Terminal transferase

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

Terminal transferase j Adds nucleotides to the 3 ends of DNA. Homopolymer tailing. 

Terminal transferase An enzyme that adds nucleotides of one type (eg, deoxyadenonucleotidyl residues) to the 3 end of DNA strands. 

Oligonucleotide probes were labeled at their 3 end using terminal transferase and digoxigenin-11 -UTP, according to the manufacturer s recommendations (Roche Pharmaceuticals). Ten ml of cells are pre-fixed in 3.7% formaldehyde for 15 min at room temperature. Five ml of cells are harvested and resuspended in 6 ml of 4% paraformaldehyde, 0.1 MKPO4 (pH 6.5), and 5 mM MgCb. After 3 h, the cells are washed twice in solution B (1.2 M sorbitol, 0.1 M KPO4 (pH 6.5)). The cells are then resuspended in 2.8 ml... 

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. 

Terminal transferase labeling was originally developed using radiolabeled (typically 32P) nucleoside triphosphates (Roychoudhury et al., 1979 Tu and Cohen, 1980). Later, the technique was extended to the use of nonradioactive nucleotide derivatives (Kumar et al., 1988). 

Of the purine nucleosides, dATP may be derivatized at its N-6 position using a long linker arm terminating in a detectable group without losing the ability to be enzymatically incorporated into DNA probes. By contrast, if modification is done at the C-8 position of purine bases, DNA polymerase cannot by used to add the labeled monomer to an existing strand. C-8 derivatives, however, can be added at the 3 terminal using terminal transferase enzyme. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Add terminal transferase to a final concentration of 50 units in the reaction mixture. 

Roychoudhury, R., Tu, C.-P.D., and Wu, R. (1979) Influence of nucleotide sequence adjacent to duplex DNA termini on 3 -terminal labeling by terminal transferase. Nucleic Acids Res. 6, 1323-1333. 

TUNEL Assay (Terminal Transferase dUTP Nick End Labelling)... 

TUNEL terminal transferase dUTP nick end labelling... 

A second tool employed by the genetic engineer is the enzyme terminal transferase that adds deoxyribonuclease residues to the 3 end of DNA strands creating 3 tails of a single type of residue. 

Other DNA polymerases, which are high fidelity enzymes that do not have terminal transferase activity, also can be used. 

Fig. 28. Synthesis of labeled DNA probes. A Labeled DNA can be generated using different enzymes (Klenow fragment of DNA polymerase or a terminal transferase) to incorporate labeled nucleotides into specific DNA sequences. Probes can be labeled using radioactive nucleotides or nucleotides labeled with an immunogenic molecule such as biotin. B The labeled probe is then hybridized to the target nucleic acid, which is either bound to a membrane or in a tissue section or cell. An antibody is then used to detect the non-radioactively-labeled probe. C The antibody may be conjugated to a fluorescent or chemiluminescent dye, or an enzyme that produces a color reaction. The target nucleic acid is thus visualized.

Schmitz, G. G. Walter, T. Seibl, R. Kessler, C. Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 1991,192(1), 222-231. 

DNA polymerase I (E. coli) Reverse transcriptase Polynucleotide kinase Terminal transferase Exonuclease III... 

Figure 26-8 Regeneration of a Pst I site by dG dC tailing with terminal transferase. See Glover." Arrowheads indicate 5 —>3 directions.

Joining DNA by complementary homopolymeric tails Terminal transferase... 

A typical processive enzyme is terminal transferase. It adds on deoxynucleo-side monophosphates randomly to exposed 3 -hydroxyl termini so that the final products are formed in a statistical distribution. The distribution follows Poisson s law.5 Suppose that the enzyme adds on an average of x residues per chain. Then the probability of a particular chain having k residues added [i.e., p(k)] is given by... 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

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