Radiolabeling of DNA by Nick Translation

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.