What is Taq Polymerase?
What is Taq Polymerase?
Taq polymerase is a thermostable DNA polymerase extracted from the thermophilic bacterium Hydrothermal Bacillus. Its main function is in the polymerase chain reaction technique, in which it automates repeated steps to amplify specific DNA sequences. The polymerase chain reaction can multiply DNA molecules by a billion-fold. This results in a large number of specific genes that are used downstream for a variety of applications.
Taq DNA polymerase belongs to the DNA polymerase family (Family a), and the DNA polymerases used in PCR are from family a and family B. Family DNA polymerases include Tth and Tma DNA polymerases and Taq, with 5'-3' exonuclease activity, but usually lacking 3'-5'. Due to the lack of 3'-5' exonuclease activity, family A polymerases are prone to errors when combining base pairs.
PCR Brief
Denaturation (94°C): After incubation, the PCR mixture is heated to separate the DNA strands
Annealing (55-65°C): This allows the primer to hybridize to the complementary region of the newly amplified DNA.
Extension (72°C): Taq polymerase-mediated enzymatic replication of primer-binding sequences. This happens at a rate of ~60 bases per second at 70°C
This process is repeated several times to increase the copy number. The use of thermophilic DNA polymerases, such as Taq polymerase, prevents denaturation of the enzyme during the heating step that separates newly synthesized strands—which subsequently simplifies the PCR technique and increases its efficiency.
In contrast, Family B DNA polymerases have high fidelity. This enables the removal of misincorporated nucleotides during DNA synthesis, which increases their accuracy relative to A-family polymerases.
Introduction to Polymerase Chain Reaction Amplification
The steps involved in PCR technology include incubating the DNA with excess primers specific to the chosen genomic sequence. DNA polymerase is responsible for extending primers using the target strand as a template.
The Taq polymerase Enzyme kinetics
Taq polymerase exhibits substantial enzymatic activity at 37°C. However, it operates best at higher temperatures (~72°C). Nucleotides are incorporated at a rate of 2 to 4 bases per minute.
However, functionality at this temperature allows for non-specific amplification, which is associated with mispriming events that occur during the initial stages of the PCR reaction. Before the first step of denaturation (at 93-95°C), the oligodeoxynucleotide primers can bind non-specifically to the template DNA, allowing extension to occur.
Mechanisms to circumvent this problem include the use of thermostable inhibitors, which block Taq polymerase activity until heat inactivation. Therefore, Taq polymerase is only active after the temperature destroys the monoclonal antibody during the initial denaturation of the PCR reaction. This antibody-mediated Taq polymerase inhibition method allows room temperature assembly of PCR reaction mixtures. Thus, non-specific amplification caused by mispriming events is eliminated or reduced.
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