Enzymes for PCR — Abundant Choices

by Liz Horton, Canon BioMedical on September 10, 2018

When thinking about the enzyme used in standard end-point PCR, typically Taq DNA polymerase, isolated from Thermus aquaticus, comes to mind. However, there are many options. There are enzymes isolated from other thermostable organisms such as Thermococcus zilligii, Pyrococcus furiosus, and Thermus filiformis. There are enzymes that have been modified, enhanced, and/or optimized with the buffer system used, for various characteristics suited for specific applications. Choosing the best enzyme depends on the properties that are most beneficial for the specific PCR application. Here are some of the many properties of commercially available PCR enzymes to be considered.

Hot Start

There are polymerases that enable hot start, meaning the enzyme is inactive until it is activated via incubation at a high temperature, generally the same as the initial denaturation temperature. This is beneficial to prevent primer dimer formation or mispriming. This has been accomplished using antibodies or aptamers that bind to the polymerase rendering it inactive until dissociated or through recombinant protein design to achieve the same effect.

Proofreading

There are polymerases that are chosen for or modified to have proofreading abilities or lack thereof. An enzyme with 3'-5' exonuclease activity, which is considered proofreading activity, can help ensure accurate synthesis. However, in some applications, lacking 3'-5' exonuclease activity is a necessity. For example, TA-cloning takes advantage of the terminal transferase activity to create 3'-A overhangs which would be removed by an enzyme with 3'-5' exonuclease activity.

Fidelity

A thermostable polymerase isolated from Pyrococcus furiosus called Pfu was shown to have higher fidelity than Taq resulting in fewer errors of nucleotide incorporation during amplification. There are commercially available versions of Pfu as well as mixtures of Taq and Pfu to take advantage of this characteristic.  There can be a trade-off with other advantages which is why sometimes a mixture results in a balance of these advantages.

Processivity

Processivity is the characteristic of a polymerase to get on and stay on. Polymerases typically will have a level of binding then dissociating. Those with high processivity will add a high number of nucleotides before dissociating. Enzymes with high processivity tend to be better at amplifying longer fragments.

Other Considerations

Some other characteristics to consider may be the active temperature of the enzyme since tough sequences such as G/C-rich stretches may do better at a higher extension temperature to prevent the formation of secondary structure.  Resistance to inhibitors may be important depending on the source of the DNA template. Speed may be important and some enzymes have modifications or enhancers added to maximize speed. 


The good news is there are many commercial choices so a scientist can find the right fit for their application. Whether amplifying a long fragment, troubleshooting a tough target, maximizing the speed of amplification, requiring the highest fidelity for sequencing, needing to minimize the potential background from contaminating DNA from the polymerase source, or simply to fit the budget, there are enzymes designed to help.

The NEXTGENPCR thermocycler has no restriction on the choice of polymerase used. You can choose the enzyme best for your application and still gain speed by not having a ramp rate. The choice is yours. Interested in seeing NEXTGENPCR for yourself? Request a demo.