Introduction
Enzymes used in PCR
Several types of thermostable DNA polymerases are available for use in PCR, providing a choice of enzymatic properties, see table DNA polymerases used in PCR.Taq DNA polymerase
Taq DNA polymerase is the most commonly used enzyme in standard end-point PCR. It is isolated from the eubacterium Thermus aquaticus. This enzyme's robustness makes it suitable for a wide variety of PCR assays. However, since Taq DNA polymerase is active at room temperature, performing a reaction setup on ice is necessary to prevent nonspecific amplification.
Several modified versions of Taq DNA polymerase have been developed to meet different needs in downstream applications, including hot-start, single-cell, high-fidelity, and multiplex PCR. While Taq DNA polymerase and its variants generally exhibit an average error rate of 1 in 10,000 nucleotides, making them less accurate than some thermostable enzymes from the DNA polymerase family B, their versatility makes them the preferred choice for most routine PCR applications. Taq DNA polymerase is also effective in several challenging PCR scenarios when used with stringent hot-start conditions.
DNA polymerases used in PCR
| Enzyme properties | DNA polymerase family A |
DNA polymerase family B |
|---|---|---|
| Available enzymes | Taq DNA polymerase | Proofreading enzymes |
| 5'–3' exonuclease activity | + | – |
| 3'–5' exonuclease activity | – | + |
| Extension rate (nucleotides/second) |
~150 | ~25 |
| Error rate (per bp/per cycle) |
1 in 103 / 104 | 1 in 105 / 106 |
| PCR applications | Standard, hot-start, reverse transcription, real-time |
High fidelity, cloning, site-directed mutagenesis |
| A-addition | + | Sometimes |
Hot-start DNA polymerase
Hot-start DNA polymerase is a specialized form of Taq DNA polymerase designed to prevent nonspecific primer binding and the formation of primer dimers when PCR setup occurs at room temperature. In traditional PCR settings, these primer dimers can be extended during the amplification cycles, producing nonspecific products and reducing the yield of the specific target product. For more challenging PCR applications, employing hot-start PCR is essential for achieving successful and specific results.
To create hot-start DNA polymerases, the activity of Taq DNA polymerase can be inhibited at lower temperatures. This is often achieved using antibodies or, more effectively, with chemical modifiers that form covalent bonds with amino acids within the polymerase. Chemical modification results in the complete inactivation of the polymerase until the covalent bonds are broken during the initial heat activation step of PCR. In contrast, antibody-mediated hot-start procedures involve antibodies that bind to the polymerase through relatively weak non-covalent forces. This method may leave some polymerase molecules in their active state, occasionally leading to nonspecific primer extension products. These unwanted products can manifest as smearing or incorrectly sized fragments on an agarose gel, complicating the analysis of PCR results.
High-fidelity DNA polymerase
High-fidelity DNA polymerase is distinguished from standard DNA polymerases, such as Taq DNA polymerase, by its ability to provide 3' to 5' exonuclease activity, which helps remove incorrectly incorporated bases during the replication process. This feature makes high-fidelity PCR enzymes particularly well-suited for applications that require a low error rate, such as cloning, sequencing, and site-directed mutagenesis.
However, if the enzyme lacks a hot-start configuration, its exonuclease activity can lead to primer degradation during PCR setup and the initial stages of the amplification process. This degradation can cause nonspecific priming, resulting in potential smearing on a gel or even complete amplification failure, especially when the template quantity is low. It is important to note that the proofreading function of high-fidelity enzymes often results in a slower operational speed compared to other DNA polymerases. Additionally, these enzymes typically have a reduced 'A-addition' function, which is necessary for direct UA- or TA-cloning, thereby necessitating the use of blunt-end cloning methods that generally exhibit lower ligation and transformation efficiencies.