Terminal deoxynucleotide transferase

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled amounts of modified nucleotides to an existing stand. However, the most common procedures utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is used with a template to add modified nucleoside triphosphates to the end of a DNA molecule or to various sites within the middle of a sequence. The terminal transferase can add modified monomers to the 3 end of a chain without a template. 

In the assay outlined earlier, apoptotic cells are detected because of lowered signal intensity resulting from DNA degradation. The finding that apoptosis involves a specific form of DNA degradation allows one to detect these unique events with a specific labeling procedure. With the aid of terminal deoxynucleotide transferase, fluorescently labeled dUTP residues are... 

Tunel (Terminal deoxynucleotide transferase dUTP nick end labeUng) assay In a modified version of the Tunel assay, apoptotic ceUs can be measured in a two-color system using flow cytometry and microscopy. In this assay, DNA breaks are labeled by deoxynucleotidyl transferase using bromodeoxyuridine triphosphate (BrdUTP) and visualized by anti-BrdU antibody. Propidium iodide is used to counterstain the total ceUular DNA. 

Terminal deoxynucleotide transferase dUTP nick and labeling 644 Testosterone 58, 1589... 

Enzo BioArray Terminal Labeling Kit (Enzo Biochem, Inc. New York, NY) 5x reaction buffer, lOx C0CI2, biotin-ddUTP, and terminal deoxynucleotide transferase. 

To label fragmented cDNA, add 20 pL 5x reaction buffer, 10 pL lOx CoClj, and 1 pL biotin-ddUTP, and 2 pL terminal deoxynucleotide transferase to 39 pL... 

From Pharmacia MuMLV reverse transscriptase (FPLC pure), T4-polynucleotide kinase (FPLC pure), terminal deoxynucleotide transferase (TdT, FPLC pure). From Boehringer Mannheim E. coli DNA polymerase (endonuclease free), T4 DNA ligase (5 U/yl), T4 DNA polymerase. 

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. 

Another common method for measuring apoptosis is the DNA labeling technique, terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). In this method, labeling of DNA strand breaks at the 3 end is achieved by using the enzyme, terminal deoxynucleotidyl transferase (TdT) and either a radioactive or a fluorescently-Iabeled deoxynucleotide. This method provides a very sensitive measurement of DNA strands breaks. However, as DNA strand... 

Terminal transferase is an enzyme that catalyzes the addition of deoxynucleotide triphosphates to the 3 hydroxyl end of double- or single-stranded DNA it can therefore be used to add tails of labeled... 

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