What is Polymerase Chain Reaction (PCR)?

What do you mean by Polymerase Chain Reaction?

Polymerase chain reaction (PCR) is a molecular technique that is used to amplify specific segments of DNA for a wide range of molecular technology applications. This technique was developed by Kary Mullis and his coworkers in 1980s and received Nobel Prize for the same. PCR technique allows for more than the billion amplifications of the specific segment of DNA – it has become instrumental in many applications such as gene cloning, diagnosis of infectious diseases, severe genetic abnormalities, and the screening of parental infants, to name only a few. This article discusses the principles and procedures of PCR technology in detail.

Principles of Polymerase Chain Reaction

Polymerase chain reaction is a technique of quick and easy amplification of DNA sequences. The basic principle of PCR is enzymatic replication of the nucleic acids. In other words, PCR is a primer-directed enzymatic replication of a specific DNA fragment. This molecular biology technique has eased molecular biology approaches to answer fundamental research questions and introduced DNA testing in a wide variety of clinical diagnostic areas.

The need for DNA amplification came several times in molecular biology experiments. For example, suppose, we’re interested in a specific segment of DNA or a gene and we have a single copy of a gene. The gene needs to be inserted into a vector DNA to form recombinant DNA and later on – used in genetic engineering experiment to make a transgenic organism.

Is it possible to handle a single gene and perform all experiments without losing the gene? Of course, no. We need many copies of a gene so that if one gene is destroyed or couldn’t work, we can use another segment of DNA (gene) to continue our experiment. This is a real-time example of why PCR is needed in molecular biology experiments.

Steps Involved in Polymerase Chain Reaction:

Suppose, we have a DNA fragment and that contains the gene of interest. We want to amplify only the specific portion of the DNA from the DNA fragment given. There are three basic steps involved in PCR:

1. Denaturation: DNA melting i.e., heating the reaction to about 95 degree Celsius to disrupt hydrogen bonds that hold the two DNA strands together.
2. Annealing: addition of primers on DNA strand at 50 to 65 degree Celsius i.e., the reaction temperature is reduced from 95 to 60-65 degree Celsius, also known as cooling.
3. Elongation: enzyme-driven replication: the reaction temperature is further increased to 75-80 degree Celsius which optimizes the activity of DNA polymerase.

Components of PCR:

• DNA template: It contains the specific region that is to be amplified
• Primers: they are oligonucleotides – complementary to the 3’-end of each target region. Both forward and reverse primers are required – one for each complementary strand of DNA.
• Free nucleotide bases: These are A, T, G, and C
• DNA polymerase enzyme: Thermo-stable analogue of DNA polymerase I such as Taq polymerase that carries out DNA replication.

After 25 cycle, the target DNA sequence amplifies by about 10⁶ fold. The results of polymerase chain reaction are visualized using agarose-gel electrophoresis. This technique separates DNA fragments on the basis of their sizes. So, we can expect a thick band of our DNA of interest due to the formation of multiple copies of it.

Conclusion

Polymerase Chain Reaction (PCR) stands as a revolutionary technique in molecular biology, offering a swift and precise method to amplify specific DNA segments. Developed by Kary Mullis and his team in the 1980s, PCR has since become a cornerstone in various fields, including gene cloning, diagnostics, and genetic research. By facilitating the replication of nucleic acids, PCR enables researchers to generate an abundance of DNA copies from a single gene, crucial for maintaining genetic integrity in experiments. The process, involving denaturation, annealing, and elongation, relies on key components like DNA templates, primers, nucleotide bases, and DNA polymerase enzymes. With its ability to amplify DNA sequences exponentially, PCR has significantly advanced molecular biology, demonstrating its versatility and importance in modern scientific endeavors.