Taq DNA Polymerase

 

Discovery

Brock and his colleagues isolated Thermus aquaticus from a thermal spring in Yellowstone National Park in 1969. Although T. aquaticus is a gram-negative, nonsporulating, nonmotile rod, it was observed that its colony morphology is affected by the growth temperature and the growth stage of the culture, that the organism can also form the long filamentous. While the growth temperature of T. aquaticus is between 40 oC- 79 oC, the optimum temperature is 70 oC. The organisms have a yellow cellular pigment and they are obligatory aerobic. The optimum pH for the growth of organism is between 7.5- 7.8. An artificial growth medium should consist of several sugars, organic acids, nitrogen source and minerals. T. aquaticus does not require vitamins and amino acids. The GC content of T. aquaticus DNA is between 65.4%-67.4%.

 

Taq DNA Polymerase

Structure of Taq DNA Polymerase

 The three–dimensional structures of a number of DNA polymerase enzymes are available. Klenow fragment of DNA polymerase I from E. coli was first structured by protease treatment. Klenow fragment has the 5‘-3‘polymerase and 5‘-3 exonuclease activity, while it lacks 3‘-5‘ exonuclease. This structure is named as right hand. Domains of the Klenow DNA polymerase are known as the fingers, the palm, and the thumb.

 

Taq DNA polymerase has both 5‘-3‘polymerase and 5‘-3‘exonuclease activities. Although Taq DNA polymerase has similar sequences of 3‘-5‘exonuclease region of E. coli, but it lost this activity. The structure, shape and domains of Taq DNA polymerase are similar to DNA polymerase Klenow fragment. 

 

Taq DNA polymerase and DNA polymerase I from E. coli have a sequence identity of 38% depending on amino acid sequence alignment. The structural alignment of Taq DNA polymerase and E. coli DNA polymerase I, on the other hand, revealed a similarity of 32%, while the similarities of 5‘-3‘exonuclease domain (residues 1 – 291), nonfunctional 3‘5‘exonuclase domain (residues 292 - 423) and 5‘-3‘polymerase (residues 424 - 832) are 32%, 14% and 49%, respectively.

 Characterization of Taq DNA Polymerase 

 Taq DNA polymerase belongs to the Family A similar to DNA polymerase I from E. coli. Thermus aquaticus DNA polymerase is a single polypeptide with 832 amino acids and a molecular weight of 94 kDa. The fidelity of the Taq DNA polymerase is affected by pH, temperature, salt concentration and concentration of metallic ions. pI value of Taq polymerase is 6.4. While Taq DNA polymerase works at a pH between 7 and 8, the optimum pH is 7.8. Taq DNA polymerase requires divalent cations such as Mg+2 and Mn+2 like other DNA polymerases as a cofactor. Most favorable condition of the Taq DNA polymerase is in the 1-2 mM Mg+2 concentration range. When considering Mn+2 effects on Taq DNA polymerase, it is partial and optimum in the 2 mM. According to studies, there is no effect of calcium ion on Taq DNA polymerase. The addition of the salt such as KCl and NaCl also affects the activity of Taq DNA polymerase. The optimal concentration of NaCl is 40 mM, whereas that of KCl is 55-60 mM. The activity of Taq DNA polymerase is inhibited above 100 mM of either KCl or NaCl. The optimum temperature for Taq DNA polymerase is between 70- 80 Degree Centigrade. It is shelf life at 97.5 Degree Centigrade for 9 min. The error rate of base substitution of Taq DNA polymerase at 70 Degree Centigrade is 1/9000 bps. 

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