Higher specficity for RT-PCR Minimized secondary structure problems Excellent performance guaranteed

DESCRIPTION
TthPlus™ DNA polymerase is isolated from the Thermus thermophilus strain. TthPlus™ DNA polymerase is a single 92 kDa polypeptide showing a 5'-3' exonuclease activity but lacking 3'-5' exonuclease activity. It catalyzes the polymerization of nucleotides into double-stranded DNA in the presence of MgCl₂. Its efficiency has been shown more particularly on large DNA fragments up to 12 kb (using lambda phage DNA as a template). TthPlus™ DNA polymerase is also capable of catalyzing the polymerization of DNA using a RNA template in the presence of MnCl₂. The ability of TthPlus™ DNA polymerase to reverse transcribe at elevated temperatures (70°C) minimizes the problems encountered with strong secondary structures in RNA since they are unstable at higher reaction temperatures. Higher temperatures also result in increased specificy of primer hybridization and extension. In coupled RT/PCR assays, TthPlus™ is about 50-100 times more efficient than Taq DNA polymerase.

TthPlus™ DNA polymerase is delivered with 10x RT-buffer, 5x amplification-buffer and separate MnCl₂ (25 mM) and MgCl₂ (50 mM) solutions. The 5x amplification-buffer contains EGTA, which binds/neutalizes Mn²⁺ from the RT-reaction. Therefore after RT it is not necessary to change the buffers. For subsequent amplification we recommend to use BioTherm™ or KlenTherm™ DNA polymerase.

FEATURES
thermostable
DNA polymerase activity in the presence of MgCl₂
reverse transcriptase activity in the presence of MnCl₂
reverse transcription at elevated temperature minimising secondary structure problems

CONCENTRATION
5 units/µl

UNIT DEFINITION
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acid-insoluble form in 30 minutes at 72°C under the following reaction conditions: 25 mM TAPS buffer (Tris-(hydroxymethyl)-methyl- amino-propanesulfonic acid, sodium salt) pH 9.3 (25°C), 50 mM KCl, 2 mM MgCl₂, 1 mM β-mercaptoethanol, 200 µM dNTPs and 10 µg of calf thymus DNA in a final reaction volume of 50 µl.

STORAGE BUFFER
10 mM K-phosphate buffer pH 7.0 (25°C), 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 50% glycerol (v/v), 0,1 mg/ml BSA

STORAGE TEMPERATURE
Store TthPlus™ DNA polymerase, preferably at -20°C, in a constant temperature freezer.

10X REACTION BUFER
670 mM Tris-HCl pH 8.8 (25°C), 166 mM (NH₄)₂SO₄, 0.1% Tween 20

5x AMPLIFICATION BUFFER
335 mM Tris-HCl pH 8.8 (25°C), 83 mM (NH₄)₂SO₄, 3.75 mM EGTA, 25% glycerol (v/v), 0.1% Tween 20

EXRTA SOLUTIONS
25 mM MnCl₂
50 mM MgCl₂

The optimal experimental conditions depend on the system used and they should be individually determined. The Mg²⁺ or Mn²⁺ concentrations and the enzyme amount are the limiting factors for an accurate result. Traditionally 5 units of enzyme and a MnCl₂ concentration of 1 mM are used for the reverse transcription in a final 50 µl reaction volume. For the amplification 2.5 MgCl₂ concentration of 1.5 mM are used for a final reaction volume of 50 µl.

Comparison of sensitivity of RT-PCR with TthPlus™ DNA polymerase and MMLV-RT

Comparison of RT-PCR with TthPlus™ DNA polymerase and MMLV-RT in 16S-rRNA system

REFERENCES
1. Ruttiman C. Cotara S.M., Zaldivar J. and Vicuna R. (1985) Eur. J. Biochemistry, 149, 41-46
2. Myers T.W. and Gelfand D.H. (1991) Biochemistry, 30, 7661-7666

PRODUCT CITATIONS
Bartl, S.; Ban, J.; Weninger, H.; Jug, G. & Kovar, H. (2003), Nucleic Acids Res 31(4), 1136--1147.
Carazo, A.; Alejandre, M. J.; Suarez, M. D. & Linares, A. (2000), Lipids 35(6), 587--593.
Torres, J. M. & Ortega, E. (2003), FASEB J 17(11), 1428--1433.