HotStart-IT Taq DNA Polymerase
Sensitive and consistent amplification polymerase for real-time and routine PCR
E. coli strain expressing a clone of Taq DNA Polymerase from Thermus aquaticus(1-3). The hot start component is a recombinant protein also expressed in E. coli.
HotStart-IT Taq DNA Polymerase uses a novel hot start method developed at Affymetrix called primer-sequestration. In general, hot start PCR methods reduce or eliminate non-specific primer-extension products formed at lower temperatures during assembly of PCR reactions. At these less stringent annealing temperatures, primers may bind non-specifically, which often leads to unwanted amplification products and primer-dimers. In order to resolve this problem, we have combined Taq DNA Polymerase with a recombinant protein which binds and sequesters primers at lower temperatures making them unavailable for use by Taq DNA Polymerase. This primer-sequestration technique effectively blocks DNA synthesis from mis-priming events at lower temperatures. Following the initial denaturation step during PCR, the protein is inactivated and the primers are free to participate in the amplification reaction. This novel hot start method enhances many complex PCR reactions by increasing both specificity and yield.
- Room temperature reaction set-up.
- High specificity and sensitivity.
- Minimizes amplification of non-specific products and primer-dimers.
Taq DNA Polymerase is a highly processive 5'→ 3' polymerase and has 5' →3' exonuclease activity. Suitable for TaqMan® assays.
Free from detectable non-specific nucleases.
20mM Tris-HCl, pH 8.5, 1mM DTT, 0.1mM EDTA, 200mM KCI, 50% glycerol, and stabilizers.
One unit incorporates 10 nmol of total nucleotides into acid-insoluble material in 30 min at 74°C in a total volume of 50 μl.
The reaction mixture (50 μl) contains 25mM TAPS, pH 9.3 (at 25°C), 50mM KCl, 2mM MgCl2, 1mM 2-mercaptoethanol, 200μM dNTPs, 250 μg/ml activated salmon sperm DNA, and cloned Taq DNA Polymerase. After incubation at 74°C for 10 min, acid-insoluble material is determined.
Polymerase Blocking Assay:
The assay compares the polymerase activity of HotStart-IT Taq DNA Polymerase relative to Taq DNA Polymerase. The reaction mixtures contain 0.625 units of polymerase, 1X PCR Reaction Buffer, 0.2mM each dNTP, and 2 pmol of overlapping, extendable oligonucleotides in a 25 μl reaction volume. Following incubation at 25°C for 4 hours, HotStart-IT Taq DNA Polymerase blocks at least 90% of the activity relative to Taq DNA Polymerase without hot start capability (Figure 2).
|Figure 2. Increased specificity of HotStart-IT Taq DNA Polymerase. The single-copy numb gene was amplified from the indicated amounts of human genomic DNA with standard Taq DNA Polymerase (-) or with HotStart-IT Taq DNA Polymerase (+). Primers were designed with a 3 bp overlap at their 3'-ends to favor primer-dimer formation during reaction set-up at room temperature. Results demonstrate a shift from mainly primerdimers to the desired product when HotStart-IT Taq DNA Polymerase is used.|
PCR with HotStart-IT Taq DNA Polymerase shifts production of primer-dimers to a specific target of 306 bp from 1 ng of human genomic DNA relative to Taq DNA Polymerase.
Functionally Tested 10X PCR Reaction Buffer (included, PN 71165):
100mM Tris-HCl (pH 8.6), 500mM KCl, 15mM MgCl2
Functionally Tested MgCl2 (included, PN 71167):
- INNIS, M. A., MYAMBO, K. B., GELFAND, D. H. AND BROW, M. A. (1988) Proc. Natl. Acad. Sci. 85, 9436-9440.
- LAWYER, F. C., STOFFEL, S., SAIKI, R. K., MYAMBO, K., DRUMMOND, R. AND GELFAND, D. H. (1989) J. Biol. Chem. 264, 6427-6437.
- INNIS, M. A. AND GELFAND, D. H. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press.
- High-specificity PCR amplification
- High-sensitivity PCR amplification
- TA-vector cloning
- Amplification prior to in vitro transcription
Shipped on dry ice. Store at -20°C.