末端转移酶/末端脱氧核苷酸转移酶/TDT

Description
Analysis Note Activity assay: 200 mM potassium cacodylate, pH 7.2, 4 mM MgCl2, 1 mM 2-mercaptoethanol, 1 mM 3H-dATP, 70 μM d（pT）6, 37 °C.
Biochem/physiol Actions Bovine terminal transferase （TdT） is a primer-dependent polymerase that catalyzes the addition of deoxynucleotides to the 3′-OH terminus of DNA molecules with the release of inorganic phosphate. TdT reacts preferentially with either single-stranded DNA molecules or double-stranded-DNA with 3′ overhangs, but procedures have been developed to label blunt ends or 3′-recessive ends. In a reaction mixture, the divalent ion （Co2+, Mn2+, Mg2+） will influence purine and pyrimidine polymerization rate. Activities of TdT are also affected by the bases （dATP, dCTP, dGTP and dTTP） present. Each reaction mixture needs to be optimized. The enzyme is commonly used to add complementary homopolymer tails to vector and insert DNA for construction of recombinant DNA in vitro.
Features and Benefits • Add homopolymers to vectors, inserts and cDNA for cloning
• Label the 3′-end of double- and single-stranded DNA with non-radioactive and radioactive labels
• Carry out in vitro mutagenesis by adding single nucleotides to DNA
• Use in TUNEL assays with deoxyuridine triphosphate （dUTP） conjugated with a fluorescent chromophore or biotin to label nicked DNA in apoptotic cells
 
Unit Definition One unit will incorporate 1 nanomole of dATP into acid-precipitable material in one hour at 37 °C using d（pT）6 as primer.
Physical form Solution in 50 mM potassium phosphate, pH 7.4, 1 mM 2-mercaptoethanol and 50% glycerol （v/v）

Properties
form buffered aqueous glycerol solution
mol wt mol wt 60 kDa
concentration ≥5,000 units/mL
foreign activity Exonuclease and endonuclease, free
shipped in dry ice
storage temp. −20°C

Safety
Personal Protective Equipment Eyeshields, Gloves, half-mask respirator （EU）, half-mask respirator （US）, multi-purpose combination respirator cartridge （US）
Safety Statements 22-24/25
WGK Germany 3

References
reference Koiwai, O., et al., Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells. Nucleic Acids Res. 14, 5777, （1986） Abstract
  Kato, K., et al., Deoxynucleotide-polymerizing enzymes of calf thymus glandIIProperties of the terminal deoxynucleotidyltransferase. J. Biol. Chem. 242, 2780, （1967） Abstract
  Tu, C.P. and Cohen, S.N., 3′-end labeling of DNA with [α-32P]cordycepin-5′-triphosphate. Gene 10, 177-183, （1980） Abstract
  , Maniatis, T., et al. Molecular Cloning: A Laboratory Manual Plainview, NY , （1989）, 148
  Deng, G. and Wu, R, Terminal transferase: use in the tailing of DNA and for in vitro mutagenesis. Meth. Enzymol. 100, 96-116, （1983） Abstract
  Gavrieli, Y., et al., Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493-501, （1992） Abstract

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