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Terminal Deoxynucleotidyl Transferase

LO certified Recombinant enzyme Thermal inactivation at 70°C in 10 min

Terminal Deoxynucleotidyl Transferase (TdT) catalyzes the template-independent addition of deoxyribonucleotides to the 3'-OH terminus of DNA molecules.
  
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Thermo Scientific Terminal Deoxynucleotidyl Transferase (TdT), a template-independent DNA polymerase, catalyzes the repetitive addition of deoxyribonucleotides to the 3'-OH of oligodeoxyribonucleotides and single-stranded and double-stranded DNA (see Reference 1). TdT requires an oligonucleotide of at least three nucleotides to serve as a primer. With RNA as template TdT shows variable performance which strongly depends upon the tertiary structure of acceptor RNA 3'-end and the nature of nucleotide. Generally, it is lower than using DNA as a template.

Highlights

  • Incorporates modified nucleotides (e.g., fluorescein-, biotin-, aminoallyl-labeled nucleotides)

Applications

  • Production of synthetic homo- and heteropolymers (see​ Reference 1)
  • Homopolymeric tailing of linear duplex DNA with any type of 3'-OH terminus (see​ References 2, 3)
  • Oligodeoxyribonucleotide and DNA labeling (see​ References 2, 4-8)
  • 5'-RACE (Rapid Amplification of cDNA Ends) (see​ Reference 9)
  • In situ localization of apoptosis (see​ Reference 10)

Note

Due to the presence of CoCl2, the TdT Reaction Buffer is incompatible with downstream applications. It is necessary to remove CoCl2 from the reaction mixture by spin column or phenol/chloroform extraction and subsequent ethanol precipitation.

  
Storage Condition-20 C
HazardousNo
5X Reaction Buffer1 M potassium cacodylate, 125 mM Tris, 0.05% (v/v) Triton X-100, 5 mM CoCl2 (pH 7.2 at 25°C)
Concentration20 U/µL
Definition of Activity Unit
  • One unit of the enzyme catalyzes the incorporation of 1 nmol of deoxythymidylate into a polynucleotide fraction (adsorbed on DE-81) in 60 min at 37°C.
  • Enzyme activity is assayed in the following mixture: 200 mM potassium cacodylate (pH 7.2), 1 mM CoCl2, 0.01% (v/v) Triton X-100, 10 µM oligo(dT)10, 1 mM dTTP, and 0.4 MBq/mL [3H]-dTTP.
InactivationInactivated by heating at 70°C for 10 min or by addition of EDTA.
InhibitionInhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions
Quality ControlThe absence of endo-, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests.
SourceE.coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase.
Storage BufferThe enzyme is supplied in 100 mM potassium acetate (pH 6.8), 2 mM 2-mercaptoethanol, 0.01% (v/v) Triton X-100 and 50% (v/v) glycerol.

References

  1. F. J. Bollum, Terminal deoxynucleotidyl transferase, The Enzymes, the third edition (Boyer, P.D., ed.3, Academic Press, New York, vol.10, 1974) pp. 145-171.
  2. G. R. Deng, R. Wu, Terminal transferase: Use in the tailing of DNA and for in vitro mutagenesis. Meth. Enzymol. 100, 96-116 (1983).
  3. W. H. Eschenfeldt et al., Homopolymeric tailing. Meth. Enzymol. 152, 337-342 (1987).
  4. C. P. Tu, S. N. Cohen, 3’-end labeling of DNA with [alpha-32P]-cordycepin-5’-triphosphate. Gene. 10, 177-183 (1980).
  5. C. Vincent et al.,Synthesis of 8-(2,4-dinitrophenyl-2,6-aminohexyl) aminoadenosine-5’-triphosphate: Biological properties and potential uses. Nucleic Acids Res. 10, 6787-6796 (1982).
  6. A. Kumar et al., Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase. Anal. Biochem. 169, 376-382 (1988).
  7. W. Gaastra, P. Klemm, Radiolabeling of DNA with terminal transferase, Methods in Molecular Biology, vol.2: Nucleic Acids (Walker, J.M., ed., Humana, Clifton, NJ, 1984) pp. 269-271.
  8. G. L. Igloi, E. Schiefermayr, Enzymatic addition of fluorescein- or biotin-riboUTP to oligonucleotides results in primers suitable for DNA sequencing and PCR. BioTechniques. 15, 486-497 (1993).
  9. M. A. Frohman et al., Rapid Production of Full-Length cDNAs from Rare Transcripts: Amplification Using a Single Gene-Specific Oligonucleotide Primer. Proc. Natl. Acad. Sci. USA. 85, 8998-9002 (1988).
  10. W. Gorczyca et al., 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 (1993).