Nick translation (or head translation), developed in 1977 by Peter Rigby and Paul Berg, is a tagging technique in

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

in which

DNA polymerase I DNA polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and the first known of any kind of polymerase). It was init ...

is used to replace some of the nucleotides of a

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in

fluorescent in situ hybridization Fluorescence ''in situ'' hybridization (FISH) is a cytogenetics, molecular cytogenetic technique that uses hybridization probe, fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence Com ...

(FISH) or

blotting In molecular biology and genetics, a blot is a method of transferring large biomolecules (proteins, DNA or RNA) onto a carrier, such as a membrane composed of nitrocellulose, polyvinylidene fluoride or nylon. In many instances, this is done after ...

techniques. It can also be used for

radiolabeling A radioactive tracer, radiotracer, or radioactive label is a synthetic derivative of a natural compound in which one or more atoms have been replaced by a radionuclide (a radioactive atom). By virtue of its radioactive decay, it can be used to exp ...

. This process is called "nick translation" because the DNA to be processed is treated with DNAase to produce single-stranded "nicks", where one of the strands is missing nucleotides. This is followed by replacement in nicked sites by

, which removes nucleotides from the 3' (downstream) end of a nick with its 3'-5' endonuclease activity and adds new, labeled dNTPs from the medium to the 5' end of the nick, moving the nick downstream in the process. 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, often using

phosphorus-32 Phosphorus-32 (32P) is a radioactive isotope of phosphorus. The nucleus of phosphorus-32 contains 15 protons and 17 neutrons, one more neutron than the most common isotope of phosphorus, phosphorus-31. Phosphorus-32 only exists in small quantiti ...

. 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 In molecular biology and biochemistry, processivity is an enzyme's ability to catalyze "consecutive reactions without releasing its substrate". For example, processivity is the average number of nucleotides added by a polymerase enzyme, such as ...

of the polymerase. This nick could be sealed by

DNA ligase DNA ligase is a type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such ...

, 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

Biochemistry detection methods Genetics techniques Laboratory techniques Molecular biology techniques {{Genetics-stub