Problems with non-specific bands after your PCR? Or, the PCR does not work, but you find lots of amplified primer dimers? No desire for hectic and cooling when pipetting your PCR and the following steps? Looking for reproducible results summer and winter? Then simply use a hot-start PCR.
Maybe you already know it: Even if you did not make a mistake when pipetting your PCR, the results can sometimes be frustrating and disappointing. But why? Conventional DNA polymerases are already active at room temperature - and this can lead to many "falsely amplified" templates, false-positive results and low yield.
To prevent this, you could e.g., add the crucial components for the PCR to the reaction only after you have heated the reaction mixture to at least 55°C. But this is complicated and time-consuming - not a good combination for successful and effective laboratory work. But what is the solution? Resourceful scientists had the idea in the late 1980s: they developed hot-start PCR - a technique in which the DNA polymerase is inhibited until a heat activation step occurs. This essentially suppresses enzyme activity during the initiation phase of PCR - amplification does not begin until the optimal temperature is reached.
Why is this crucial?
The DNA is amplified exponentially during the PCR cycles. If errors already occur during the first cycles, they are accumulated in each PCR cycle. Therefore, it is particularly important to avoid incorrect amplification at the beginning of the PCR. The hot-start technique can make a significant contribution here. The amplification can thus run as error-free as possible, especially in the first PCR cycles - and it is precisely these first PCR cycles that are crucial for achieving accurate and reliable results.
Over time, 3 principal inhibition techniques have become established, each with their own advantages and disadvantages. In this blog post, we will compare the most common inhibition techniques for enzyme inhibition and highlight the advantages and disadvantages of each.
What are the different methods of enzyme inhibition?
In hot-start PCR, enzyme activity can be inhibited at room temperature by various inhibitors. Upon subsequent heating during the PCR cycles, the DNA polymerase detaches from the inhibitor and begins polymerization.
Method #1: Chemical inhibition
Chemical inhibition of DNA polymerase involves covalently linking the enzyme to chemical groups to block enzyme activity at room temperature. The advantages of chemical enzyme inhibition are a low risk of contamination and high stability of the chemical inhibitors. This also leads to a consistent performance, so that usually time-consuming optimization steps of the protocol are omitted. Another advantage of chemical inhibition is the possibility of a stepwise activation of the enzyme since part of the DNA polymerase remains inactive during the first heat activation and is only finally activated during the further cycles. Enzyme is gradually "added" and thus the reaction is intensified. This can lead to a higher efficiency. While other polymerases tend to weaken during the PCR, the chemically modified DNA polymerase can also show a higher performance in later PCR cycles due to the gradual activation.
Disadvantages of this method include, above all, the need for a longer activation time. For example, it can sometimes take more than 10 minutes to release the chemical inhibitor from the DNA polymerase. During this time, your template DNA could also be damaged by the heat. Therefore, this method is usually unsuitable for fragments longer than 3kb. In addition, it is usually also not possible to completely detach the chemical inhibitor from the polymerase, so that the reaction cannot take place to its full extent.
Method #2: Antibody-modified DNA polymerases
In antibody-modified hot-start polymerases, a monoclonal antibody binds to the active site of the enzyme, blocking its activity. The hot-start denatures the antibody, detaching it from the active site of the DNA polymerase, which is thus activated. After this heat activation step, the enzyme then behaves in principle like a conventional standard Taq polymerase. Compared to chemical inhibition, this has the advantage that it is a faster process, since the denaturation step of the PCR activates the DNA polymerase completely. After this short denaturation step (1-3 minutes), the DNA polymerase is fully activated. The disadvantage of this method is the mostly animal origin of the antibodies as well as a possible contamination due to the higher proportion of antibodies in the reaction, which could play a role especially when using target DNA from mammals.
Method #3: Aptamer inhibition
In principle, aptamer inhibition of DNA polymerase works similarly to inhibition with antibodies. However, instead of antibodies, specific aptamers, i.e., oligonucleotides, bind to the active center of the enzyme until it is released again by the heat activation step. The biggest advantage of this method is the very fast activation time (usually only 30 seconds). In addition, the oligonucleotides are not of animal origin. The disadvantage, however, is that the oligonucleotides usually bind less stringently, which can lead to non-specific amplifications.
Do you also want to benefit from the advantages of hot-start PCR? Ask us and together we will find the right Hot-Start solution for you: info@genaxxon.com
