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PCR at High Temperature

Figure 5: Yellowstone Hot Spring which Thermus aquaticus is isolated

Reference:

Elizabeth van Pelt-Verkuil,  Alex van Belkum and John P. Hays (2008) Chapter 7: Taq and Other Thermostable DNA Polymerases. Principles and Technical Aspects of PCR Amplification. Springer, pp 103-118.

Table 2: Comparison between Pfu and Taq DNA polymerases on error rate, accuracy and proofreading activity

 

Figure 6Thermus aquaticus under the microscope. Left- light microscope. Its morphology is slender and long, rod shape. Right- cell structure is seen using electron microscope, its morphology is indifferent with many other bacteria that strive in conventional temperature. Little that we know it exhibits such unique physiological characteristic.

 

Previously, the original PCR technique utilized the DNA polymerase originated from Escherichia coli Pol III, however, due to fact that high temperature is needed to denature the double-stranded DNA template, the enzyme will also be denatured and had to be replenished every cycle. Hence, the discovery of thermostable DNA polymerase isolated from thermophilic hot spring bacterium Thermus aquaticus is able to solve this problem.

 

Thermus aquaticus is discovered by Thomas D. Brock in Yellowstone Hot Spring which is an environment that is approximately 73˚C (T. D. Brock, 1997). Taq polymerase is stable to 95˚C and so it is unaffected by the denaturation step which employs about 92-94˚C in the PCR reaction. Another crucial function of Taq polymerase is that it increases the specificity of PCR products because the DNA is copied at 72˚C rather than 37˚C. At such higher temperature, non-specific hybridization of primers to non-target DNA can be prevented, thus resulting in products of PCR are more homogenous than those obtained using E.coli enzyme. On the contrary, during annealing of primers to target DNA will promote non-specific binding.

 

Another Thermostable DNA polymerase ?

 

Pfu polymerase from Pyrococcus furiosus which exhibits hyperthermophilic nature, able to grow optimally at 100˚C. As compared to Taq polymerase, Pfu polymerase is more thermostable than former. Futhermore, Pfu polymerase has proofreading activity (ability of removing wrongly inserted nucleotides) which resulting in highly specific PCR products. Hence, the error rate of Pfu polymerase is far more lower than Taq polymerase. 

 

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