Hot start PCR is the modification of the conventional PCR which reduces the non-specific bindings by limiting one of the reagents until the heating step of the PCR.
Non-specific binding is the major problem of any of the PCR reaction. The non-specific bindings increase the chance of false results.
Primer dimer and misprimed or false primed targets are the major reason for the non-specific bindings.
In this article, we will discuss the reason for non-specific binding and how to overcome it by using the hot start PCR.
So let’s begins,
The PCR is originally developed by Kary Mullis and coworker in the year 1989. The PCR technique is the in vitro process of replication in which multiple copies of DNA can be generated using Taq DNA polymerase in a cyclic manner.
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See the figure,
Successful amplification is the prime goal of an amplification reaction. However, non-specific binding is the major barrier in the PCR reaction.
The major topics of the present article are,
Reason for non-specific binding:
Non-specific binding is the common problem of PCR reaction. There are several reasons why non-specific bindings occur in PCR reaction.
The annealing temperature:
If the annealing temperature of the reaction is lower than its original annealing temperature, it results in non-specific bindings.
The specificity of the primer:
If the primer binds at multiple locations in the genome, that might amplify more than one fragments of the template DNA results in more than one bands.
Check the primers, whether it is specific to your target DNA or not. Read our primer design guideline: PCR primer design guidelines
The concentration of primers:
Primer concentration is another factor facilitates non-specific bindings. Here if the concentration of the primers is higher than the desired range, the primer binds other than its specific location on the template DNA and results in non-specific bindings.
Higher concentration of PCR additives:
PCR additives play a crucial role in achieving amplification for the impossible templates. However, the higher concentration of any of the additives such as MgCl2, KCl, DMSO or other facilitates non-specific bindings.
An early action of Taq DNA polymerase:
The reaction preparation time is the major factor that induces non-specific bindings. At the room temperature, the Taq DNA polymerase actively involves in the non-specific amplification.
Even if, you performing the reaction on ice, it will cause non-specific bindings.
The reason for that is the early reactivity of the Taq DNA polymerase. All the components such as dNTPs, template DNA, PCR additives and primers are there in the PCR tube, before putting it in the PCR machine, the Taq actively starts the synthesis process by adding dNTPs randomly.
due to the non-specific bindings, the yield of the original result, as well as our target amplicon, is decreased. That is why the results are not useful in downstream applications such as DNA sequencing and restriction digestion.
By designing the hot start PCR reaction we can avoid non-specific bindings.
What is a hot-start PCR?
The hot start PCR is the most advanced modification of conventional PCR in which one of the PCR reagents is activated only after heating (in PCR).
“ Hot start PCR = One of the components starts its activity under the hot condition of PCR.”
The non-specific bindings and primer dimers decrease the yield of the reaction and our DNA of interest amplifies less.
The aim of the Hot start PCR is to limit the reaction at an early stage, by limiting Taq DNA polymerase in the reaction.
There are several ways we can avoid the non-specific bindings:
1. By using the enzyme-linked antibodies.
It is a very novel way to inactivate the Taq DNA polymerase. Here, the polymerase is linked with the specific antibody which makes it busy. Hence it cannot amplify any DNA early before the reaction.
The antibody used here is temperature-sensitive, once the temperature reaches above 70°C, the antibody is degraded and destroyed, the Taq DNA polymerase is released into the reaction and activated.
After that, it synthesises template DNA.
The limitation of the antibody linked enzyme method is the cost of the reaction. The use of the antibody increases the overall cost of the reaction. Also, every time for different enzymes different types of antibody is needed.
2. Another method is preheating the PCR machine.
In this method heat the PCR machine at 95°C. Prepare all the reaction on the ice at 4°C and immediately put the tubes into the PCR machine.
This will decrease the chance of Taq activation. Also, the mispaired primers are released immediately.
3. Freezing the PCR reaction.
Another method is deep-freezing the PCR mixture. Once the dNTPs, primers, water and template are added into the reaction, immediately the reaction mixture is frozen. After that, the Taq DNA polymerase and PCR additives such as MgCl2 are added on the froze surface of the reaction.
Then the tubes are placed into the PCR machine. by doing this non-specific binding are avoided.
4. The addition of Taq DNA polymerase separately.
Once the reaction is prepared, it is placed into the PCR machine and when the temperature is achieved at 95°C, the Taq is added into the reaction tube.
However, the method is not more reliable because opening the PCR machine in between increases the chance of the cross-contamination and reaction failure.
5. Wax beads:
Another interesting method of hot start PCR is the use of wax beads. The temperature depended-wax beads create a barrier between Taq DNA polymerase and other PCR components.
The dNPTs, water, primers and template DNA are added in the bottom of the PCR tube, followed by the barrier of the wax bead.
After that, the enzyme and other reagents such as MgCl2 are added on the surface of the wax bead.
Once the temperature is achieved above 70°C, the wax bead is melted and Taq DNA polymerase is added into the reaction, immediately starts the amplification reaction.
6. High specific oligonucleotides
Below, the temperature of 50°C, the Taq DNA polymerase remains inactive in the presence of highly specific oligonucleotides. After the temperature above 50°C, the oligonucleotides are detached from the Taq and the Taq release it into the reaction.
These are some of the best methods used for the hot start PCR. Nonetheless, the best-adapted method for the hot start PCR is an enzyme-linked antibody, wax bead and specific oligonucleotide method.
In the amplification of low base pair amplification such as 80bp or 90bp, due to the early amplification (non-specific bindings), the chance of the false-positive results are very high. Because in the presence of the Taq, the primer can bind to 4 or 5 specific bases and can produce short products of same base pairs.
One of the amazing use of the hot start PCR is its use in the TB-PCR.
In the TB-PCR, the chance of the infection is always high, while we are performing the DNA extraction.
Hot start PCR helps to overcome this problem.
The sample can directly be added into the PCR tube along with all other reagents
The first step of the PCR is extended up to 10 minutes for 94°C, the bacterial cell membrane is degraded and DNA comes out from the bacterial cell.
Immediately followed by the annealing step, the available template DNA is amplified.
The method is rapid, safe and accurate.
Also, the accuracy of the results in very good. I personally used this method in our lab.
The hot start PCR technique decreases the nonspecific bindings.
Also, it prevents mis-priming and primer dimer formation.
By using the hot start Taq DNA polymerase, the reaction can even be prepared at room temperature.
It increases the yield and accuracy of the results.
The overall cost of the reaction is increased, due to the use of the antibody and the was beads.
Another disadvantage is that it can not amplify the larger DNA templates (more than 2kb).
The heating step is predominant in the hot start PCR, hence due to the higher temperature for a longer time the template DNA can damage or break down badly.
Hot Start Taq DNA polymerase:
The important player of the Hot start PCR technique is obviously, the HotstartTaq. the Hot start Taq DNA polymerase is different in comparison with the normal Taq DNA polymerase.
Commercially available all the Hot start Taq are chemically modified.
The hot start Taq DNA polymerases are either, enzyme-linked, oligonucleotide-linked or chemically modified inactive enzyme. However, the chemistry of the Hot start Taq varies from the manufacturer to the manufacturer and they never reveal it.
From the 200U stock, 2U of hot start Taq is sufficient for 30μL reaction.
The commercial kit of it contains the 10X reaction buffer with MgCl2, KCl and other additives. Use a 1X buffer.
It facilitates specificity, decreases non-specific bindings and primer-dimer formations.
The Hot-start PCR is one of the best variations of the conventional PCR method which gives best and accurate results. The overall idea for developing the hot start PCR is to improve the performance of the reaction. The technique is one of the best choices for the diagnosis of inherited disease.