Nick translation
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.
- OSTI ID:
- 6617939
- Journal Information:
- Methods Enzymol.; (United States), Vol. 152
- Country of Publication:
- United States
- Language:
- English
Similar Records
DNA-repair reactions by purified HeLa DNA polymerases and exonucleases
How Neurospora endo-exonuclease might act in recombinational double strand break DNA repair
Related Subjects
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