Deoxynucleotidyl terminal

The enzymatic monoaddition of blocked substrates to primers catalyzed by polynucleotide phosphorylase (217) or deoxynucleotidyl terminal transferase (218)-, and... 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Kumar, A., Tchen, P., Roullet, F., and Cohen, J. (1988) Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase. Anal. Biochem. 169, 376-382. 

Gorczyca, W., Gong, J., and Darzynkiewicz, Z., Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick... 

Many other enzymes can be used to remove or add groups to the ends of the DNA molecule. The three major ones are alkaline phosphatase, which removes a phosphate group from the 5 terminus polynucleotide kinase, which adds a phosphate group to a free 5 terminus and terminal deoxynucleotidyl transferase, which adds one or more deoxynucleotides to the 3 terminus. 

Terminal deoxynucleotidyl transferase normally adds homopolydeoxynucleotide tails to single-stranded DNA primers in the presence of a deoxynucleoside triphosphate and magnesium. If cobalt is used instead, not only does double-stranded DNA become an acceptable substrate, but ribonucleotides or homopolymer deoxyribo-nucleotide tracts may be added to all forms of duplex DNA at their 3 -ends, regardless of whether these are staggered or even.162 This allows terminal labelling for sequence analysis at the cleavage sites of restriction endonucleases,162- 183 or tail formation for in vitro studies on recombinant DNA.162... 

Apoptosis Nuclear condensation and lobulation, caspase activation, phosphatidyl serine externalization, annexin, immuno-cytochemistry, DNA fragmentation and labeled-dUTP incorporation by terminal deoxynucleotidyl transferase... 

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling... 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

There is an alternative method of generating cohesive tails. The enzyme calf thymus terminal (deoxynucleotidyl) transferase adds deoxynucleoside monophosphate residues from 5 -deoxynucleoside triphosphates to protruding 3 -hydroxyl termini in the absence of a template. For example, as shown in equations 14.5 to 14.7,... 

Terminal deoxynucleotidyl transferase (TdT) buffer, 5X concentration 500 mM sodium cacodylate, 10 mM cobalt chloride, 1 mM dithiothreitol Add water to the sodium cacodylate, cool to 0°C on ice, add dithiothreitol, and then cobalt chloride dropwise, stirring continuously. Store in aliquots at —20°C... 

Talbot, David, 190 Tandem dyes, 71-72, 255 Tape back-up, 43 Temporal resolution, 17 Terminal deoxynucleotidyl transferase (TdT), 153... 

TUNEL The TUNEL assay represents a historical low point in attempts to coin acronyms. The Terminal deoxynucleotidyl transferase mediated dC/TP Nick End-Labeling assay labels the ends of DNA with fluorescent UTP. Apoptotic cells often have fragmented DNA these fragments will provide more substrate for the enzyme terminal deoxynucleotidyl transferase. Apoptotic cells can therefore be enumerated by flow cytometry according to their increased intensity in the TUNEL assay. 

Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay examining apoptosis in smooth muscle cells and HL60 leukemia cells treated with paclitaxel. Abbreviations HL60, human leukemia 60. PTX, paclitaxel SMC, smooth muscle cells Tunel, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. Source From Ref. 49. 

TUNEL stands for terminal deoxynucleotidyl transferase x-dUTP nick end labeling. This assay is based on the detection of DNA fragments marked by an enzyme that incorporates modified nucleotides to the 3 -OH ends of the fragments, which can be then specifically detected. The enzyme is a deoxynucleotidyl transferase, which can act in absence of a complementary strand. Among the nucleotides, there is one specifically marked with a fluorochrome, an enzyme, or an antigen. This allows different methods of detection. 

Biochemically, apoptosis is characterized by the internucleosomal degradation of chromosomal DNA to form a series of double-stranded fragments that are multiples of 180 200 base pairs in length. These fragments give a characteristic DNA ladder pattern on gel electrophoresis [91, 92] and can be detected by several cytochemical methods, the most extensively used being the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) [93-95], The detection of ladder pattern and TUNEL positivity has been adopted as a marker of apoptosis. 

Terminal transferase Terminal deoxynucleotidyl transferase. Enzyme... 

Figure 29.8. Kidney tissue from a rat treated with para-aminophenol (300mg/kgip). Tissue was stained with hematoxylin and eosin and was subjected to terminal deoxynucleotidyl transferase-diaminobenzidine (TUNEL) staining to reveal DNA strand breaks. Darker gray areas represent tubules that stain positive for DNA strand breaks. Final magnification was lOOx (left panel) and 360x (right panel).

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