3 questions to ask when choosing a PCR enzyme

With so many PCR enzymes available, choosing the right one can be challenging. The various enzymes used to amplify DNA each differ in their accuracy, speed, and specificity. The following three questions can help you sort out which factors to focus on when selecting your PCR enzymes.

Question #1: Do you need to preserve sequence accuracy?

Sometimes you only need to detect a PCR product or estimate its size, such as, when you’re genotyping mice or screening for recombinant clones. For this type of routine PCR, you should use a standard thermostable DNA polymerase (like Taq DNA polymerase) to confirm the presence or absence of target DNA.

If, however, you are performing cloning experiments or next-generation sequencing (NGS), then accuracy matters. To ensure an exact DNA copy, make sure that you are choosing a DNA polymerase that has high-fidelity. High-fidelity PCR enzymes have the ability to proofread the DNA sequence being amplified through 3'-5' exonuclease activity. When a mismatched base pair is incorporated, the DNA polymerase stalls which creates a delay in synthesis. This delay allows the mismatched nucleotide to be removed and replaced with the correct nucleotide.

An excellent choice for a DNA polymerase that preserves sequence accuracy is Thermo Scientific Phusion DNA polymerase. This enzyme features high-fidelity that is 52x more accurate than Taq DNA polymerase, with high product yields.

Question #2: Are you working with long DNA templates or looking to cut down on your cycling time?

When you’re working with long DNA templates, you need a PCR enzyme that can go the distance. In this case, choose a polymerase with a high processivity and high extension rate. Processivity represents the number of nucleotides that can be incorporated during a single binding event by the DNA polymerase. Highly processive DNA polymerases help with amplification of long templates due to the number of nucleotides that can be processed. In addition, DNA polymerases with high extension rates are able to amplify DNA in a shorter amount of time. PCR enzymes with high processivity and extension rates will ensure efficient DNA synthesis of long templates and cut down on your cycling time.

Question #3: Are you encountering non-specific amplification?

Maybe you are seeing bands on your gel that should not be there. These extra bands could be an example of non-specific amplification (see Figure 1). To help achieve specific target band amplification with high yields, choose a hot-start DNA polymerase. Hot-start DNA polymerases begin amplification only when the initial denaturation step reaches 90°C. This feature helps ensure that the PCR reaction does not initiate too soon  resulting in undesirable off-target products. This feature also helps prevent primer-dimer formation from occurring, which is particularly helpful when using multiple primers for multiplex and high-throughput PCR.

non-specific amplification (left) vs specific amplification using a hot-start DNA polymerase (right)

Figure 1. Non-specific amplification (left) vs specific amplification using a hot-start DNA polymerase (right).

How to choose the right format for your DNA polymerase

There’s more to consider. Selecting the right polymerase format can simplify your workflow. PCR enzyme format options include ready-to-use master mixes, buffers with dyes for direct gel loading, and kits that assemble all the necessary components for direct PCR.

With a master mix, you can just add your template and primers, then start your PCR. If you want to further minimize pipetting steps, use a polymerase with a buffer and loading dye that allows for direct gel loading of PCR products (note: ensure the dye is compatible with downstream applications). Direct PCR kits can also help save time by allowing you to skip purification of DNA and go straight to DNA amplification. Despite these available options, if you’re looking to optimize specific components of your reaction, stick with a stand-alone DNA polymerase.

As you evaluate the level of accuracy, speed, and specificity you need for your PCR experiments, you can confidently navigate the crowded marketplace of PCR enzymes and choose the right DNA polymerase for your molecular biology applications.