An Overview of Taq DNA Polymerase in PCR

What is the Origin of Taq DNA Polymerase?

DNA polymerase is an essential enzyme involved in the processes of chromosome replication, repair, and recombination within cells, ensuring accurate replication and transmission of genetic information to daughter cells and offspring. Based on the analysis of DNA polymerase amino acid sequences and structures, it can be classified into seven major families: A, B, C, D, X, Y, and reverse transcriptase (RT) family. Heat-resistant DNA polymerases discovered so far belong to either the A or B group. Among them, Taq DNA polymerase from group A was the first to be discovered and applied in PCR. Heat-resistant DNA polymerase is a core component of the PCR reaction system, and its performance determines the sensitivity, specificity, and success rate of PCR detection. In 1976, Taq DNA polymerase was isolated and purified from the thermophilic bacterium Thermus aquaticus YT-1 in 1988, Taq DNA polymerase was first applied in PCR technology, marking a milestone in the development of PCR techniques.

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What is the Structure and Function of Taq DNA Polymerase?

Taq DNA polymerase consists of 832 amino acid residues with a molecular weight of 94 kDa. It primarily comprises three main structural domains, as illustrated in the three-dimensional structure diagram. The N-terminal region spanning amino acids 1-291 forms the 5'-3' exonuclease activity domain, which is utilized in the hydrolysis of TaqMan probes. Amino acids 292-423 constitute the 3'-5' exonuclease domain, but this domain lacks activity. Amino acids 424-832 form the 5'-3' polymerase activity domain, whose three-dimensional structure resembles a right hand with distinct regions: the thumb, fingers, and palm. The thumb region is responsible for maintaining tight binding between DNA polymerase and the primer-template complex, further aiding in DNA polymerase extension. The fingers region is involved in binding dNTPs. The palm region serves as the active site for divalent metal ion binding, regulating the chemical environment surrounding the bound dNTPs and the primer 3'-OH end, thus mediating and catalyzing the reaction.

Structure of Taq DNA polymerase showing the conserved motifs. (Laos, R.; et al, 2014)

What are the Characteristics of Taq DNA Polymerase

Good Thermal Stability

Taq DNA polymerase exhibits optimal catalytic activity at 75-80°C, retaining over 50% of its activity even after exposure to 95°C for half an hour. It is a Mg²⁺-dependent polymerase, with the optimal concentration range being 2-4 mM. Taq DNA polymerase also requires monovalent cations and exhibits significant activity in KCl.

5'-3' Polymerase Activity

This characteristic is highly sensitive to temperature. At 70°C, Taq DNA polymerase can polymerize DNA chains at a rate of 60 nt/s, which decreases to 24 nt/s at 55°C, 1.5 nt/s at 37°C, and essentially halts at 22°C.

5'-3' Exonuclease Activity

Taq DNA polymerase is one of the few DNA polymerases that possess this activity. This activity can be utilized to excise 5'-end radiolabeled DNA probes, enabling specific detection of PCR products through changes in radioactive signals. It laid the foundation for the development of qPCR techniques, with Taq DNA polymerase becoming the designated enzyme for probe-based qPCR assays.

Absence of 3'-5' Proofreading Activity

Although Taq DNA polymerase shares high structural similarity with E. coli polymerase I (including the 3'-5' exonuclease domain), it lacks 3'-5' exonuclease activity, reducing its fidelity in PCR amplification.

Tolerant to deoxyuridine triphosphate (dUTP)

Therefore, Taq DNA polymerase can be used for establishing dUTP/UDG (deoxyuridine triphosphate/uracil-DNA glycosylase) contamination prevention systems in PCR. Additionally, it exhibits non-template-dependent activity, generating PCR products with a single adenine overhang at the 3' end.

How to Choose Taq DNA Polymerase

Currently, there are various types of Taq DNA polymerases available on the market to meet diverse experimental needs. So, how do you choose the most suitable Taq DNA polymerase? Based on its characteristics such as specificity, fidelity, thermostability, amplification rate, maximum fragment length, ability to amplify complex templates, and ease of optimization, the main categories of Taq DNA polymerases can be classified as follows, along with their properties and uses.

HighSpecificity Taq DNA Polymerase

Highly specific amplification of the required fragments is the fundamental requirement of PCR, and there are many factors that influence PCR specificity, including template quality, primer properties, and control of reaction conditions. The emergence of high-specificity Taq DNA polymerases has greatly reduced the cumbersome experimental process of adjusting conditions, laying the foundation for the rapid and effective purification of PCR products (direct purification of PCR products). The most typical representative of this type of enzyme is the hot-start Taq DNA polymerase. It is well known that before the first denaturation cycle of PCR, there is a heating process during which some non-specific pairing occurs between the primers and the template. If Taq DNA polymerase is active at this time, non-specific amplification is easily generated. Since the template amount is very low at the beginning of the cycle, the non-specific bands generated are exponentially amplified in subsequent cycles, which severely interfere with the amplification of the target fragments and may even prevent specific bands from being amplified. In contrast, hot-start Taq DNA polymerase is an enzyme that must be activated at high temperature, so it is not active before denaturation in the initial cycle, which greatly improves the specificity of PCR amplification.

