Terminal Deoxynulceotidyl Transferase (TdT)

• OEM by QIAGEN offers customized bulk manufacturing of this enzyme.
• Useful for 3' labeling incorporates dATP and dTTP with a fivefold higher efficieny than dCTP and dGTP

Terminal Deoxynulceotidyl Transferase (TdT) efficiently extends blunt, 5′-overhanging and single-stranded DNA, and RNA. Useful for 3′ labeling. this template-independent DNA polymerase catalyzes the addition of deoxynucleotides to the 3′ hydroxyl terminus of single- or double-stranded DNA molecules. The presence of 1 mM Co²⁺ stimulates the tailing of the 3ʹ ends of DNA fragments. This construct is sold as an N-terminal truncation of the terminal transferase gene attached to an N-terminal fusion tag.

The enzyme is supplied in 50 mM KPO₄, 100 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, <0.1% Triton X-100 and 50% glycerol; pH 7.3 at 25°C.

The enzyme is provided with 10x Green Buffer (cat. no. B0120) and 2.5 mM CoCl₂ (cat. no. B0220). 10x Green Buffer contains the following: 200 mM Tris-acetate, 500 mM potassium acetate and 100 mM magnesium acetate; pH 7.9 at 25°C.

Properties

• Storage temperature: –25°C to –15°C
• Molecular weight: 82,598 Daltons

Reference:

1. Deng, G.R., and Wu, R. (1983) Meth. Enzymol. 100:96.

Source of recombinant enzyme protein
The protein is produced by an E. coli strain that carries the cloned terminal deoxynucleotidyl transferase gene from calf thymus.

Unit definition:
One unit is defined as the amount of polymerase required to convert 1 nmol of dTTPs into acid insoluble material in 1 hour at 37°C.

Instructions for non-templated addition of dNTPs to 3ʹ termini of DNA
Reaction setup:

1. Incubate at 37°C for 30 minutes.
2. Inactivate the Terminal Deoxynulceotidyl Transferase (TdT) and stop the reaction by heating to 70°C for 10 minutes.

Notes
Co²⁺ increases the nucleotide incorporation efficiency of pyrimidines, and at blunt and 3ʹ recessed ends. However, the addition of dNTPs to 3ʹ-overhanging ends is more efficient than with 3ʹ -recessed or blunt ends. Terminal Deoxynulceotidyl Transferase (TdT) requires a free 3ʹ-hydroxyl group to make a non-templated nucleotide addition.

With limited efficiency, Terminal Deoxynulceotidyl Transferase (TdT) will incorporate ribonucleotides, biotinylated and dideoxynucleotides in the presence of Co²⁺.

Terminal Deoxynulceotidyl Transferase (TdT) incorporates dATP and dTTP with a fivefold higher efficiency than dCTP and dGTP, as evidenced by the following K_m values for nucleotides:

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1x reaction buffer and added to 50 µL reactions containing oligo dT 20mer DNA, 1x reaction buffer, 0.25 mM CoCl₂ 3H-dTTP and 100 µM dTTPs. Reactions were incubated 10 minutes at 37°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• 3ʹ labeling
• Extend blunt 5 ʹ overhangs