Non disponible en dehors du Royaume-Uni et de l'Irlande

Application

Thermus thermophilus (Tth) DNA Polymerase might be used:to amplify DNA fragments by polymerase chain reaction (PCR) due to its resistance towards prolonged incubations at high temperatures (95 °C)to label DNA fragments with either radiolabeled nucleotides, digoxigenin, or biotin, since this enzyme accepts modified deoxyribonucleotides as substrates to efficiently transcribe RNA targets into cDNA due to its intrinsic Mn-dependent reverse transcriptase (RT) activityfor real time PCR

Biochem/physiol Actions

Thermus thermophilus (Tth) DNA Polymerase is a thermostable DNA polymerase with intrinsic reverse transcriptase (RT) activity. The enzyme is a highly processive 5′-3′ DNA polymerase and lacks 3′-5′ exonuclease activity (proof reading). It was found to possess a very efficient intrinsic reverse transcriptase (RT) activity in the presence of manganese ions – much higher than that demonstrated for Escherichia coli DNA polymerase I and Taq DNA Polymerase. The RT activity is not associated with RNase H activity. The elevated temperatures of Tth DNA Polymerase (optimum +55°C to +70°C, maximum +95°C) activity overcomes the problems posed by RNA secondary structure. Resulting cDNA can be amplified by PCR using the same enzyme in the presence of Mg²⁺-ions. The ability of Tth DNA Polymerase to perform both reverse transcription and DNA amplification at elevated temperatures allows this enzyme to be used for quantitative RT-PCR, cloning, and gene expression analysis of cellular and viral RNA. Tth DNA Polymerase is used for RT-PCR amplification of RNA up to 1kb.

Features and Benefits

Tth DNA Polymerase: Ensures optimized polymerase chain reaction (PCR) product size for at least up to 1,000 bp in a RT-PCR reaction Accepts modified desoxyribonucleoside triphosphates as substrates Has no association with RNase H activity Has high thermostability to overcome the problem, typically associated with the high degree of secondary structure present in RNA

General description

Tth DNA Polymerase is isolated from the thermophilic eubacterium Thermus thermophilus HB8, and is purified to be free of nonspecific DNases and RNases. The enzyme is a highly processive 5′-3′ DNA polymerase, and lacks 3′-5′ exonuclease activity. The enzyme exhibits its highest activity at a pH of approximately 9 (adjusted at +25°C), and temperatures around +75°C. It is resistant to prolonged incubations at high temperatures (+95°C).

Legal Information

Use of this product is covered by US patent claims and corresponding patent claims outside the US. The purchase of this product includes a limited, non-transferable immunity from suit under such patent claims for using only this amount of product for the purchaser′s own internal research. No right under any other patent and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser′s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patent claims require a separate license from Roche. 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.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Packaging

1 kit containing 3 components

Quality

Each lot of Tth DNA Polymerase is assayed for activity on activated DNA. A function test is performed using ?DNA, as well as human genomic DNA. Performance in RT/PCR is assayed using total mouse RNA and primers specific for ?-actin. Each lot of Tth DNA Polymerase is tested for the absence of contaminating activities like exo- and endonucleases and nicking activities according to the current Quality Control procedures. A unique self-priming assay ensures the absence of contaminating DNA according to the current Quality Control procedures.

Unit Definition

One unit of Tth DNA Polymerase is defined as the amount of enzyme which catalyses the incorporation of 10 nmol total dNTPs into acid precipitable DNA within 30 minutes at +70 °C under the assay conditions stated in "unit assay".Unit Assay: Incubation buffer for assay on activated DNA67 mM Tris-HCI, pH 8.8 (+25 °C), 16.6 mM (NH₄)₂SO₄, 6.7 mM MgCl₂, 10 mM 2-mercaptoethanol, 0.2 mM dATP, dCTP, dGTP, dTTP each.Incubation procedure12.5 µg activated herring sperm DNA and 0.1 µCi [α³²P] dCTP are incubated with 0.01 to 0.1 units Tth DNA Polymerase in 50 µl Incubation buffer with a paraffin oil overlay at +70 °C for 30 min.The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation followed by scintillation counting.Volume Activity: 0.5 to 5 U per reaction of PCR (optimal)2.5 U per reaction of PCR (standard)Determined in the assay on activated DNA described under "unit assay".