Taq Polymerase Enzyme

 

Taq polymerase is a thermostable DNA-dependent DNA polymerase that catalyzes the template-directed polymerization of dNTPs at high temperatures. Taq Polymerase was first isolated in 1976 from Thermus aquaticus strain YT-1.

The Properties of Taq Polymerase Enzyme

Taq Polymerase catalyzes the DNA-dependent polymerization of dNTPs, one unit of the enzyme is defined as the amount of enzyme that will incorporate 10 nmol of radioactively labeled dTTP into acid insoluble material at 80^oC in 30 minutes. The enzyme could be purified to a specific activity of 200000 U/mg.
The activity of the enzyme is dependent on bivalent cations, such as Mg²⁺. The optimum concentration of₂ is 2 mM. Monovalent cations also have an effect on the activity of Taq Polymerase. The monovalent cation is K⁺ in a form of KCl when it is using with the optimum concentration 50 mM, when KCl concentration more than 75 mM it can inhibit the activity of taq polymerase. Another monovalent cations, such as NaCl, NH₄Cl and NH₄acetate, cannot substitute KCl without a decrease in specific activity. MgCl
Maximum polymerization rates of Taq Polymerase are obtained with 0.7-0.8 mM dNTPs. While at dNTP concentrations of 4-6 mM, substrate inhibition is observed. Denaturing agents, detergents, and organic solvents in low concentration are tolerated by Taq Polymerase, while at higher concentrations, the inhibition of enzyme activity is observed.
The major distinguishing feature of Taq Polymerase is its extreme thermal stability. The enzyme can withstand temperatures in excess of 95^oC for prolonged periods, and in fact, its optimum for reaction is 75^oC. The rate of reaction is reduced to 50% at 60°C, and to 10% at 37^oC.

Taq Polymerase and PCR Technique

The major application of Taq polymerase at present is in the polymerase chain reaction (PCR). This technique is a simple method of amplifying minute quantities of DNA for a variety of subsequent procedures, including cloning, sequencing, hybridization, and genome mapping. The application of taq polymerase to the PCR was the basis for the success of the technique. It is caused by these following factors:

• Taq polymerase enzyme is stable up to 95^oC, so it is not necessary to replenish the enzyme after each PCR cycle.
• The activity of the enzyme is maximum at the temperature between 70 and 75^oC, which minimizes secondary structures of the template, resulting in high polymerization yield.
• The annealing can be chosen from 30-70^oC, allowing an optimal adaptation of cycle parameters to appropriate annealing temperatures of the primers; therefore, by products are hardly generated.

The enzyme from Thermus thermophilus (when used in a manganese-containing buffer) has an additional reverse transcriptase activity, which extends the use of PCR directly to cDNA synthesis. Another use of Taq polymerase 1s directly in DNA sequencing, where the high temperatures employed help reduce problems caused by secondary structure in the template and allow an increase in the stringency of primers used.