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, isolated from the eubacterium Thermus aquaticus, is the most commonly used enzyme for standard end-point PCR. The robustness of this enzyme allows its use in many different PCR assays. However, as this enzyme is active at room temperature, it is necessary to perform reaction setup on ice to avoid nonspecific amplification.

A number of modifications of the original “PCR polymerase” — Taq DNA polymerase — are now available for different downstream application needs, such as hot-start, single-cell, high-fidelity, or multiplex PCR. With an average error rate of 1 in 10,000 nucleotides, Taq DNA polymerase and its variants are less accurate than thermostable enzymes of DNA polymerase family B. However, due to its versatility, Taq DNA polymerase is still the enzyme of choice for most routine applications and when used with a stringent hot-start, is suitable for several challenging PCR applications.

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

When amplification reaction setup is performed at room temperature, primers can bind nonspecifically to each other, forming primer–dimers. During amplification cycles, primer–dimers can be extended to produce nonspecific products, which reduces specific product yield. For more challenging PCR applications, the use of hot-start PCR is crucial for successful specific results. To produce hot-start DNA polymerases, Taq DNA polymerase activity can be inhibited at lower temperatures with antibodies or, more effectively, with chemical modifiers that form covalent bonds with amino acids in the polymerase. The chemical modification leads to complete inactivation of the polymerase until the covalent bonds are broken during the initial heat activation step. In contrast, in antibody-mediated hot-start procedures, antibodies bind to the polymerase by relatively weak non-covalent forces, which leaves some polymerase molecules in their active state. This sometimes leads to nonspecific primer extension products that can be further amplified during PCR. These products appear as smearing or incorrectly sized fragments when run on an agarose gel.

High-fidelity DNA polymerase

Unlike standard DNA polymerases (such as Taq DNA polymerase), high-fidelity PCR enzymes generally provide a 3' to 5' exonuclease activity for removing incorrectly incorporated bases. High-fidelity PCR enzymes are ideally suited to applications requiring a low error rate, such as cloning, sequencing, and site-directed mutagenesis. However, if the enzyme is not provided in a hot-start version, the 3' to 5' exonuclease activity can degrade primers during PCR setup and the early stages of PCR. Nonspecific priming caused by shortened primers can result in smearing on a gel or amplification failure — especially when using low amounts of template. It should be noted that the proofreading function often causes high-fidelity enzymes to work more slowly than other DNA polymerases. In addition, the A-addition function required for direct UA- or TA-cloning is strongly reduced, resulting in the need for blunt-end cloning with lower ligation and transformation efficiency.