Frontiers of Science丨What is the magic of high-fidelity DNA polymerase?

Background

Taq polymerase is the tried and true standard when it comes to PCR, but that doesn’t mean it’s the most accurate polymerase for high-sensitivity applications. Conversely, alternative enzymes, called high-fidelity polymerases, provide greater accuracy in PCR-generated amplicons due to a combination of more stringent base reading and proofreading activities¹.

The discovery and development of high-fidelity polymerases have been the focus of Vazyme Biotech Co., Ltd for many years. For experiments in which the success of the experimental results mainly depends on whether the DNA sequence is correct, high-fidelity PCR enzymes with high accuracy in DNA amplification play a crucial role in the experiments (e.g. cloning, mutation analysis and sequencing).

Figure 1: Cloning, mutation analysis and sequencing

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What is a high-fidelity DNA polymerase?

High-fidelity DNA polymerase contains polymerase domain and exonuclease domain². The polymerization domain has 5′-3′ polymerase activity, which is responsible for polymerization; the enzyme cleavage domain has 3′-5′ exonuclease activity (also known as proofreading activity), which is responsible for excising unpaired nucleotides.

In the process of PCR amplification, when the primer is completely complementary to the template, it enters the polymerization domain. Under the action of the polymerase activity, the free deoxynucleotides are sequentially bound to the 3′ end of the primer by the principle of complementary pairing, and the new strand DNA from 5′ to 3′ for extension (Fig. 1-a). When the nucleotide bound at the end of the primer is not paired with the template, the mismatched nucleotide will cause structural perturbation (Fig. 1-b). The new strand of DNA cannot continue to extend forward and then enter the exonuclease domain. Under the action of exonuclease activity, mismatched nucleotides are excised (Fig. 1-c). After excision, the nucleotide will be recombined correctly, and the new strand of DNA can continue to extend from 5′ to 3′ (Fig. 1-d). Ultimately, the new strand of DNA can be replicated with precision.

Figure 2：Schematic of the working principle of high-fidelity DNA polymerase

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What is the fidelity of DNA polymerases?

The fidelity of a DNA polymerase refers to its ability to accurately replicate a template³. Specifically, this involves multiple steps: DNA polymerase is able to read the template strand, select the appropriate nucleoside triphosphate and insert the correct nucleotide at the end of the 3′ primer, maintaining the standard Watson-Crick base pair⁴. The rate of misincorporation (incorporation of incorrect nucleotides) is called the “error rate” of the polymerase⁵. In addition to effectively discriminating between correct and incorrect nucleotide incorporations, some DNA polymerases have 3′-5′ exonuclease activity. This activity, also known as “proofreading” , is used to excise misincorporated single nucleotides and then replace them with the correct nucleotide. Overall, high-fidelity PCR is able to utilize high-fidelity DNA polymerases that combine low misincorporation rates with proofreading activity for reliable replication of target DNA.

Application direction of high-fidelity DNA polymerase

Gene function research

Case 1: Studies indicate that the A gene may be involved in the metabolism of anthocyanins in Arabidopsis thaliana. To test this hypothesis, the A gene was first amplified from Arabidopsis thaliana, and then constructed into an overexpression or suppression expression vector. Subsequently, it was transformed into Arabidopsis thaliana by the Agrobacterium-mediated method, so that the A gene was overexpressed or inhibited in Arabidopsis thaliana. Finally, the morphological indicators, physiological indicators and gene expression levels were used to determine whether this hypothesis was established. 

Gene Sequencing

Case 2: To explore the similarities and differences of different genes in the B protein family in Arabidopsis thaliana, it is necessary to amplify all the genes in the B protein family from Arabidopsis thaliana. Then, the base sequences of these genes were obtained. Finally, their similarities and differences are preliminarily analyzed by bioinformatics analysis.

The above cases are only the tip of the iceberg of related research, but they all require the use of PCR technology to accurately amplify the target gene from the experiment. If the amplified gene sequence is mutated, subsequent analysis will be inaccurate. Therefore, in order to ensure the accurate amplification of the target gene, it is necessary to use a high-fidelity DNA polymerase in the PCR amplification process.

Figure 3: Functional studies of Genome-Wide Association Studies⁶ 

Theatre Q&A

Q1: The amplification product of high-fidelity DNA polymerase is blunt-ended. How to add A to the end of the product for subsequent TA cloning?

A1: Reaction system: 10 µl / Reaction program: 72°C, 10 min

① Enzyme only: 1 µl 10×Taq Buffer (MgCl₂ Plus), 0.5 µl dNTP Mix (10 mM), 0.2 µl Taq DNA Polymerase, 1-7 µl purified PCR fragment, ddH₂O supplemented to 10 µl.

② Mix：5 μl 2×Taq Master Mix, 1-5 μl purified PCR fragment, ddH₂O supplemented to 10 μl.

Q2: Are the fragments amplified by the high-fidelity enzyme DNA polymerase guaranteed not to be mutated?

A2: High-fidelity DNA polymerase has 3′-5′ exonuclease activity, which reduces the probability of nucleotide mismatches during PCR amplification. The higher the fidelity of the DNA polymerase, the lower the probability of nucleotide mismatch, but it does not mean that nucleotide mismatch will never occur during the amplification process. Therefore, in order to improve the success rate of the experiment, it is recommended to pick multiple (>3) single clones from the same sample for sequencing.

Q3: Is the fidelity of high-fidelity DNA polymerases from different companies comparable?

A3: The fidelity of high-fidelity enzymes is usually expressed as dozens of times that of Taq enzymes. Common fidelity assay methods include blue-white screening assay and next-generation sequencing. Different brands of DNA polymerase have different methods for determining fidelity. To compare the fidelity of different brands of DNA polymerases, parallel comparisons must be performed using the same fidelity assay.

What can Vazyme support?

In order to help you complete your PCR experiments well and quickly, Vazyme solemnly recommends the following fast high-fidelity enzyme products.

Reference

1. Chloe. (2018, December 12). High-Fidelity DNA Polymerases & When to Use Them. Clent life science. https://www.clentlifescience.co.uk/high-fidelity-dna-polymerases-and-when-to-use-them/

2. Kim, S. W., Kim, D. U., Kim, J. K., Kang, L. W., & Cho, H. S. (2008). Crystal structure of Pfu, the high fidelity DNA polymerase from Pyrococcus furiosus. International journal of biological macromolecules, 42(4), 356-361.

3. Echols, H., & Goodman, M. F. (1991). Fidelity mechanisms in DNA replication. Annual review of biochemistry, 60(1), 477-511.

4. Kool, E. T., Morales, J. C., & Guckian, K. M. (2000). Mimicking the structure and function of DNA: insights into DNA stability and replication. Angewandte Chemie International Edition, 39(6), 990-1009.

5. Hestand, M. S., Van Houdt, J., Cristofoli, F., & Vermeesch, J. R. (2016). Polymerase specific error rates and profiles identified by single molecule sequencing. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 784, 39-45.

6. Lichou, F., & Trynka, G. (2020). Functional studies of GWAS variants are gaining momentum. Nature communications, 11(1), 1-4.