What Is Polymerase Chain Reaction And PCR Test?

The polymerase chain reaction, or PCR, was originally developed by Karry Mullis who won the Nobel Prize for this in 1993.PCR is a laboratory technique that is used to generate large quantities of specific DNA copies. The polymerase chain reaction produces the selective amplification of a specific type of DNA- fragment for cloning.

The polymerase chain reaction is the cell-free amplification technique that is used to synthesize multiple identical copies of any DNA of interest in the genes. It is called the rapid in vitro method of the amplification process of DNA copies. The primers are used in the whole process of PCR which helps in the process of DNA amplification. The PCR is used to amplify DNA sequences in a given reaction mixture. 

PCR  is executed to observe the selective amplification of the DNA or deoxyribonucleotide acid sequences within the organism.   For this, there should be some prior knowledge of the DNA sequences with the researcher to get the actual copies of the required DNA.

Principle Of PCR

The DNA or genes of interest is denatured by the action of restriction endonuclease which separates the two strands of the DNA. The strand is hybridized with a primer in the process called renaturation. The complex of the resultant duplex is used further for DNA sequencing. The primers used in the polymerase chain reactions should be appropriate, not too long nor too short. Here., the primers are called amplimers which facilitate the amplification process during the PCR.

The three steps of denaturation, renaturation and synthesis take place again and again to produce copies of target DNA or deoxyribonucleic acid.

The Techniques Of PCR

The following are the essentials of a polymerase chain reaction:

1. A target DNA.
2. Two primers (oligonucleotides of 17-30 nucleotides that are complementary to the target DNA)
3. 4- deoxyribonucleotides (d-ATP, d-TTP, d-GTP, d-CTP)
4. A DNA polymerase that can withstand the temperature up to 95 degrees Celsius.
5. PCR machines or thermal cyclers 
6. PCR plates
7. Reverse transcriptase enzyme
8. 30 cycles of repetitive PCR are done to acquire the desired DNA mixture. 

Working on the mechanism of PCR :

The actual mechanism of PCR involves the following steps which include the repeated cycles of amplification of target DNA.

Primer Designing

This is the most important part of the PCR. For primer designing, some sort of DNA sequences should be known from the target DNA sequences. This information is needed to design the two primers which are called amplimers. These amplimers are specific to the DNA or deoxyribonucleotide acid sequences of the target DNA.  The primers are added to the target DNA sequences so that they can bind to them.

Denaturation:

This step is known as the melting of the target DNA. In this stage of denaturation, the DNA of interest is subjected to a high-temperature range from 94 to 96 degrees Celsius, resulting in the separation of the two strands of sequences.  Every single strand of the target DNA or deoxyribonucleic acid then acts as the template for DNA synthesis.

Renaturation Or Annealing

As the temperature of the above mixture is slowly cooled to about 55 degrees Celsius, the primer's base pairs with the complementary region flanking the DNA target strands. The two oligonucleotides’ primers anneal or hybridize to each of the single-stranded DNA. This step operates at a low-temperature range of 40 to 60 degrees Celsius, depending upon the length and sequence of the primers.  This process is called renaturation or annealing. The high concentration of primers ensures annealing between each DNA strand and the primers rather than the two strands of DNA. The primers designed in the first step are used in this level of PCR.  The annealing temperature should be 5⁰ less than the calculated temperature of the PCR process.

Synthesis Or Extension Of Polymer Chains

Starting of the synthesis step occurs in the 3’-hydroxyl parts of each primer. The primers get elongated with the addition of the complementary strands or bands.  The synthesis process in PCR is quite comparable to the DNA replication of the leading strand. DNA replication happens in this process of polymerase chain reaction. 

The last step is the extension, wherein Taq DNA polymerase (thermophilic bacterium Thermes aquatics) synthesizes the DNA region between, using dNTPs (deoxynucleotides triphosphates) and magnesium ions.  The optimum temperature for the step is 72 degrees Celsius.

Like the above protocol, all the steps are repeated again and again to give multiple copies of the target DNA.

All these 4 steps make the core of the polymerase chain reaction. The PCR steps can be carried out repetitively by changing the temperature of the reaction mixture used.   The thermostability of polymerase enzymes makes this process more feasible.

What Is A PCR Test?

A PCR test is done for determining the viral infections caused by viruses, including the covid-19 virus.  The test also confers the contagious nature of the viruses. PCR test is also helpful in the prevention of the transmission of the virus and other bacterial infections. Polymerase chain reaction detects the RNA or ribonucleic acid of the coronavirus which implies the infection. 

PCR test is the series of the above steps in the right amount and duration.  The denaturation, annealing, renaturation, and synthesis are done repetitively to get the right amount of DNA or deoxyribonucleic acid. The target DNA is taken in the reaction which amplifies the DNA, to get the exact copies of DNA. Extending DNA samples is used in this process for making different copies. 

A Typical PCR Test Is Done By The Following Steps :

• The sample is taken from the nasal or swab of the infected person. The saliva can also be used in the polymerase chain reaction.
• Then, the sample is loaded in the PCR kits for the denaturation, annealing,  and renaturation reactions at the optimum temperature.  The samples are treated with primers, oligonucleotides, and taq polymerases for the reaction of PCR.
• After, the whole process is processed and analyzed for the viral DNA chunks. For the analyses process,  the PCR uses specific chemicals and a PCR machine which is known as thermal cyclers.  These cyclers are known for causing rapid copies of the DNA in one go.  In this step, a fluorescent dye can also be used to detect the presence of viral infections. 

The decision of infection is made after the whole of polymerase chain reaction cycle.  However, some antibody reactions or serological reactions can also be used for the detection of viral infections in the person.

In the light of covid-19 testing, the PCR test is called the boon.  It is a very effective test to detect the covid-19 virus infections in a population.  To make the tests in handy, PCR test kits are used by the researchers in the lab. 

The whole of process of PCR testing is rapid and can be continued within a few hours.  All this is done by thermocyclers which are programmed to execute the steps of PCR.  Moreover, they are programmed to alter the temperature for the levels of polymerase chain reaction. These cyclers are useful in the steps to allow DNA denaturing and synthesis. 

Conclusion

The polymerase chain reaction is useful in detecting viral infections in the population. PCR cycles are the schematic diagram of denaturation, annealing, and renaturation to detect the DNA copies.   For the whole process of PCR, the enzymes, primers all are useful to execute it.  PCR cycles are useful in amplifying the DNA copies.