Nick translation

Current analytical methods have difficulty detecting picogram levels of nucleic acids, particularly when high levels of other biopolymers (e.g., proteins) are present. The most widely used assay method employed by the pharmaceutical industry involves a nick translation DNA hybridization method (1). This assay offers high sensitivity and selectivity but has a number of drawbacks. 

DNA polymerase 1 j Synthesizes double-stranded DNA from j single-stranded DNA. Synthesis of double-stranded cDNA nick translation generation of blunt ends from sticky ends. 

DNase 1 j Under appropriate conditions, produces j single-stranded nicks in DNA. Nick translation mapping of hypersensitive sites mapping protein-DNA interactions. 

Nick translation A technique for labeling DNA based on the ability of the DNA polymerase from E colt to degrade a strand of DNA that has been nicked and then to resynthesize the strand if a radioactive nucleoside triphosphate is employed, the rebuilt strand becomes labeled and can be used as a radioactive probe. 

Purify the labeled probe by ethanol precipitation according to steps 4-7 of the protocol previously described for nick translation. 

Isolate the labeled probe by alcohol precipitation as described previously for nick translation. 

The modified DNA may be recovered by alcohol precipitation according to the method in Section 1 (this chapter) described previously for nick-translation modification. Alternatively, dialysis or gel filtration may be done to remove excess reactants. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 (this chapter) for nick-translation modification of DNA probes. Alternatively, dialysis, gel filtration, or w-butanol extraction may be used to remove excess reagents. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 for nick translation (this chapter). 

Rigby, P.W.J., Dieckmann, M., Rhodes, C., and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I./. Mol. Biol. 113, 237-251. 

DNA polymerase 1. For most applications DNA polymerase 1 from E. coli is used. The enzyme attaches to a short single-stranded region in a dsDNA molecule and then synthesizes a new strand of DNA, degrading the existing strand as it proceeds. When used in vitro this incubation is carried out at 12-15 °C to prevent more than one round of replication occurring. It is used for in vitro labelling of DNA by the nick translation method (described below). 

Nick translation uses the enzyme DNA polymerase 1. Most pieces of DNA contain some nicked areas. DNA polymerase 1 can attach here and... 

Gorczyca, W., Gong, J., and Darzynkiewicz, Z. (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 53, 1945-1951. 

Hapten-labeled DNA probes for FISH assays can be used for BISH assays and those probes can be purchased from various vendors. If commercial hapten-labeled DNA probes are not available, DNA probes can be labeled with haptens by nick translation or by PCR in a laboratory. Nick translation kit can be utilized for labeling DNAprobes with hapten (10976776001, Roche Applied Science, Germany). New probes must be analyzed for the specificity by FISH or BISH assays using a comparative genomic hybridization metaphase control slide. 

A sample of total DNA derived from the cells in which the product is produced is then radiolabelled with using the process of nick translation. It is heated to 90°C (promotes... 

Clemens DL, Johnson PJ (2000) Failure to detect DNA in hydrogenosomes of Trichomonas vaginalis by nick translation and immunomicroscopy. Mol Biochem Parasitol 106 307-313... 

No evidence has been found for the mitosomal genome in any organism so far. An earlier study on E. histolytica reported the presence of DNA in Entamoeba mitosomes (Ghosh et al. 2000) and synonymized these organelles with DNA-containing structures named kinetoplast-like organelles (EhKO) (Orozco et al. 1997). However, work by Leon-Avila and Tovar (2004) later showed that EhKO and mitosomes are not related structures. Moreover, in situ nick translation coupled with immunofluorescence microscopy failed to detect the presence of DNA in Entamoeba mitosomes (Leon-Avila and Tovar... 

Snyder, R.D. Matheson, D.W. (1985) Nick translation—a new assay for monitoring DNA damage and repair in cultured human fibroblasts. Environ. Mutag., 1, 267-279... 

Neurotransmitter(s) 29, 553 receptors for 479 Neutron diffraction 137 Neutrophils 26,188 Nick translation 257 Nickel 877-882... 

Most of these compounds displayed very potent cytotoxic activity against several tumor cells. Dercitin (435) was also active in vivo, prolonging the life of P388-bearing mice with a T/C of 170 at 5 mg/Kg. Compound 435 inhibited both DNA and RNA synthesis, DNA polymerase and I/DNase nick translation, bound to calf thymus DNA, and relaxed supercoiled <()X174DNA [348]. The compounds also showed... 

The nick translation method can be used in conjunction with cell-cycle analysis to determine which phases of the cell-cycle the apoptotic cells are in. To do this, add 5 pg/mL propidium iodide to the samples 30 min prior to analysis. 

Gorczyca, W, Melamed, M. R., and Darzyrikiewicz, Z. (1993) Apoptosis of S-phase HL-60 cells induced by topoisomerase inhibitors detection of DNA strand breaks by flow cytometry using the in situ nick translation assay Toxicol Lett 67, 249-258. 

Gold, R, Schmied, M., Rothe, G, Zischler, H., Breitschopf, H, Wekerle, H, and Lassmann, H, (1993) Detection of DNA fragmentation in apoptosis- application of in situ nick translation to cell culture systems and tissue sections J Histochem Cytochem 41, 1023-1030... 

In the original system, biotin is attached to the deoxy analog of rUTP via a spacer arm. Biotinylated dUTP is incorporated into DNA strands by a conventional labeling reaction, nick translation, which was also widely used to pre-... 

In addition to biotin, a digoxigenylated derivative of dUTP was also synthesized. This derivative of dUTP can be incorporated into DNA by Pol I (or the Klenow fragment of Pol I). Therefore, digoxigenin-labeled DNA probes can be prepared by nick translation or random primed-labeling methods developed for the biotin system. It is almost certain that more nonradioactive alternatives to biotin and digoxigenin will be developed in the future. Chemiluminescent methods for nonradioactive probe detection are now widely being used... 

---