Hot start PCR is a method of DNA amplification. It is a modified form of traditional Polymerase Chain Reaction method. However this technique reduces the production of non specific DNA.
It is also a convenient method to follow as it can be set up at normal room temperature and does not require special optimization in this regard.
As the potential of traditional PCR was acknowledged, scientists pivoted towards modification of the original technique in order to achieve maximum yield with enough efficacy. The results of their research led them to Hot PCR.
Mechanism of Hot Start PCR
It works on the same mechanism just like normal PCR by using DNA polymerase enzyme for synthesizing new strands of DNA from a single strand of template DNA but with a few advancements.
These modifications involve the usage of excessive heat and the methods of separation. The separation method includes alterations in Taq polymerase activity by inactivating its binding when required or adding it late into the sample. It leads to a higher yield with increased specificity.
Prevention of non Specific binding
When the reaction mixture is completed after denaturation, then it reduces the chances of non specific binding of primers, priming or the development of primer dimmers. The methods which can be adopted to complete the reaction mixture at such high temperatures include certain modifications and alterations which could prevent the activity of DNA polymerase at low temperature range.
Other than that, modification of dNTPs or adding an essential reagent after denaturation has taken place would give the required results.
Applications of Hot Start PCR
This method has many applications in medical and industrial field. The examples include the usage of PCR in Forensics where DNA fingerprinting techniques are used.
There Amplification of DNA is required to catch criminals, testing to detect parents, cloning, and detection of any mutation, genetic testing, bio-defense, and sequencing of DNA.
Reduction in Non Specific Amplification
When the temperatures are low, the amplification of DNA leads to non specific DNA production. But, Hot start PCR reduces this amplification which was initially a major problem in normal PCR method but it got eliminated by this modification.
The alterations in this method make sure that the enzymes which are present in the mixture are not active until the normal binding temperature is attained.
When the production of non specific products is inhibited in the start, the overall specificity of the PCR process is substantially increased. This feature makes it the most important component of diagnosis procedures.
Taq DNA Polymerase Inactivation
- Enzyme linked Antibodies:
The Taq DNA polymerase can be inactivated by enzyme linked antibodies. These antibodies bind thus preventing unnecessary amplification of early stages of lower temperature.
The antibodies will denature and detach from the polymerase once the annealing temperature has been attained and the enzyme gets released into the mixture. Once it enters freely, the process of amplification starts after that.
The commercially available and used Taq polymerases involve Platinum and AccuStart Taq DNA polymerase which comes with a mixture of associated monoclonal anti-bodies.
- Wax beads:
With a help of wax beads, a physical barrier is developed between polymerase and the rest of the mixture components.
These wax beads are melted away when temperature is increased and polymerase gets released into the mixture to start amplification. The wax moves up and becomes a vapor barrier.
Aptamers are high specificity oligonucleotides which bind with polymerase and only detaches when the temperature is high enough.
This method along with antibodies is the most preferred methods of inactivation however other methods also include:
Late Addition of Taq DNA Polymerase
Heating of PCR machine when the mixture is on ice and then its transfer on to the machine eliminates the need of warm up which is required prior to PCR and also reduces non specific binding.
When the components are freezed even before they can anneal together, non specific binding and amplification can be stopped. This method works like the wax beads creating a physical barrier.
- Late Addition of Taq:
The taq DNA polymerase added into the mixture when the required temperature has been reached. This method is however avoided because it can contaminate the components of mixture.
- Modifications of dNTP:
The dNTPs used in Hot start PCR can be modified by adding a protecting group at 3 prime terminal which is sensitive to heat. This helps in preventing the nucleotides from binding to eachother before the required temperature.
When the temperature is attained, these groups get detached and the amplification starts.
- Secondary structure:
When the oligonucleotides are added which are structurally like a hairpin, then they cannot function as a primer.
Their shape is changed when the annealing temperature is attained hence amplification starts when the primers anneal to the targeted portion of DNA strands.
- Photochemically removable cages:
A protecting group like caged thymidine phosphoramidities, can be introduced in the primer. Then the activation and inactivation of primers can thus be controlled using a UV radiation.
Magnesium controlled addition:
Taq polymerase requires magnesium to start the PCR because it acts as a co factor for it. When the amount of magnesium and phosphates are increased then a magnesium precipitate is formed.
That means there is no available magnesium for the enzyme to work. So, when the optimal temperature is reached, the magnesium gets dissolved back into the mixture and becomes available.
Advantages of Hot Start PCR
- It requires less handling.
- Has a reduced risk of contamination.
- It can be modified chemically or can be antibody based which provides many advantages.
- Chemically modified one can be performed at room temperature.
- Reduces non specific amplification.
- Increased specificity and sensitivity of procedure.
- Increases the overall yield of process.
- Requires less time for activation of polymerase.
Limitations of Hot Start PCR
- When heat is provided for longer periods of time, the chances of DNA template denaturation is increased.
- Heating at increased temperatures means the process is not suitable for One tube and Reverse transcriptase PCR which are performed at low temperatures.
- Due to an increased re activation time required, this can have negative effects on the PCR process.
- If the DNA template is too long, this can also affect the procedure.
- It’s a costly procedure because each enzyme has a different antibody.
Hot PCR is a modification of normal PCR which allows the amplification of required template region. It eliminates the production of non specific DNA regions hence results in an increased yield of product.
It has many advantages in diagnostic and forensic fields due to its specificity but also has some drawbacks like it’s a costly process and along with that, it requires higher exposure of heat towards DNA which could result in its damage.