“In a single experiment, The best PCR amplification can be achieved by determining the optimum annealing temperature using gradient PCR.”
The gradient PCR is not actually a type of true PCR, it is a variation of the conventional PCR which facilitates the optimization of PCR reaction by determining the exact annealing temperature.
The PCR facilitates the amplification of infinite copies of the gene of our interest, called amplicons which later used in the downstream molecular application.
It is further used in inherited disease diagnosis, evolutionary studies, phylogenetic analysis and DNA barcoding. However, the main objective to perform PCR is to amplify the DNA efficiently, with high precision and with a great yield. Also, the specificity and the sensitivity matter a lot for any PCR reaction.
For achieving our objectives, we must have to optimize the reaction using the temperature variations, MgCl2, buffer and different cyclic conditions.
In this article, we will understand how we can use gradient PCR to optimize the polymerase chain reaction.
Before going into the article, we have to understand the importance of the annealing temperature in the PCR reaction.
The annealing temperature is a very crucial factor for PCR, the annealing temperature is a temperature at which the primer binds to its complementary sequence on the DNA.
While the melting temperature is a temperature at which the half of the DNA strand denatured or opened. Commonly, the annealing temperature is 5°C to 7°C lower than the melting temperature.
Still, it is just an assumption not correctly work all the time. The melting temperature can be calculated using the following formula,
Tm= 4 (G + C) + 2 (A + T)
Softwares are available to calculate the melting temperature as well as the annealing temperature for a particular PCR reaction, nowadays.
The annealing temperature of the primers between 55°C to 65°C is ideal for PCR reaction, deviation of annealing temperature above or below this range can cause non-specific bindings or reaction failure.
If the annealing temperature is too high, the primer can not bind properly to the template DNA, on the other side if the annealing temperature is too low, it facilitates more bindings, more bands and non-specific amplification during the PCR reaction.
Tips: It is better to use a touch-down PCR protocol to do the amplification which facilitates accurate amplification. Read our touch down PCR protocol: What is touchdown (TD)-PCR?
On the other side, the non-specific binding is one of the biggest problems in any of the PCR. At a lower annealing temperature, once the primer matches 3 to 4 bases, it binds to it and produced non-specific bands.
This 3 or 4 bases might be some other DNA sequences or a primer itself.
All this happened due to the lower annealing temperature. Therefore the annealing temperature is one of the biggest factors responsible for problems in the PCR.
Now, The annealing temperature is just a rough assumption based on the melting temperature of the DNA hence it might work or might not.
Suppose if we put one reaction at 58°C temperature having the melting temperature of 63°C and not getting any results than what happened?
We prepared a reaction for half an hour, done amplification for 3 hours, gel preparation and gel run for 1 hour, total ~4 to 5 hours are wasted, and still, amplification is not obtained.
Also, the reagents, chemicals and other utilities are wasted too.
We can say the entire day is ruined if a single reaction is failed and on the next day we have to perform the same experiment with another annealing temperature.
Therefore, optimising PCR reaction with the first generation traditional PCR is quite a tedious job.
But what if all the temperature can be tested in a single run? or
All the optimization can be done in a single run or using minimum steps?
Gradient PCR helps to do this.
Actually, the gradient PCR is not a modification, but it is actually a different machine.
The first generation PCR machines are conventional, simple and very basic. The grid of the machine contains uniform temperature in all wells.
Contrary, the gradient PCR machine contains different temperature gradients. Each pair of the column contains a different temperature zone. See the figure below,
We have explained different types of PCR methods very preciously. Read the articles here,
- What is in situ PCR?
- Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications
- What is colony PCR?
Now let’s understand the importance of gradient PCR,
What is gradient PCR?
“The factual determination of annealing temperature can be achieved using a series of optimization experiments in a single run by the gradient PCR machine.”
In the gradient PCR, not only the annealing temperature but other modifications such as an optimum concentration of MgCl2, buffer and primers can be decided using the gradient PCR.
“Gradient: Something increasing or decreasing”
Structurally, the gradient PCR machine contains the same parts as the conventional PCR. It has the power button, the display, upper heating lid and lower heating block.
The PCR has the power to change the temperature at a rapid speed. It switches between each step within a fraction of second.
The lower heating block of the gradient PCR machine is different than the conventional PCR. The lower heating block of it contains different heating columns.
Out of 12 columns, each pair of the column contains different heating blocks. >0.5°C and <5°C temperature change can be achieved between each pair of heating blocks.
However, the temperature of the rows remains the same as the respected columns.
Therefore, we can analyse 6 different annealing temperature for a PCR reaction in a single run and this is the power of gradient PCR.
It is better to understand the mechanism with an example.
Suppose theoretical annealing temperature of our DNA is 58°C, we have selected 6 different temperature as shown into the figure,
(The selected temperature: 57°C, 58°C, 59°C, 60°C, 60.5°C, 61°C)
Ideally or theoretically, the annealing temperature is 58°C but the best results are obtained at the temperature 60°C temperature. See the hypothetical gel image below,
Although the DNA band is observed at 58°C the bands are very dense and thick, hence there is a chance if one of the reagent concentration is changed, non-specific bads might observe at 58°C.
So practically safer and best annealing temperature for our PCR is 60°C.
Now let’s take another set of optimisation with MgCl2.
The MgCl2 is important ingredient into the PCR reaction which helps to achieve amplification even at a lower temperature.
We had covered an entire article on how MgCl2 help in optimising PCR reaction. Please read it here: Role of MgCl2 in PCR reaction
Here the temperature range is the same as the previous experiment but 4mM Mgcl2 added additionally to the reaction.
The hypothetical agarose gel electrophoresis results are shown into the figures below,
The results of the PCR reaction shows that the 60°C annealing temperature is still the best choice for our PCR reaction, lower than this temperature non- specific bindings and primer dimers are observed.
ProFlex PCR system: Innovation at its peak
The ProFlex PCR system is the product of ABI (Applied BioSystem a registered trademark of Thermo scientific). This machine is not only a gradient PCR but it is a combination of three different PCR machines in one.
In this PCR machine, we can run three different protocols anytime. Also, three of each different grid has gradient platform which facilitates even more optimisation in a single experiment.
Now lets quickly answer some of the questions regarding gradient PCR:
What is the difference between conventional PCR and gradient PCR?
The conventional PCR contains only a single heating block which distributes a single temperature in all the wells. Whereas the gradient PCR has different temperature zones in different pairs of a column. Total 6 different annealing temperature can be optimized in a gradient PCR.
When to use gradient PCR?
The gradient PCR is basically utilized for new protocols and new primer optimization. If we designed primers for our experiment, we must have to optimize it before doing the actual experiment. In those conditions, 6 different temperatures can be optimized for our new set of primers.
Furthermore, it is the unmatched choice for high CG rich template DNA.
How much time the gradient PCR consume to complete reaction?
The gradient PCR consume the same time as the conventional PCR but it can perform 6 different protocols in a single run, means 6X less time than conventional PCR.
Where the gradient PCR is applicable?
The gradient PCR is a great tool for the development of new protocols and SOPs in quick time. It is mainly utilised in molecular diagnostic and microbial genetics.
The gradient PCR is the lifeline for the tough templates such as high GC rich DNA and longer amplicon.
Read more on PCR: A Complete Guide of the Polymerase Chain Reaction
The process of amplification is a time-consuming and costly method. So it is important to reduce cost and time with high-end optimisation. The gradient PCR facilitates optimisation by saving time, reagents and consumables. The temperature-dependent optimisation fulfils the requirement of additional PCR or reagent.