Nick translation
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Nick translation is a tagging technique from molecular biology in which endonucleases are used to remove some of the nucleotides of a DNA sequence. Then, Klenow fragment of E. coli DNA polymerase I uses the gaps or nicks as a starting point for replication of DNA. During this elongation process, one or more radioactively or fluorescently labelled nucleotides are used as building blocks for the new DNA synthesized, thus creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization or blotting techniques.
Nick translation involves the use of restriction enzymes to remove certain nucleotides in a DNA sequence. After the removal, polymerase and ligase enzymes are used to repair the sequence with tagged nucleotides. This results in a hybrid DNA with a tagged sequence which can be used in various ways.
This process is called nick translation because the DNA to be so processed is treated with DNase to produce single-stranded "nicks." This is followed by replacement in nicked sites by DNA polymerase I. Nucleotides that are removed and replaced in this way are the ones referred to as "junk DNA", that is, DNA that does not code for any proteins or have any other function we can discern. Courtesy of Iscid.org
In Labeling of smaller nucleic acid fragments In this method, two activities of DNA Polymerase I, namely the 5'-3' exonuclease activity and the template dependent DNA polymerase activity are utilized. In order to start the labeling procedure the double stranded DNA is treated with the enzyme DNAse I. The resulting nicked double stranded DNA is subsequently treated with DNA polymerase I enzyme. This polymerase eliminates a few nucleotides from the nick by its 5'-3' exonuclease activity and then fills up the gap as a polymerase with dNTPs (deoxyribonucleotide triphosphates). 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.
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