Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) in simple words polymerase chain reaction or PCR is a technique that is used to make several copies of a small fragment of DNA or RNA. if understand the meaning of the words in PCR. The name itself PCR is made up of two words polymerase and chain reaction.

Polymerase means an enzyme that makes polymers of any other the molecule which in this technique is the DNA chain reaction. this is a type of reaction that progresses in an exponential way in simple terms if the first reaction produces two molecules the second reaction will make four and the third will make sixteen copies and then 32, 64, 128, and 512 copies, and so on so in a matter of just a few a hundred reactions we can synthesize billions of copies of a single fragment of Deoxyribonucleic acid.

Billion DNA copy is used for the diagnosis of different types of virus infections, crime investigation, and genetic research.

Requirement for Polymerase chain reaction (PCR)

Materials required for PCR:

• Two primers, each about 20 bases long with a sequence complementary to the sequence immediately adjacent to the DNA segment of interest.
• DNA polymerase (e.g., TAQ polymerase) can sustain high temperatures. TAC polymerase is a type of DNA polymerase and like DNA replication in any organism PCR requires a DNA polymerase enzyme that makes new strands of DNA using existing strands as templates the DNA polymerase typically used in PCR is called dark polymerase after the heat-tolerant bacteria from which was isolated named Thermos Aquaticus this material lives in hot springs and it’s DNA polymerase is very heat stable and it is most active at around 70 degrees this heat stability is ideal as high temperatures are needed for this reaction

• A large number of free deoxynucleotides (dNTPs)
• The target DNA fragment.
• Thermal cycler

Polymerase chain (PCR) Technique

The hydrogen bonds between the bases of DNA are broken by the high temperature (92 to 94 degrees Celsius), which causes the two DNA strands to separate. The priming sequences for the desired region are added, and the temperature is then dropped. This is done because all enzymes that replicate DNA need short sequences known as primers.

Primer: DNA polymerase Required primers to start the reaction as the polymerase cannot initiate this reaction but can only propagate it. PCR primers are short sequences of nucleotides usually around 20 nucleotides in length primers provide a starting point for DNA synthesis. primers are also important as they help to select the right part of DNA that will be amplified and we use such primers in each PCR reaction.

The DNA’s individual strands’ sequences and the primers anneal together. TAC polymerase then synthesizes a new DNA strand that is bound to the primers and complementary to each of the original strands by adding nucleotides.

The sample is then heated again in order to split the strands and release the primer, after which it is once again cooled to allow for more DNA duplication. The quantity of DNA strands is doubled after just a few minutes of each round. The process is repeated many more times The high temperature (92 to 94 degrees Celsius) disrupts the hydrogen bonds between the bases of DNA and results in the separation of the two DNA strands.

The primer sequences for the desired region are added, and the temperature is then decreased, as all enzymes that replicate DNA need short sequences known as primers. The primers anneal with sequences on each strand of the DNA, and Tag then uses added nucleotides to synthesize it new strand of DNA that is attached to the primers and complementary to each of the original strands.

A PCR has three main processes that are performed for 30 or 40 cycles. This is carried out using an automated cycler, which can quickly heat and cool the tubes containing the reaction mixture.

1. Denaturation at 94 °C for 1 minute:

Denaturation is the process of DNA separation into fragments. Through this process, DNA’s two strands are split apart. The double strand of DNA melts open to form single-stranded DNA during denaturation. The two DNA strands separate as a result of the hydrogen bonds between DNA bases being broken by the high temperature (94 °C).

2. Annealing at 54 °C for 45 seconds:

The process of Heating and then cooling is basically annealing. Synthetic oligonucleotide primer pairs are permitted to bind to the corresponding base sequences on the separated DNA helical. The single-stranded primer and the single-stranded template are continually forming and breaking ionic bonds. The polymerase may attach to that small portion of double-stranded DNA (template and primer) and begin duplicating the template because the more stable bonds remain a little while longer (primers that fit perfectly). The ionic link between the template and the primer becomes so strong after a few bases have been added that it no longer breaks.

3. Extension at 72 °C for 2 minutes:

The polymerase performs best at this temperature. The ionic attraction between the template and the primers, which contain a few bases, is already stronger than the forces that break these attractions. Because of the high temperature, primers that are in areas where there is no exact match become loose once more and fail to extend the fragment. When the polymerase adds dNTPs from 5′ to 3′, reading the template from 3′ to 5′ sides, bases are added that are complementary to the template. These bases are attached to the primer on the 3′ sides.

Read Also: Voltammetry and Polarography Explained

The number of copies of the gene increases exponentially during PCR because both strands are copied. Suppose there is only one copy of the wanted gene before the cycle starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, the third cycle makes 8 copies, and so on. So the number of copies produced in PCR after x cycles will be given by 2x.