The three phases of a PCR

Finally it is time to bake the cake; we take the test tube and insert it in the thermocycler. What happens next? The baking consists of subjecting the test tube to a certain number of thermal variation cycles. Each thermal cycle is made up of three phases. Periods of heating are alternated with periods of cooling.
1st phase: denaturation
In this first phase of a PCR reaction the total separation of the two DNA filaments of the target sample that has to be replicated takes place. Usually the temperature applied to the test tube must reach 94°C for 30-60 seconds. By warming the DNA the unwinding of the double helix and the separation of the two filaments takes place; it is said that the DNA is denatured.
2nd phase: hybridization of primers
This is the most delicate moment in which the primers have to ‘stably hybridize’ to the DNA template. What does this mean? It means that the primers that have been added to the mixture have to bind to the single separated strands of the original DNA target sequence. Usually the procedure involves various experimental attempts in order to find the ideal temperature that promotes the binding of the primer strands to the target strands according to the type of DNA sample that has to be replicated. Usually the temperature is lowered to about 30 – 55 °C.
3rd phase: the extension of the new DNA strand
The ideal temperature for polymerization, i.e. the construction of DNA filaments identical to the DNA target depends on the DNA polymerase enzyme utilized. At present an enzyme called Taq polymerase is used and a temperature of 65 – 72°C is applied for 5 minutes. In this phase the polymerase enzyme physically attaches the building blocks of the filament (the nucleotides) to the primers that are coupled and complementary to the target filament. In practise, the copying of each single target filament takes place. DNA polymerase takes care of correcting any mistakes in the event that a ‘wrong’ nucleotide is incorporated, an eventuality that can take place since the mechanism is never perfect. Thus the DNA polymerase is capable of eliminating an error and creating a correct pairing with the corresponding nucleotide present on the filament that is being copied and replicated.
During the first cycle of the PCR reaction new filaments are synthesized which, after denaturation i.e. the division of the double helix and separation of the parallel strands, can bind themselves to the primers. These products accumulate arithmetically but it is only from the second cycle on that two single helix products are formed that will make up a fragment identical to the double helix target DNA.
Thus with repeated denaturation cycles, hybridization of primers and their extension an exponential increase of the copies of the target DNA segment is obtained.
During each ‘cooking’ cycle the number of fragments of DNA that have to be copied (i.e. those present between two primers) doubles. After just 32 repeated cycles, millions of copies of double helix DNA fragments identical to the target are already formed.
At this point the cake is ready and we can take it out of the oven. It will have risen and cooked and will be ready to be tasted.  In the same way the test tube removed from the thermocycler at the end of all the thermal cycles of ‘cooking’ and cooling to which it has been subjected, is ready, the PCR is over. The numerous copies of DNA, identical to those of the original sequence we wanted to replicate, are available to be subsequently analysed and utilized in different ways according to the requirements of the experiment.

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