Taq DNA polymerase:-
Taq DNA polymerase is a DNA dependent DNA polymerase, first isolated from the hot spring bacterium, Thermus aquatics in 1976 and in 1989. Due to its wide use in molecular biology (primarily PCR), it is termed as ‘Molecule of the Year’. This thermophilic DNA polymerase encodes an 832-amino acid, 94 kDa protein, which consists of two domains.
1. -NH2 domain: Similar to 5’-3’ exonuclease domain of members of polymerase I family of DNA polymerase
2. -C terminal domain contains a catalytically inactive 3’-5’ exonuclease and a polymerase sub domain, similar to klenow of DNA polymerase I.
The thermal stability of Taq DNA polymerase is attributed to its hydrophobic core and stable electrostatic interactions and high density of proline residues on the surface of the enzyme.
The optimal activity is at 75-800C temperature and at 600C, the activity is reduced by a factor of 2 and at 370C, its activity is reduced to only 10%.
To initiate DNA synthesis, like other DNA polymerases, it also requires a primer that is annealed to the template strand and caries an extensible 3’-OH group.
Taq DNA polymerase requires Mg2+ for its optimal activity. Phosphate buffers inhibit Taq DNA polymerase and therefore should be avoided. The reaction is usually carried out in the presence of Tris buffer at pH 8.3. Because of the lack of a proofreading function, the rate of misincorporation of dNTPs is high in PCR reactions which are catalyzed by Taq polymerase (or any other DNA polymerase that does not have editing domains).
Several mutant forms of native polymerases, are also available like Pfu And vent. Both of them have proofreading activities contributed by 3’-5’ exonuclease.
Pfu polymerase is therefore known to generate lowest errors while vent is probably intermediate between Taq and Pfu.
Pfu polymerase is isolated from Pyrococus furiosus Vent is isolated from Thermococcus litoralis (also known as Tli polymerase)
Taq DNA polymerase is a DNA dependent DNA polymerase, first isolated from the hot spring bacterium, Thermus aquatics in 1976 and in 1989. Due to its wide use in molecular biology (primarily PCR), it is termed as ‘Molecule of the Year’. This thermophilic DNA polymerase encodes an 832-amino acid, 94 kDa protein, which consists of two domains.
1. -NH2 domain: Similar to 5’-3’ exonuclease domain of members of polymerase I family of DNA polymerase
2. -C terminal domain contains a catalytically inactive 3’-5’ exonuclease and a polymerase sub domain, similar to klenow of DNA polymerase I.
The thermal stability of Taq DNA polymerase is attributed to its hydrophobic core and stable electrostatic interactions and high density of proline residues on the surface of the enzyme.
The optimal activity is at 75-800C temperature and at 600C, the activity is reduced by a factor of 2 and at 370C, its activity is reduced to only 10%.
To initiate DNA synthesis, like other DNA polymerases, it also requires a primer that is annealed to the template strand and caries an extensible 3’-OH group.
Taq DNA polymerase requires Mg2+ for its optimal activity. Phosphate buffers inhibit Taq DNA polymerase and therefore should be avoided. The reaction is usually carried out in the presence of Tris buffer at pH 8.3. Because of the lack of a proofreading function, the rate of misincorporation of dNTPs is high in PCR reactions which are catalyzed by Taq polymerase (or any other DNA polymerase that does not have editing domains).
Several mutant forms of native polymerases, are also available like Pfu And vent. Both of them have proofreading activities contributed by 3’-5’ exonuclease.
Pfu polymerase is therefore known to generate lowest errors while vent is probably intermediate between Taq and Pfu.
Pfu polymerase is isolated from Pyrococus furiosus Vent is isolated from Thermococcus litoralis (also known as Tli polymerase)
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