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Nick translation is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization or blotting techniques.
Additional recommended knowledge
This process is called nick translation because the DNA to be so processed is treated with DNase to produce single-stranded "nicks." This is followed by replacement in nicked sites by DNA polymerase I, which elongates the 3' hydroxyl terminus, removing nucleotides by 5'-3' exonuclease activity, replacing them with radiolabeled dNTPs. When DNA polymerase I eventually detaches from the DNA, it leaves another nick in the phosphate backbone. The nick has "translated" some distance depending on the processivity of the polymerase. This nick could be sealed by DNA ligase, or its 3' hydroxyl group could serve as the template for further DNA polymerase I activity.
This process could cause a break in double-stranded DNA, if DNA polymerase I encounters another nick on the opposite strand. In this case, both phosphate backbones will be incomplete, and the DNA will essentially be cleaved into two shorter dsDNA fragments with blunt ends. This can, of course, be reversed by ligation.
To radioactively label a DNA fragment for use as a probe in blotting procedures, one of the incorporated nucleotides provided in the reaction is radiolabeled in the alpha phosphate position.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Nick_translation". A list of authors is available in Wikipedia.|