Nick translation (or head translation) developed in 1977 by Rigby and Paul Berg. This is the oldest method of nucleic acid labeling and is still the most commonly used. This technique is quite flexible with respect to probe size, specific activity and concentration. It is particularly suited for the production of large quantities of probes for use in multiple hybridization reactions or where a high probe concentration is required. The procedure for nick translation involved the preparation DNA to be labeled is treated with DNase I for a very short time. This induces nick in the DNA molecules, the positions of the nick being random. A ‘nick’ is the point in one strand of a double stranded DNA molecule where the phosphodiester bond has been broken. Thus each nick has a free 3’ OH group. The nicked sample is subjected to E.coli DNA Polymerase I which successively adds new nucleotides to the free 3’OH group. As the new segment of DNA is synthesized in this way, the existing homologous strand in the duplex is progressively displaced and digested away by the 5’→3’ exonuclease activity of the enzyme. In this reaction usually one of the deoxynucleoside triphosphates is radiolabelled (eg. with P32). But all four nucleotides could be labeled. The labeled nucleotides are progressively incorporated into the newly synthesized segment. Since this segment is synthesized by complementary base pairing, it has the same sequence as the strand it replaces.
The process is called ‘nick translation’ because there is a movement (translation) of the ‘nick’ along the DNA duplex due to the activities of E.coli DNA Polymerase I. The term ‘translation’ in its name does not refer to the synthesis of proteins. Nick translation can be used with a variety of labels to generate probes suitable for most hybridization applications. It has been used to generate probes having high enough specific activity to detect single copy genes on Southern Blots of mammalian DNA.
What is Nick translation and its effects
Author: Dr. Leena Kansal