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Title: Nick translation

Abstract

Nick translation, or more precisely nick translocation, is a specific procedure for incorporating radioactive nucleotides into double-stranded DNA. The method takes advantage of the ability of Escherichia coli DNA polymerase I to combine the sequential addition of nucleotide residues to the 3'-hydroxyl terminus of a nick with the elimination of nucleotides from the adjacent 5'-phosphoryl terminus. Linear, supercoiled, nicked, or gapped circular double-stranded molecules can be labeled to specific activities > 10/sup 8/ cpm/..mu..g with deoxynucleotide 5'-(..cap alpha..-/sup 32/P)triphosphates by this technique. Since the nicks are introduced at random sites in the duplex, the method generates a population of radioactive fragments which partially overlap each other. At saturating levels of nucleotide triphosphates the size of the fragments is determined by the DNase concentration. While experiments consistent with hyperpolymer formation of nick-translated probes have been reported, the reproducibility and extent of hyperpolymer formation seem to be difficult to obtain, probably because of the critical dependence on probe size.

Authors:
;
Publication Date:
OSTI Identifier:
6617939
Resource Type:
Journal Article
Journal Name:
Methods Enzymol.; (United States)
Additional Journal Information:
Journal Volume: 152
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; DNA; LABELLING; DNA POLYMERASES; BIOCHEMISTRY; DNA SEQUENCING; DNA-ASE; ESCHERICHIA COLI; GENE RECOMBINATION; PHOSPHORUS 32; RECOMBINANT DNA; BACTERIA; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CHEMISTRY; DAYS LIVING RADIOISOTOPES; ENZYMES; ESTERASES; HYDROLASES; ISOTOPES; LIGHT NUCLEI; MICROORGANISMS; NUCLEI; NUCLEIC ACIDS; NUCLEOTIDYLTRANSFERASES; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; PHOSPHODIESTERASES; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; POLYMERASES; RADIOISOTOPES; STRUCTURAL CHEMICAL ANALYSIS; TRANSFERASES; 550601* - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats

Meinkoth, J, and Wahl, G M. Nick translation. United States: N. p., 1987. Web. doi:10.1016/0076-6879(87)52012-2.
Meinkoth, J, & Wahl, G M. Nick translation. United States. https://doi.org/10.1016/0076-6879(87)52012-2
Meinkoth, J, and Wahl, G M. 1987. "Nick translation". United States. https://doi.org/10.1016/0076-6879(87)52012-2.
@article{osti_6617939,
title = {Nick translation},
author = {Meinkoth, J and Wahl, G M},
abstractNote = {Nick translation, or more precisely nick translocation, is a specific procedure for incorporating radioactive nucleotides into double-stranded DNA. The method takes advantage of the ability of Escherichia coli DNA polymerase I to combine the sequential addition of nucleotide residues to the 3'-hydroxyl terminus of a nick with the elimination of nucleotides from the adjacent 5'-phosphoryl terminus. Linear, supercoiled, nicked, or gapped circular double-stranded molecules can be labeled to specific activities > 10/sup 8/ cpm/..mu..g with deoxynucleotide 5'-(..cap alpha..-/sup 32/P)triphosphates by this technique. Since the nicks are introduced at random sites in the duplex, the method generates a population of radioactive fragments which partially overlap each other. At saturating levels of nucleotide triphosphates the size of the fragments is determined by the DNase concentration. While experiments consistent with hyperpolymer formation of nick-translated probes have been reported, the reproducibility and extent of hyperpolymer formation seem to be difficult to obtain, probably because of the critical dependence on probe size.},
doi = {10.1016/0076-6879(87)52012-2},
url = {https://www.osti.gov/biblio/6617939}, journal = {Methods Enzymol.; (United States)},
number = ,
volume = 152,
place = {United States},
year = {Thu Jan 01 00:00:00 EST 1987},
month = {Thu Jan 01 00:00:00 EST 1987}
}