“Nick translation”的意思、由来-开放百科全书

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Nick translation

释义

References

{{Refimprove|date=September 2009}}Nick translation^[1] (or head translation), developed in 1977 by Rigby and Paul Berg, 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 (FISH) or blotting techniques. It can also be used for radiolabeling.^[2]

This process is called nick translation because the DNA to be processed is treated with DNAase 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 dNTPs. 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. Similarly, a fluorophore can be attached instead for fluorescent labelling, or an antigen for immunodetection. 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. Proprietary enzyme mixes are available commercially to perform all steps in the procedure in a single incubation.

Nick translation could cause double-stranded DNA breaks, if DNA polymerase I encounters another nick on the opposite strand, resulting in two shorter fragments. This does not influence the performance of the labelled probe in in-situ hybridization.

References

1. ^{{cite journal |vauthors=Rigby PW, Dieckmann M, Rhodes C, Berg P |title=Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I |journal=J. Mol. Biol. |volume=113 |issue=1 |pages=237–51 |date=June 1977 |pmid=881736 |doi= 10.1016/0022-2836(77)90052-3|url=}}
2. ^{{cite journal |author=Mathew CG |title=Radiolabeling of DNA by nick translation |journal=Methods Mol. Biol. |volume=2 |issue= |pages=257–61 |year=1985 |pmid=21374202 |doi=10.1385/0-89603-064-4:257 |url=}}

3 : Genetics|Molecular biology|Laboratory techniques

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