Radiolabeling of DNA by Nick Translation
- C. G. P. Mathew
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Nick translation is the name given to a reaction that is used to replace cold nucleoside triphosphates in a double-stranded DNA molecule with radioactive ones (1,2). Free 3′-hydroxyl groups are created within the unlabeled DNA (nicks) by deoxyribonuclease 1 (DNAse 1). DNA polymerase 1 from E. coli will then catalyze the addition of a nucleotide residue to the 3′-hydroxyl terminus of the nick. At the same time, the 5′- to 3′-exonuclease activity of this enzyme will eliminate the nucleotide unit from the 5′-phosphoryl terminus of the nick. Thus a new nucleotide with a free 3′-OH group will have been incorporated at the position where the original nucleotide was excised, and the nick will have been shifted along by one nucleotide unit in a 3′ direction. This 3′ shift, or translation, of the nick will result in the sequential addition of new nucleotides to the DNA while the pre-existing nucleotides will be removed. If radioactively labeled deoxyribonucleoside triphosphates are used as substrates, up to 50% of the residues in the DNA can be labeled.Furthermore, Rigby et al. have shown (2) that the DNA is labeled throughout at a uniform specific activity, which is an important requirement if the DNA is to be used as a probe in molecular hybridization experiments.
- Maniatis, T., Jeffrey, A., and Kleid, D. G. (1975) Nucleotide sequence of the rightward operator of the phage λ Proc. Natl. Acad. Sci. USA 72, 1184–1188. CrossRef
- Rigby, P. W. J., Dieckmann, M., Rhodes, C, and Berg, P. (1977) Labelling DNA to high specific activity in vitro by nick translation with DNA polymerase 1. J. Mol. Biol. 113, 237–251. CrossRef
- Mathew, C. G. P. (1983) Labelling DNA in vitro—Nick translation, in Techniques in Molecular Biology (Walker, J. M., and Gaastra, W., eds.) pp. 159–166. Croom Helm, London and Canberra.
- The Radiochemical Centre, Amersham (1980) Labelling of DNA with 32P bv nick translation. Technical Bulletin 80/3.
- Radiolabeling of DNA by Nick Translation
- Book Title
- Nucleic Acids
- pp 257-261
- Print ISBN
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- Series Title
- Methods in Molecular Biology
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- Humana Press
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- Humana Press
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