Nick translation
Encyclopedia
Nick translation was developed in 1977 by Rigby and Paul Berg. It is a tagging
technique in molecular biology
in which DNA Polymerase I
is used to replace some of the nucleotides of a DNA
sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization
or blotting
techniques.
This process is called nick translation because the DNA to be processed is treated with DNase to produce single-stranded "nicks." This is followed by replacement in nicked sites by DNA polymerase I
, which elongates the 3' hydroxyl terminus, removing nucleotides by 5'-3' exonuclease activity, replacing them with dNTPs. 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. 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
of the polymerase. This nick could be sealed by DNA ligase
, 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.
Fluorescent tag
In molecular biology and biotechnology, a fluorescent tag is a part of a molecule that researchers have attached chemically to aid in detection of the molecule to which it has been attached. The tag is some kind of fluorescent molecule...
technique in molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
in which DNA Polymerase I
DNA polymerase I
DNA Polymerase I is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg in 1956, it was the first known...
is used to replace some of the nucleotides of a DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
or blotting
Blot (biology)
In molecular biology and genetics, a blot is a method of transferring proteins, DNA or RNA, onto a carrier . In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane...
techniques.
This process is called nick translation because the DNA to be processed is treated with DNase to produce single-stranded "nicks." This is followed by replacement in nicked sites by DNA polymerase I
DNA polymerase I
DNA Polymerase I is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg in 1956, it was the first known...
, which elongates the 3' hydroxyl terminus, removing nucleotides by 5'-3' exonuclease activity, replacing them with dNTPs. 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. 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
Processivity
In molecular biology, processivity is a measure of the average number of nucleotides added by a DNA polymerase enzyme per association/disassociation with the template. DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have...
of the polymerase. This nick could be sealed by DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
, 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.