Thermus

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

Taq DNA polymerase from Thermus aquaticus) has made it unnecessary to add fresh enzyme for each round of synthesis. Because the amount of target DNA theoretically doubles each round, 25 rounds would increase its concentration about 33 million times. In practice, the increase is actually more like a million times, which is more than ample for gene isolation. Thus, starting with a tiny... 

Yokoyama, A., G. Sandmann, T. Hoshino, K. Adachi, M. Sakai, and Y. Shizuri. 1995. Thermozeaxanthins, new carotenoid-glycoside-esters from thermophilic eubacterium Thermus thermophilus. Tetrahedron Lett. 36 4901 1904. 

Angelini, S., Moreno, R., Gouffi, K. et al. (2001) Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli. FEBS Letters, 506 (2), 103-107. 

Hagen, W.R., Dunham, W.R., Johnson, M.K., and Fee, J.A. 1985a. Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin. Possible new fingerprints for three iron clusters. Biochimica et Biophysica Acta 828 369-374. 

M.S. Lah, M.M. Dixon, K.A. Pattridge, W.C. Stallings, J.A. Fee, and M.L. Ludwig, Structure-function in Escherichia coli iron superoxide dismutase comparisons with the manganese enzyme from Thermus thermophilus. Biochemistry. 34, 1646-1660 (1995). 

One of the most important of these extremophiles is a bacterium (Thermus acquaticus) discovered in 1965 in a Yellowstone National Park hot spring where the temperature is a constant 73 °C (Centigrade or Celsius) or 163 °F (Fahrenheit). About twenty years... 

To overcome this shortcoming, biologists turned to an enzyme that could survive the PCR hot cycle. They replaced the original heat-sensitive enzyme with DNA-polymerase from Thermus acqua-ticus, the Yellowstone extremophile. The new enzyme is unscathed... 

Taq polymerase is a thermostable DNA polymerase which was originally isolated from the bacterium Thermus aquaticus, which lives in hot springs. 

RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

Tanner, J J., R.M. Hecht, and K.L. Krause. 1996. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceralde-hyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. Biochemistry 35 2597-2609. 

Salminen, T., A. Teplyakov, J. Kankare, B.S. Cooperman, R. Lahti, and A. Goldman. 1996. An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases. Protein Sci 5 1014-1025. 

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