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Terminal Deoxynucleotidyl Transferase

#EP0161 Supplied with: 5X Reaction Buffer	500 u (20 u/µl) 0.4 ml
#EP0162 Supplied with: 5X Reaction Buffer	2500 u (20 u/µl) 2x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0161, #EP0162 MSDS (English) MSDS (English-USA) MSDS (German)

 

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

Description
The Terminal Deoxynucleotidyl Transferase (TdT), a template-independent DNA polymerase, catalyzes the repetitive addition of deoxyribonucleotides to the 3’-OH of oligodeoxyribonucleotide and single-stranded, and double-stranded DNA (1). The TdT requires an oligonucleotide of at least three nucleotides to serve as a primer.

Source
E.coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase.

Applications

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

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases, phosphatases and ribonucleases confirmed by appropriate quality tests.

Concentration
20 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.

Storage Buffer
The 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.

5X Reaction Buffer
1 M potassium cacodylate, 0.125m Tris, 0.05% (v/v) Triton X-100 and 5 mM CoCl₂ (pH 7.2 at 25°C).

Inhibition and Inactivation

• Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions.
• Inactivated by heating at 70°C for 10 min or by the addition of EDTA.

Related Products

dNTP Set M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP 0.5 M EDTA, pH 8.0 Water, nuclease-free

References

1. Bollum, F.J., Terminal deoxynucleotidyl transferase, The Enzymes, the third edition (Boyer, P.D., ed.), Academic Press, New York, vol.10, 145-171, 1974.

2. Deng, G.R., Wu, R., Terminal transferase: Use in the tailing of DNA and for in vitro mutagenesis, Meth. Enzymol., 100, 96-116, 1983.
3. Eschenfeldt, W.H., et al., Homopolymeric tailing, Meth. Enzymol., 152, 337-342, 1987.
4. Tu, C.-P.D., Cohen, S.N., 3’-end labeling of DNA with [alfa-³²P]cordycepin-5’-triphosphate, Gene, 10, 177-183, 1980.
5. Vincent, C., 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. Kumar, A., et al., Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase, Anal. Biochem., 169, 376-382, 1988.
7. Gaastra, W., Klemm, P., Radiolabeling of DNA with terminal transferase, Methods in Molecular Biology, vol.2: Nucleic Acids (Walker, J.M., ed.), Humana, Clifton, NJ, 269-271, 1984.
8. Igloi, G.L., Schiefermayr, E., Enzymatic addition of fluorescein- or biotin-riboUTP to oligonucleotides results in primers suitable for DNA sequencing and PCR, BioTechniques, 15, 486-497, 1993.
9. Frohman, M.A., 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. Gorczyca, W., 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.

Updated vasario 27, 2008 13:21