TTH DNA POLYMERASE

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) activity
• for real time PCR

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).

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.