ligate the said DNA strand to the primer; d) removing the first nucleic acid from the synthesised DNA strand; and e) removing said liquid therefrom. Advantageously the nucleic acid to be mutagenised is attached to the insoluble support by hybridisation to a nucleic acid probe attached to said support which probe is covalently attached to said insoluble support. Preferably, the insoluble support consists of magnetic particles which are monodisperse and/or superparamagnetic.</p> |
