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TrueStart Hot Start Taq DNA Polymerase


DNA polymerase designed for hot start PCR featuring improved specificity and sensitivity.
  
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Thermo Scientific TrueStart Hot Start Taq DNA Polymerase is designed for hot start PCR, a technique shown to improve specificity, sensitivity and yield of DNA amplification during PCR (see References 1,2,3,4,5). TrueStart Hot Start Taq DNA Polymerase is chemically modified by adding proprietary heat-labile blocking groups to amino acid residues. TrueStart Hot Start Taq DNA Polymerase is inactive at room temperature because of this modification, avoiding extension of non-specifically annealed primers or primer dimers.

TrueStart Hot Start Taq DNA Polymerase activates rapidly during the initial denaturation step of PCR. The lack of additional activation step differentiates it from other hot start DNA polymerases, making this enzyme an ideal tool for both automatic hot start PCR and fast PCR. Activated TrueStart Hot Start Taq DNA Polymerase catalyzes 5'→3' synthesis of DNA, lacks detectable 3'→5' exonuclease (proofreading) activity, but possesses low 5'→3' exonuclease activity. These two activities are not detectable before activation.

Highlights

  • Fastest activating hot start Taq DNA polymerase
  • High PCR specificity – reduced effects of mis-priming and primer-dimer formation
  • Enhanced PCR sensitivity
  • Convenient room temperature PCR set-up
  • Generates PCR products with 3’-dA overhangs

Applications

  • High throughput hot start PCR
  • Highly specific amplification of genomic and cDNA targets up to 3 kb
  • Amplification of low copy DNA targets
  • Multiplex PCR

Includes

  • TrueStart Hot Start Taq DNA Polymerase (5 U/µL)
  • 10X TrueStart Taq Buffer
  • 25 mM MgCl2
  
Storage Condition-20 C
HazardousNo
10X TrueStart Taq Buffer200 mM Tris-HCl (pH 8.3 at 25°C), 200 mM KCl, 50 mM (NH4)2SO4.
Definition of Activity Unit
  • One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 74°C.
  • Enzyme activity is assayed in the following mixture: 25 mM TAPS (pH 9.3 at 25°C), 50 mM KCl, 2 mM MgCl2, 0.2 mM of each dATP, dGTP, dTTP, 0.1 mM dCTP, 0.75 mM activated salmon milt DNA, and 0.4 MBq/mL [3H]-dCTP.
InactivationInactivated by phenol/chloroform extraction.
InhibitionInhibitors: ionic detergents (deoxycholate, sarkosyl and SDS) at concentrations higher than 0.06, 0.02 and 0.01%, respectively (6).
Molecular Weight94 kDa monomer
Quality Control
  • The absence of endo-, exodeoxyribonucleases, and ribonucleases confirmed by appropriate quality tests.
  • Functionally tested in PCR.
Storage BufferThe enzyme is supplied in 20 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.5% (v/v) Tween 20, 0.5% (v/v) Nonidet P40, and 50% (v/v) glycerol.
Comparison of PCR specificity with TrueStart Hot Start <em>Taq</em> DNA Polymerase or other commercial Hot Start PCR systems on a complex target.</strong><br />

Comparison of PCR specificity with TrueStart Hot Start Taq DNA Polymerase or other commercial Hot Start PCR systems on a complex target.

Comparison of PCR specificity with TrueStart Hot Start <em>Taq</em> DNA Polymerase or other commercial Hot Start PCR systems on a complex target.</strong><br />

A 2 kb DNA fragment of the human beta-globine gene was amplified according to the manufacturer’s recommendations.

M – GeneRuler100 bp Plus DNA Ladder
1 to 2 – TrueStart Hot Start Taq DNA Polymerase
3 to 4 – Taq DNA Polymerase (chemical modification), Vendor A
5 to 6 – Taq DNA Polymerase (temperature-dependent inhibitor), Vendor B
7 to 8 – Taq DNA Polymerase (antibody-based), Vendor C
9 to 10 – Taq DNA Polymerase (not hot start), Thermo Scientific


References

  1. R. T. D’Aquila et al., Maximizing sensitivity and specificity of PCR by preamplification heating. Nucl. Acids Res. 19, 3749 (1991).

  2. D. E. Kellogg et al., TaqStart antibody: “Hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques. 16, 1134-1137 (1994).

  3. R. M. Horton et al., AmpliGrease: “Hot Start” PCR using petroleum jelly. BioTechniques. 16, 42-43 (1994).

  4. C. Dang S. D. Jayasena, Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR. J. Mol. Biol. 264, 268-278 (1996).

  5. T. Moretti et al., Enhancement of PCR amplification yield and specificity using AmpliTaqGold DNA polymerase. BioTechniques. 25, 716-722 (1998).

  6. R. S. Weyant et al., Effect of ionic and nonionic detergents on the Taq polymerase. BioTechniques. 9, 308-309 (1990).

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