No one can refute that science is playing a major role in our daily lives. Science has seen new inventions and techniques that help us in our everyday life. No one would have agreed that we can amplify our desired segment of DNA into millions of copies prior to 1983, but science allowed that too. We may call this a gift to human being, which has been used in a wide spectrum of science, including microbiology, molecular biology and clinical microbiology field to understand, revise and to discover the mysteries of life.
Kary Mullis, an employee of Cetus Corporation a biotechnology firm located near Berkeley, California, discovered the PCR technology in 1983 and was awarded Nobel Prize during 1993. The polymerase chain reaction i.e. PCR originally used DNA polymerase from E. coli, but it was really a major concern of scientists that this polymerase couldn't withstand on higher temperature. They had to find some alternative for that. Eventually, it lead to the discovery of Taq polymerase an enzyme separated from Thermus aquaticus that lives in hot spring. This Taq polymerase is not inactivated by higher temperature.
PCR technique is nothing but cloning a particular piece of DNA in vitro. It may be in individual test tubes, Strip tubes with separate strip caps or PCR plates with markings to easily identify wells and to avoid loading mistakes and PCR plates sealing films also available to prevent the contamination during process.
PCR technique requires the following:
DNA template i.e. the desired part of DNA that has to be amplified.
Primer or oligonucleotides which are precisely complementary to the sequence at the 3' end of each strand of the DNA we wish to amplify.
DNA polymerase is an enzyme that is used for synthesis of DNA in the presence of primer.
Nucleotides. There are four types of nucleotides. i.e. adenosine triphosphate (dATP), guanosine triphosphate (dGTP), cytosine triphosphate (dCTP), and thymidine triphosphate (dTTP).
PCR involves three main steps:
1. Denaturation of DNA
The process begins with the heating of the vial which contains the DNA template i.e. the DNA which contains the target sequence. While heating the vial at 95 degree C the DNA gets separates. This process is called as denaturation of DNA.
2. Annealing of primers
After this process the temperature reduced to 50-65 degree C, so that the primers hybridize with the DNA, specifically at 3' end of the desired part. This step is called as annealing.
3. Elongation of DNA
The temperature of the vial increased to 72 degree C in the next step which allows DNA elongation. These primers then directed by Taq polymerase to synthesize the complementary sequence using the free nucleotides that has been added in the vial.
Taq polymerase can synthesize only the sequence that has the primer. So, In the whole DNA, only the targeted sequence will be copied. At the end of the first cycle we get two more copies of DNA. The cycle repeatsThe number of copies increase up to millions...Taq polymerase cannot be inactivated by higher temperature which made PCR technique easier than earlier. Deoxyribonucleotide triphosphates and magnesium an appropriate buffer also important ingredients for PCR. The concentration of enzyme and primers, the annealing time, extension time, and number of PCR cycles all were found to affect the specificity of the PCR.
This PCR can make millions of copies in a matter of hours. So this technique has been considered as a cornerstone in science and widely used in molecular biology, microbiology, genetics, diagnostics, clinical laboratories, forensic science, environmental science, hereditary studies, paternity testing, and many other applications.
Nowadays, many new types of PCR being introduced in science, like Real-Time PCR (RT-PCR)which permits the analysis of the products while the reaction is actually in progress. And Quantitative PCR (Q-PCR), as this technique is known, is used to measure the quantity of a PCR product. It is used to quantitatively measure starting amounts of DNA, cDNA or RNA.
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