• Antibody-Based Hot-Start Enzyme

It is generally considered to use DNA polymerase antibodies to block enzyme activity so that it is inactive at low temperatures. When heated to the denaturation temperature, the antibody denatures, releasing the activity, which can greatly avoid nonspecific amplification caused by mismatches or primer dimers. On the other hand, for low-abundance genes, blocking antibodies can increase the efficiency of primer annealing with the correct template by avoiding nonspecific binding of the enzyme and template before pre-denaturation, ensuring the effective detection of low-abundance genes and greatly enhancing the sensitivity and efficiency of the reaction.

• Chemically Modified Hot-Start Enzyme

The chemical modification of DNA polymerase generally involves the use of small chemical molecules such as acid anhydrides to interact with side-chain amino acids (lysine) on the polymerase through chemical reactions. This renders the enzyme inactive at low temperatures. As the temperature rises, the chemical bond between the acid anhydride and the amino group breaks, releasing the enzyme activity, thereby achieving the hot-start effect. Chemical modification can effectively block enzyme activity, is easy to operate, but the release of enzyme activity is slow, it depends on temperature to activate DNA polymerase, can effectively suppress nonspecific products, can be prepared at room temperature, and has higher requirements for PCR reaction buffer. In addition, since almost all Taq DNA polymerase products come with corresponding reaction buffers, the quality of the buffer also plays an important role in ensuring the specificity of Taq DNA polymerase amplification.

• Ligand-Based Hot-Start Enzyme

Ligand-based modification mainly relies on nucleic acid ligands to block enzyme activity. As the name suggests, nucleic acid ligands generally refer to a class of single-stranded nucleic acid molecules with specific recognition functions, which can be RNA or DNA, with a length generally ranging from 25 to 60 nucleotides. As a recognition molecule for oligonucleotide sequences, its target molecule range is wide, ranging from small molecules of inorganic metals to large molecules in the biological field. These ligands can form specific spatial conformations, thereby resembling antibodies in their apparent functions. They exhibit high specificity and affinity for proteins or other small molecules. Nucleic acid ligands bind to DNA polymerase through non-covalent bonds, thereby inhibiting the polymerase's polymerization reaction at non-permissive temperatures. The screening of nucleic acid ligands generally requires a short cycle, and the entire process can rely on automation, making it simple and fast. In addition, hot-start Taq DNA polymerase is also the basis for achieving one-step RT-PCR.

High-Fidelity Taq DNA Polymerase

For downstream applications such as gene screening, sequencing, mutation detection, molecular diagnostics, etc., users often require high PCR fidelity. A common criterion for fidelity is the error rate, with lower error rates indicating better fidelity. The error rate of ordinary Taq DNA polymerases is in the range of 2-5 × 10^-5 bases/cycle, while the error rate of high-fidelity Taq DNA polymerases can reach the order of 10^-6, greatly reducing the likelihood of errors. The principle is mainly because high-fidelity Taq DNA polymerases have the activity of 3'-5' exonuclease (Proofreading), which can cut off mismatched bases generated during amplification, ensuring the accuracy of amplification. Because this enzyme has proofreading activity, it often has a lower amplification efficiency, and some are prone to degrade primers. The products are generally not suitable for cloning using TA or UA cloning methods.

High-Thermostable Taq DNA Polymerase

For some templates with high denaturation temperatures and long reaction times, a high thermostable Taq DNA polymerase may be required. For example, Taq DNA polymerase isolated from deep-sea hydrothermal vents has a thermostability of more than three times that of ordinary Taq DNA polymerases.

Ultra-Long Fragment Amplification Taq DNA Polymerase

For applications such as genome mapping, sequencing, and molecular genetics research, amplification of ultra-long fragments may be required. Ultra-long fragment amplification Taq DNA polymerases have proofreading enzymes and hot-start antibodies, and can amplify 10 kb fragments from complex templates and up to 40 kb from simple templates.

The Application of Taq DNA Polymerase in POCT qPCR

Compared to conventional qPCR, the design of instruments for molecular POCT testing must meet requirements such as compactness, portability, integration of sample processing, amplification, and detection, as well as rapid detection speed. Therefore, the demands on Taq DNA polymerase are higher, requiring enzymes with faster reaction rates, greater thermostability, stability at room temperature for storage, and higher tolerance to inhibitors. For a specific molecular POCT instrument, extensive screening of Taq DNA polymerases is often necessary to find one or several suitable ones. Furthermore, specific optimization of the selected enzymes and reaction systems is required for particular detection targets. Additionally, the stability of Taq DNA polymerase and other reagents after freeze-drying or air-drying on chips also needs to be considered.

Reference

1. Laos, R.; et al. DNA Polymerases Engineered by Directed Evolution to Incorporate Nonstandard Nucleotides. Frontiers in Microbiology. 2014, 5: 565.