With the advent of thermocyclers and the accessibility of PCR reagents, running PCR in the lab has become relatively simple. Still, many times, researchers can encounter problems. Sometimes the yield is too low or the amplified product isn’t suitable for the downstream application. One possible explanation for hiccups like these is choice of DNA polymerase.
Factors to consider when choosing an enzyme
Not all DNA polymerase enzymes are created equal. Different enzymes have different strengths, so the first step to choosing which enzyme to use is to determine what your particular experimental needs are.
“The primary factors to consider when choosing a PCR enzyme are fidelity and processivity,” says Nick Hapshe, Support Scientist at Roche. “How accurate does the enzyme need to be, and can it work through difficult and complex templates? It's also important to consider the enzyme speed and the length of template that can be amplified. These different considerations can become more or less important depending on the downstream applications.”
According to Rod Pennington, Senior Research Scientist at Promega, “For most routine PCR applications, factors such as ease of use or cost may be paramount, and a standard thermostable DNA polymerase such as Taq works very well. However, for some application scenarios, basic Taq polymerase might not be your first choice. These scenarios, to mention a few, might include amplification with less-than-pristine samples, amplification of long or GC-rich amplicons, or applications where high-fidelity amplification is required. In these instances, enzyme features such as error rate, processivity, or resistance to inhibitors may be of elevated importance and a polymerase other than Taq may be optimal.”
Types of polymerases
There are a number of different types of DNA polymerases that are appropriate for different experimental scenarios. Listed here are four common classes of polymerases and what they are suited for.
Standard thermostable polymerases: Many routine applications of PCR, including identifying whether there is a product or what the product size is, do not require a specialized polymerase. Applications such as these should do fine with a less-expensive, standard thermostable enzyme like Taq.
Hot-start (HS) polymerases: As regular Taq can be active even on ice, primers sometimes anneal nonspecifically to each other and to the DNA templates during reaction setup. This allows for amplification of nonspecific products and decreased yield. Hot-start polymerases remain chemically inactivated until the temperature rises, preventing nonspecific amplification from occurring before the reaction starts. These enzymes are beneficial when the amount of DNA template is low or when there are multiple primers, as in multiplex PCR.
High-fidelity (Hi-Fi) polymerases: If the amplified DNA product is going to be used for cloning, sequencing, or mutagenesis studies, accuracy is key. Hi-Fi polymerases exhibit proofreading activity that corrects misplaced nucleotides as they’re incorporated into the growing strand, greatly improving the accuracy of amplified product.
Highly processive polymerases: Processivity of a DNA polymerase refers to how many nucleotides the polymerase is able to incorporate before it dissociates from the DNA template. If your target DNA product is long, it will be necessary to use a highly processive polymerase.
Enzyme mixes
Sometimes enzymes can be mixed in order to combine their strengths. “Enzyme mixtures may be useful where no particular polymerase, working alone, provides all the functionalities that are needed,” Pennington explains. “Good examples would include enzyme blends used for amplification of very long templates. For instance, the enzyme blend included in Promega’s GoTaq® Long PCR Master Mix contains both high-performance Taq and a quantity of a thermostable proofreading polymerase. The proofreading enzyme performs the task of repairing DNA mismatches, allowing the Taq enzyme, which is highly processive, to elongate the DNA much further and more accurately than would be possible with either enzyme alone.”
New advances in polymerases
“Fidelity requirements for downstream PCR applications are becoming more and more stringent,” Hapshe says. “This increased need for fidelity has led to some advancements in PCR enzymes, such as directed evolution.” According to Hapshe, ‘directed evolution’ is a method of protein engineering done by directing natural selection in a lab. Using this approach, scientists are able to improve the enzyme fidelity and robustness of DNA polymerases by enhancing enzyme functions like thermostability, specific activity, processivity, proofreading capability, and resistance to inhibitors.
Another avenue for advancing polymerases is to look beyond Taq. “These advances often derive from the exploitation of polymerases found in thermophilic organisms other than Thermus aquaticus, the source of Taq,” Pennington adds, “and in many cases, innovations in buffer formulation can be as beneficial as changes in the enzyme.
Optimizing the buffer
“Typically, PCR enzymes are accompanied by a reaction buffer when purchased, and you can be confident that they will work well together for all routine uses,” says Pennington. But if you’re performing a more demanding application, there are various buffer modifications that may improve enzyme performance.
“Ensuring buffer optimization starts with addressing the requirements for the enzyme and the application involved,” Hapshe explains. “Important factors to consider are pH, the mass of input DNA, and how GC- or AT-rich it is. Depending on the polymerase in question, different salt concentrations may be preferred and the base buffer may need to be changed (e.g., Tris-HCl vs KCl). Based on the different factors at play, additives such as DMSO, PEG, or various detergents may also be needed to improve performance. The MgCl2 concentration should be optimized as well to ensure proper enzyme activity.”
The takeaway
Before selecting a DNA polymerase, know your experiment and its downstream applications. Most reputable vendors will have a guide that will help you understand which of their enzymes are appropriate for various scenarios, and they’ll also have a technical support team that can help you design enzyme mixtures or optimize your buffer for more complicated applications.