Abstract
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.
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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.
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The Radiochemical Centre, Amersham (1980) Labelling of DNA with 32P bv nick translation. Technical Bulletin 80/3.
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© 1984 The Humana Press Inc.
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Mathew, C.G.P. (1984). Radiolabeling of DNA by Nick Translation. In: Walker, J.M. (eds) Nucleic Acids. Methods in Molecular Biology, vol 2. Humana Press. https://doi.org/10.1385/0-89603-064-4:257
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DOI: https://doi.org/10.1385/0-89603-064-4:257
Publisher Name: Humana Press
Print ISBN: 978-0-89603-064-0
Online ISBN: 978-1-59259-489-4
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