Question

We usually think of enzymes as being most active at around \(37^{\circ} \mathrm{C}\) yet in PCR the DNA polymerase is subjected to multiple exposures of relatively high temperatures and seems to function appropriately at \(65-75^{\circ} \mathrm{C}\). What is special about the DNA polymerase typically used in PCR?

Short answer

Answer: The DNA polymerase used in PCR, such as Taq polymerase, is unique due to its thermostability, allowing it to maintain its structural integrity and remain active at high temperatures. This enzyme is derived from thermophilic microorganisms like Thermus aquaticus that thrive in extreme temperature conditions and possess heat-resistant enzymes. Its specific amino acid composition and protein structure contribute to its resistance to denaturation.

Step-by-step solution

Background of PCR enzymes

The DNA polymerase enzyme typically used in PCR (Polymerase Chain Reaction) is special due to its tolerance towards high temperatures. This is essential because PCR involves repeated cycles of high temperatures (in the range of 65-75°C) to denature the DNA strands for amplification. In typical conditions, enzymes like DNA polymerase would lose their activity at such high temperatures.

The source of DNA polymerase for PCR

The DNA polymerase typically used in PCR is derived from thermophilic microorganisms, such as Thermus aquaticus. These microorganisms usually thrive in extreme temperature conditions, such as hot springs, and hence have enzymes that are stable and functional at high temperatures. The DNA polymerase from Thermus aquaticus is called Taq polymerase and is widely used in PCR.

Stability at high temperatures

Taq polymerase, derived from the thermophilic bacteria, has special properties that allow it to maintain its structural integrity and remain catalytically active at high temperatures. Its increased thermostability at temperatures encountered during PCR (65-75°C) is due to its specific amino acid composition and protein structure, which make it resistant to denaturation.

Other thermostable DNA polymerases

Apart from Taq polymerase, there are other thermostable DNA polymerases used in PCR, such as Pfu, Vent, and Phusion. These enzymes also come from thermophilic microorganisms and have similar properties that make them suitable for use in PCR. Comparatively, they have higher fidelity (lower error rates) and can tolerate higher temperatures, up to 98°C.

Importance of heat-resistant DNA polymerase in PCR

The thermostable nature of these DNA polymerases is essential for the PCR process, as it allows the enzyme to withstand denaturing conditions and maintain activity during the entire reaction. If a heat-labile enzyme were used, the reaction would be inhibited after a few cycles, rendering the PCR process ineffective.

Key concepts

Polymerase Chain Reaction

The Polymerase Chain Reaction, or PCR, is a revolutionary technique used in molecular biology to amplify a single or few copies of a fragment of DNA across several orders of magnitude, creating thousands to millions of copies of a particular DNA sequence. This is exceptionally useful for various applications, such as genetic fingerprinting, cloning gene fragments, diagnosing hereditary diseases, and detecting infectious agents.

The PCR process consists of three main steps: denaturation, annealing, and extension. During denaturation, the double-stranded DNA is heated to around 95°C to separate it into two single strands. In the annealing step, the temperature is lowered to around 50-65°C to allow short DNA primers to attach to the DNA template. Finally, during the extension step, the temperature is raised to about 72°C, the ideal temperature for Taq polymerase to synthesize a new DNA strand by adding nucleotides to the primers.

Importantly, PCR must be repeated through multiple cycles to achieve significant amplification of the target DNA.

Taq polymerase

Taq polymerase is a specific type of DNA polymerase that is often used in PCR because of its ability to remain stable and active at high temperatures. It is named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated. This enzyme is instrumental in the synthesis of DNA strands during the extension phase of PCR.

One distinctive feature of Taq polymerase is its 'thermostability', which allows it to withstand the high temperatures required for the denaturation phase of PCR. This is critical because repetitive heating and cooling cycles would degrade most enzymes, but Taq polymerase remains functional throughout the entire PCR process. While there are other polymerases with higher fidelity, Taq polymerase is widely used due to its robustness and effectiveness in most standard PCR procedures.

Thermophilic microorganisms

Thermophilic microorganisms are a fascinating group of extremophiles that thrive in hot environments, such as hydrothermal vents, hot springs, and geysers, where temperatures can exceed 80°C. These organisms have evolved to not only survive but also to function optimally at such high temperatures, which would typically inhibit or denature essential biomolecules in other organisms, such as enzymes and proteins.

Enzymes derived from thermophilic microorganisms, like Taq polymerase, have amino acid sequences and three-dimensional structural configurations that confer exceptional stability at elevated temperatures. This characteristic makes such enzymes ideal for industrial applications that require robustness in extreme conditions, including PCR, where sustained enzyme activity at high temperatures is vital for DNA amplification.

Enzyme thermostability

Enzyme thermostability refers to the ability of an enzyme to maintain its activity at high temperatures. Thermostable enzymes like Taq polymerase are critical in biotechnological processes such as PCR because of their resistance to the denaturing effects of heat. Heat stability is typically a result of specific adaptations in the enzyme's structure, such as increased hydrogen bonds, salt bridges, and hydrophobic interactions, which together contribute to the enzyme's robustness against thermal inactivation.

Different factors can influence enzyme thermostability, including the presence of particular amino acids that increase temperature resistance, and the overall compactness of the protein structure. These adaptations enable the enzyme to operate in environments that would typically degrade less-stable proteins.

High-fidelity DNA polymerases

High-fidelity DNA polymerases are enzymes that replicate DNA with a high degree of accuracy. They are characterized by their ability to insert the correct nucleotides with minimal error during the replication process, which is crucial in applications where the precise amplification of DNA is necessary, such as in cloning and genomic sequencing.

These polymerases possess proofreading activity; a 3' to 5' exonuclease activity that allows them to remove incorrectly inserted nucleotides and replace them with the correct ones before proceeding with DNA synthesis. This proofreading ability significantly reduces the rate of errors, or mutations, ensuring the generation of DNA copies that are almost identical to the original template. Examples of high-fidelity DNA polymerases include Pfu polymerase from Pyrococcus furiosus and Phusion polymerase, which combine the processivity of Taq with proofreading capabilities for applications that require higher accuracy in DNA amplification.