Terminal Deoxynucleotidyl Transferase (rTdT), Recombinant
(Nucleosidetriphosphate: DNA deoxynucleotidylexotransferase, EC 2.7.7.31)
Source:
E. coli clone expressing bovine TdT
Description:
Terminal Deoxynucleotidyl Transferase (TdT) catalyzes the template-independent addition of unlabelled or radio-, fluorescent-, biotin-, or digoxygenin-labelled deoxynucleotides, dideoxynucleotides, or ribonucleotides to the 3'-hydroxyl termini of single or double-stranded DNA. TdT requires an oligodeoxynucleotide primer with at least three phosphate groups and a free 3'-hydroxyl end, and a divalent cation for activity. TdT also catalyzes the depolymerization of DNA (pyrophosphorolysis) and pyrophosphate exchange(1,2). Historically, TdT is used to label DNA probes (either non-isotopic or radiolabeled), in apoptosis detection and quantification in the terminal deoxynucleotidyl transferase dUTP nick end labeling method (TUNEL) (also referred to as ISEL, in situ end labeling), in DNA sequencing,(1,3) and to create ‘sticky’ ends by 3’ tailing DNA prior to annealing and transformation(4). For 5’ RACE, TdT is used to add homopolymer tails to the 3'-end of the first strand cDNA(5). Recently, TdT has been used to clone unknown sequences adjacent to known sequences in genomic DNA(6) and for TdT-dependent PCR(7,8) (TDPCR). TDPCR is used in RNA analysis(9,10,11), transcription analysis(12,13), DNA damage analysis(14), and DNA footprinting(7).
Properties:
Molecular Weight: 60 kDa
Purity:
Tested for contaminating exonucleases and endonucleases.
Storage Buffer:
50mM potassium phosphate, pH 6.4, 100mM NaCl, 1mM DTT, 0.1% Tween® 20 and 50% glycerol.
Assay Conditions:
The reaction mixture (50 μl) containing 25mM sodium cacodylate (pH 7.2), 0.5mM radiolabelled dATP (100 - 150 cpm/pmol), 10mM MgCl2, 0.1mM β-mercaptoethanol, 0.01mM
d(pA)35, and enzyme is incubated at 37°C for varying amounts of time. DNA is captured and incorporated counts determined.
Unit Definition:
One unit is the amount of enzyme required to polymerize 1 nmol of dATP residues into DNA in 60 minutes at 37°C under standard assay conditions.
Concentration:
30 units/μl
Tested User Friendly™ Functional Test:
Functionally tested by 3'-end labeling of a fluorescently labeled oligonucleotide.
Functionally Tested 5X TdT Reaction Buffer (1 ml included, PN 72035):
500mM sodium cacodylate, pH 6.8, 5mM cobalt chloride and 0.5mM DTT.
References:
- ANDERSON, R. S., BOLLUM, F. J., AND BEATTIE, K. L. (1999) Nucl. Acids Res. 27, 3190-3196.
- KRAYEVSKY, A. A., VICTOROVA, L. S., ARZUMANOV, A. A., AND JASKO, M. V. (2000) Pharm & Therapeutics 85, 165-173.
- TechTip 201, Use of Terminal Deoxynucleotidyl Transferase (TdT) to resolve BAFLs, USB Corporation.
- SAMBROOK, J. AND RUSSELL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 11.110.
- SAMBROOK, J. AND RUSSELL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 8.54 – 8.60.
- LIU, X. AND BAIRD, W. V. (2001) Plant Molec. Biol. Reporter 19, 261-267.
- KOMURA, J. AND RIGGS, A. D. (1998) Nucl. Acids Res. 26, 1807-1811.
- DAVIES, N. P. AND MURRAY, V. (2000) BioTechniques 29, 1168-1172.
- SCHMIDT, W. M. AND MUELLER, M. W. (1996) Nucl. Acids Res. 24, 1789-1791.
- ALBUQUERQUE-SILVA, J., HOUARD, S., AND BOLLEN, A. (1998) Nucl. Acids Res. 26, 3314-3316.
- CHEN, H., CASTANOTTO, D., LEBON, J. M., ROSSI, J. J., AND RIGGS, A. D. (2000) Nucl. Acids Res. 28, 1656-1664.
- BUETTNER, V. L., LEBON, J., GAO, C., RIGGS, A. D., AND SINGER-SAM, J. (2000) Nucl. Acids Res. 28, e25.
- TAGOH, H., HIMES, R., CLARKE, D., LEENEN, P., RIGGS, A. D., HUME, D., AND BONIFER, C. (2002) Genes & Development 16,1721-1737.
- ARLT, V. M., SCHMEISER, H. H., AND PFEIFER, G. P. (2001) Carcinogenesis 22, 133-140.
Applications:
- 5’ RACE.
- Creating isotopic, fluorescent, biotinylated, or digoxygenin labeled probes.
- Creating fluorescent 3’ labeled sequencing primers.
- Elimination of background bands in manual DNA sequencing.
- TDPCR.
- Detection of apoptotic DNA damage (TUNEL assay).
Storage:
Shipped on dry ice. Store at -20°C.
MSDS / Material Safety Data Sheets
Terminal Deoxynucleotidyl Transferase (rTdT), Recombinant_MSDS
Protocol, Brief
Terminal Deoxynucleotidyl Transferase (rTdT), Recombinant_Brief Protocol