PCR Essentials to Elevate Your Research
Selecting the Right PCR Enzyme for Your Research
With a wide selection of thermostable DNA polymerase choices for PCR, it can be difficult to determine which enzyme best meets your needs (Figure 1). The key to choosing the right PCR enzyme is to understand what problems can arise during PCR setup. During this time, two undesired DNA syntheses could happen while the reaction mixture sits at room temperature: mispriming of less specific sites on template sequences, and the formation of primer dimer, where the primers are extended by serving each other as templates. Even if the setup is performed on ice, Taq DNA polymerase remains active resulting in unwanted misprimed or primer-dimer products. Each of these events, occurring at a temperature below the cycling conditions, can compromise the success, yield, and/or the specificity of amplified products.
Figure 1. Workflow for PCR process
Hot Start for Optimal Synthesis
The most useful way to prevent undesired DNA synthesis and optimize PCR is via ‘hot start.’ Hot-start PCR is a technique used to reduce or prevent DNA synthesis before the reaction has been warmed to the normal DNA extension temperatures (62–72 °C) or higher. At high temperature, the stringency of primer annealing is increased and little to no mispriming occurs.
Various manual hot-start PCR methods have been developed, all with similar limitations and shortcomings. All general manual hot-start PCR methods follow the same basic protocol. One or more critical components, such as primer, enzyme, and/or dNTPs, are added to the reaction only after the reaction is heated to 60–65 °C. Besides being tedious and prone to error, this manual method is subject to cross-contamination of PCR samples.
The use of hot-start PCR enzymes provides an automated approach for optimizing PCR, which is less labor intensive and less prone to errors. Hot-start PCR enzymes are prelabeled with a blocking element, such as chemical modification, or a blocking antibody that prevents primers from bonding prematurely before the optimum annealing temperature has been met. The DNA polymerase enzyme can be maintained in an inactive state through chemical modification and is reactivated by heating in a special preincubation step. The bonds are disrupted at the higher temperatures, releasing the functional DNA polymerase. The two most abundant types of hot-start Taq DNA polymerases are chemically modified and antibody based.
Although both types of hot-start PCR enzymes will accomplish your PCR needs, there are a few considerations. Chemically modified hot-start enzymes, like AmpliTaq Gold® DNA polymerase, are best for high-throughput applications. This enzyme withstands longer periods at room temperature. However, the activation time is longer and the read lengths are shorter. In general, a chemically modified hot start would be best for target sizes of <3 kB.
Antibody-mediated hot-start enzymes, like Platinum® Taq DNA polymerase, help deliver fast activation times and minimal template degradation, which promotes the amplification of long fragments. However, antibody-mediated chemistries tend to exhibit reduced specificity and involve longer setup times. In general, an antibody-mediated hot start would be best for target sizes between 3–10 kb.
Getting the Right PCR Enzymes
Select the antibody- or chemical-mediated hot-start technology for ideal results for everyday PCR applications such as gene expression, cloning, and sequencing. In fact, many PCR analyses targeting samples with a very low copy number (i.e., those requiring many cycles (>35) or where the DNA template is denatured) could benefit from hot-start PCR. However, when a hot-start enzyme isn’t needed, you can get cost-effective results from the popular and trusted AmpliTaq® enzyme, which offers proven reliability to amplify a wide range of targets (Table 1).
Table 1. Thermostable DNA polymerase enzyme features.
|Enzyme||AmpliTaq® DNA Polymerase||AmpliTaq® Gold (<3 kb) DNA Polymerase||Platinum®Taq (3-10 kb) DNA Polymerase|
|Recommended use||Everyday standard PCR with general-purpose Taq Polymerase for cloning and sequencing applications||Hot start applications for targets, <3 kb; for short activation time ideal for HTP experiments; gene expression, genotyping, cloning, and sequencing applications||Hot start applications for targets 3–10 kb; gene expression, genotyping, cloning, and sequencing applications|
|Activation time||Immediate||10 minutes||30 seconds|