The initial PCR method was conducted in 1980s was a labor intensive and a slow process. Scientists have to add fresh polymerase enzyme into reaction tube for each cycle of polymerase chain reaction (PCR), because DNA polymerase enzyme isolated from E. coli lose all activity during denaturation steps (90 °C) of the cycles. This problem was resolved with an improvement that transformed molecular biology laboratories in worldwide forever. That was the discovery of heat-stable (thermostable) DNA polymerases for PCR amplification.
Thermus aquaticus is a thermophilic bacterium that was first discovered in hot springs of Yellowstone National Park by Thomas Brock in 1965. Thus, living conditions in higher temperature has evolved the bacterial proteins and enzymes to survive in hot springs. So, the DNA polymerase enzyme was derived from Thermus aquaticus (also commonly known as Taq polymerase) can tolerate up to 95 °C. This enzyme very stable at higher temperatures, this gives unique future to PCR reaction needs in heating steps that would inactivate other natural forms of DNA polymerases (such as E. Coli DNA polymerase).
Extremophiles are archaebacterial live in extreme conditions. The genus Thermus is famous for high resistance to hot water. There are two other group extremophiles; methanogens and halophiles have distinct preferences against harsh environment. Methanogens are anaerobes organisms “produce methane” gas. Halophiles can survive in excessive salt concentrations, like salt lakes. Thus, the enzymes are extracted by these organisms also can tolerate these extreme conditions. Research and development facilities of biotechnology industries eagerly work on organisms live at extraordinary conditions (high pressure, hot springs, Dead sea, deep sea hydrothermal vents etc.) to discover valuable enzymes.
Taq polymerase is one the of the most commonly used polymerase enzyme for PCR amplification, Optimal temperature for Taq polymerase activity is about 72 °C. Like other polymerase enzymes Taq polymerase can elongate growing DNA strand in a 5′ → 3′ direction”
Taq polymerase does not have any proofreading capacity, it can insert wrong nucleotide bases during DNA synthesis. Not only Taq polymerase but other polymerase enzymes could randomly introduce a new base-pair mutation. However, Taq polymerase lack of proofreading activity, when it is compared with other polymerase with proofreading activity, it has higher error chance. The possible error take place at one nucleotide every about 10 kb nucleotides of new synthesized DNA strand. But, when you consider 30 cycles of PCR reaction, error rate would be one base pair for 300 bp of final PCR product.
In PCR amplification cycles, this error will be inherited to new DNA molecules, and adding more mutations during those cycle. Therefore, error rate would be a big problem for cloning applications, because each of these mutations will be preserved in all the offspring of that single clone. So, when low error rate is necessary, instead of suboptimal Taq polymerase, a high fidelity polymerase with 3′ → 5′ proofreading function can be employed.
Table: Comparison of DNA polymerases with proofreading activity:
|DNA Polymerase||Origin of enzyme||Product Ends||Proofreading activity|
|Taq||Thermus aquaticus||Stick End 3′||–|
|Pfu||Pyrococcus furiosus||Blunt end||+|
|Psp (Deep vent)||Pyrococcus sp.||Blunt end||+|
|Pwo||Pyrococcus woesi||Blunt end||+|
|Tli||Thermococcus litoralis||Blunt end||+|
|Tma||Thermotoga maritima||Blunt end||+|
|Tth||Thermus thermophilus||Stick End 3′||+|
3′ Adenine Overhangs
Another important future of Taq polymerase adding adenosine residue to the 3′ ends of the DNA product. This reaction could be quite useful for cloning purposes, because PCR product will be sticky-ended instead of blunt-end.
Brock, T.D., and H. Freeze. 1969. Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J. Bacteriol. 98:289–297.
Saiki, R.K., S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, and N. Arnheim. 1985. Enzymatic amplifi cation of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle-cell anemia. Science 230:1350–1354.
Saiki, Randall K., et al. “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.” Science 230.4732 (1985): 1350-1354.