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TrueStart™ Taq DNA Polymerase

#EP0611 Supplied with: 10X TrueStart™ Taq Buffer 25 mM MgCl2	200 u (5 u/µl) 1.25 ml 1.25 ml
#EP0612 Supplied with: 10X TrueStart™ Taq Buffer 25 mM MgCl2	500 u (5 u/µl) 2x1.25 ml 2x1.25 ml
#EP0613 Supplied with: 10X TrueStart™ Taq Buffer 25 mM MgCl2	5x500 u (5 u/µl) 10x1.25 ml 10x1.25 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0611, #EP0612, #EP0613 MSDS (English) MSDS (English-USA) MSDS (German) Flyer
Protocol for PCR with TrueStart™ Taq DNA Polymerase

 

Features

• Fastest to activate chemically modified hot start Taq DNA polymerase.
• High PCR specificity - reduced effects of mispriming and primer-dimer formation.
• Enhanced PCR sensitivity.
• Convenient room temperature PCR set-up.
• Generates PCR products with 3'-dA overhangs.

TrueStart™ and Hot Start  Taq DNA Polymerases, flyer in pdf, 182 KB

Description
TrueStart™ Taq DNA Polymerase is designed for hot start PCR, a technique that has been shown to improve specificity, sensitivity and yield of DNA amplification during PCR (1-5). In addition, the enzyme provides the convenience of room temperature reaction set-up.
TrueStart™ Taq DNA Polymerase is a Taq DNA polymerase, which has been chemically modified by the addition of proprietary heat-labile blocking groups to its amino acid residues. Due to this modification, TrueStart™ Taq DNA Polymerase is inactive at room temperature (Fig.1), avoiding extension of non-specifically annealed primers or primer dimers and providing higher specificity of DNA amplification (Fig.3). TrueStart™ Taq DNA Polymerase is the fastest to activate chemically modified hot start Taq DNA polymerase in the market. It requires only 1 min at 95°C to retain functional activity (Fig.2). The possibility to use TrueStart™ Taq DNA Polymerase without an additional activation step differentiates it from other hot start DNA polymerases and makes the enzyme an ideal tool for high throughput hot start PCR. Activated TrueStart™ Taq DNA Polymerase maintains the same functionality as Taq DNA polymerase: it catalyzes 5'=>3' synthesis of DNA, has no detectable 3'=>5' exonuclease (proofreading) activity, but possesses low 5'=>3' exonuclease activity. Before activation the both activities are not detectable.

Applications

• High throughput hot start PCR.
• RT-PCR.
• Highly specific amplification of complex genomic and cDNA targets.
• Amplification of low copy DNA targets.
• Real-time PCR.
• Multiplex PCR.
• Generation of PCR products for TA cloning.

Molecular Weight
94 kDa monomer.

Concentration
5 u/µl

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested in hot start PCR.

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: 67 mM Tris-HCl (pH 8.8 at 25°C), 6.7 mM MgCl₂, 1 mM 2-mercaptoethanol, 50 mM NaCl, 0.1 mg/ml BSA, 0.75 mM activated calf thymus DNA, 0.2 mM of each dNTP, 0.4 MBq/ml [³H]-dTTP.

Storage Buffer
The enzyme is supplied in: 20 mM Tris-HCl (pH 9.0), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.5% (v/v) Tween 20 and 50% (v/v) glycerol.

10X TrueStart™ Taq Buffer
200 mM Tris-HCl (pH 8.3 at 25°C), 200 mM KCl, 50 mM (NH₄)₂SO₄.

Inhibition and Inactivation
Inactivated by phenol/chloroform extraction.

Figure 1. TrueStart™ Taq DNA Polymerase is inactive at low temperatures.
Enzyme activity assays were performed at 25°C in one hour intervals with TrueStart™ Taq DNA Polymerase and Taq DNA polymerase.

Figure 2. TrueStart™ Taq DNA Polymerase is the fastest to reactivate at 95°C.
Hot start Taq DNA polymerases (chemically modified) from different suppliers were assayed for polymerase activity after the incubation at 95°C in the reaction buffer supplied with enzyme.

Figure 3. Higher PCR specificity with TrueStart™ Taq DNA Polymerase compared to other commercial hot-start PCR systems.
50ng of human genomic DNA were used as a template for amplification of 2 kb fragment of beta-globine gene using different DNA polymerases. 35 cycles of PCR was performed in thin-wall 0.2-ml tubes using GeneAmp® PCR System 9700 according to the manufacturer's recommendations.
M - GeneRuler™ 100 bp Plus DNA Ladder
1-2 - TrueStart™ Taq DNA Polymerase
3-4 - Taq DNA Polymerase (chemical modification), Vendor A
5-6 - Taq DNA Polymerase (temperature-dependent inhibitor), Vendor B
7-8 - Taq DNA Polymerase (antibody-based), Vendor C
9-10 - Taq DNA Polymerase (not hot start), Fermentas

Related Products

PyroStart™ Fast PCR Master Mix (2X) dNTP Mixes dNTP Set Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP CloneJET™ PCR Cloning Kit InsTAclone™ PCR Cloning Kit First Strand cDNA Synthesis Kit RevertAid™ First Strand cDNA Synthesis Kits M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ H Minus M-MuLV Reverse Transcriptase FastRuler™ DNA Ladders, ready-to-use O'RangeRuler™ DNA Ladders, ready-to-use GeneRuler™ DNA Ladders T4 DNA Polymerase Genomic DNA Purification Kit DNA Gel Extraction Kit Agarase Water, nuclease-free

References

1. D’Aquila, R.T., et al., Maximizing sensitivity and specificity of PCR by preamplification heating, Nucleic Acids Res., 19, 3749, 1991.
2. Kellogg, D.E., et al., TaqStart antibody: "Hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase, BioTechniques, 16, 1134-1137, 1994.
3. Horton, R.M., et al., AmpliGrease: "Hot Start" PCR using petroleum jelly, BioTechniques, 16, 42-43, 1994.
4. Dang, C. and Jayasena, S.D., Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR, J. Mol. Biol., 264, 268-278, 1996.
5. Moretti, T., et al., Enhancement of PCR amplification yield and specificity using AmpliTaqGold™ DNA polymerase, BioTechniques, 25, 716-722, 1998.

Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights  (such as 5' Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel.  Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. For complete license disclaimer, see.

Updated kovo 18, 2008 10:10