Why hot start is so hot?

Imagine you are preparing your samples for PCR on a hot summer day. You have already mixed all the necessary components together, now you just have to insert your samples into the PCR machine. Suddenly your co-worker shows up and says that they need only a few minutes of your time to discuss a very important matter. As per usual a few minutes then turns into fifteen minutes. Finally, you turn to your samples and insert them into the PCR machine. A little while later it’s time to analyze the results, but for some reason, they are messy and don’t make sense. You sigh since you have wasted a perfectly good summer day, but gotten nothing out of it. So how to make sure this doesn’t happen again?

First, let’s talk about what went wrong. Presuming that there was no contamination and all the primers and PCR mix components were working properly, there was only the issue of letting your samples sit at room temperature for fifteen minutes. During that time your primers probably randomly bound to DNA templates or to each other forming primer dimers and starting non-specific amplification, because there was nothing to stop them from doing that. In the end, you ended up with lower yield and non-specific results, since part of the PCR efficiency went to amplifying unnecessary segments [1]. 

To avoid that the easy answer is using the hot start PCR technique. With this technique, you don’t have to worry about leaving your samples on the table while talking to a colleague or worry that once you get to the last samples the first ones might already be messed up. The main idea behind a hot start is that the reaction needs a hot start to work [1][2]. No non-specific amplification will occur during the reaction setup since the specially modified DNA polymerase won’t work until heated at 95 °C in the PCR machine [1][2]. So using the hot start PCR technique means that not only can you work calmly on your samples at a room temperature, but you will also end up with higher yields, better specificity, and sensitivity [1][2].  

There are still some things to consider when using hot start since there isn't only a yes and no option. There are many different hot start variants: antibody, chemical, oligo, wax bead, etc., each with its advantages and disadvantages.

• Let's start with the antibody-mediated hot start. A monoclonal antibody is bound to the Taq DNA polymerase's active site and prevents catalysis [2][3]. By raising the temperature the antibody denatures leaving the active site free. The advantage of this is that denaturing the antibody is a rapid process (takes 1-3 minutes) and the antibody doesn't alter the polymerase [2]. The disadvantage is that antibodies can contaminate the reaction when detecting mammalian DNA targets since antibodies are mostly produced in hybridoma cells of mammalian origin. [4]. Also, antibodies are predominantly produced in vivo using animals (rabbits, mice, camelids) which sets strict rules for animal welfare and ethical questions, as well as increases the overall production cost of antibody hot start DNA polymerases.

• Chemically modified Taq polymerase has a small organic compound attached to it, which prevents its activity. The advantages of this are that there are no contamination risks and chemical inhibitors are generally more stable than antibodies, which can degrade over time. Also, chemical inhibitors are more consistent in their performance, so there is generally less need for optimization compared to, for example, antibody-based methods. In the case of chemically modified Taq polymerase, a portion of polymerase remains inhibited after the initial activation step. During cycling additional enzyme molecules are activated hence enhancing the reaction. In addition, chemical hot start is generally less expensive than antibody-based methods since the large-scale chemical synthesis of the blocking agent is significantly less expensive than the in vivo production of anti-Taq antibodies. The disadvantages of this option are that getting rid of the compound can take over ten minutes and during this time the DNA you are researching may get heat-damaged, which is why this method is not good for long targets. Also, it may be impossible to completely remove the organic compound from the polymerase and therefore it may not work at full capacity. [2] To maximize the availability of the active  enzyme, very carefully designed chemical treatment protocols are used by the manufacturers in the production of chemically modified Hot start enzymes. It is advised to follow manufacturer’s instructions to achieve the  best results in a given assay, but also test different enzymes on the market to find the best match for your needs.

• Oligo (aptamer) hot start uses oligonucleotides to block the Taq polymerase until raising the temperature will free the polymerase. In general, it works just like the antibody-mediated hot start. The advantages of this method are that oligonucleotides aren't as strong as small organic compounds in chemical hot start, so enzyme activation will only take around 30 seconds (sometimes longer incubation times are used to also enable longer target DNA molecules to denature). Using oligonucleotides is also more ethical than using antibodies. The disadvantages are that since oligonucleotides aren't very strongly attached to the enzyme, and if the conditions are not optimal there could be some nonspecific amplification taking place. [2] This could limit the application of oligo hot start enzymes for instance in one-step RT-qPCR reactions.

• Wax bead based hot start is probably the strangest technique. It has Taq polymerase inside wax spheres. Once the wax melts around the polymerase it can be used for action. The advantage of this is again that the polymerase activation process doesn't take long. The disadvantage is that using wax is messy, it's not soluble in water, and can make removing the amplified DNA difficult. [2]

For your convenience, most Solis BioDyne PCR and qPCR mixes already contain reagents required for the hot start. We use chemical and oligo hot start methods to keep our enzymes inactive during reaction setup. In addition, due to Stability TAG technology, all our enzymes and master mixes have enhanced stability at room temperature with no activity loss for up to 1 month, so you don’t have to worry about not using ice while preparing your samples. Also, be sure to check out our SolisFAST® line products that are not only thermostable but so fast that you can repeat your perfect result and still have time left to enjoy the summer.