RT-PCR: Definition, Principle, Enzymes, Types, Steps, Uses


Polymerase chain reaction (PCR) is a technique that allows scientists to make many copies of a specific DNA segment from a small amount of DNA. This technique is useful for studying DNA sequences, identifying genetic variations, diagnosing diseases, and detecting pathogens. PCR works by using a DNA polymerase enzyme that can synthesize new DNA strands from existing ones, following the rules of base pairing. However, the DNA polymerase enzyme needs a primer, a short piece of DNA that matches the beginning of the target DNA segment, to start the synthesis. Therefore, PCR requires two primers that flank the target DNA segment on both strands. By repeatedly heating and cooling the mixture of DNA, primers, and DNA polymerase, PCR can produce millions to billions of copies of the target DNA segment in a few hours.

Reverse transcriptase polymerase chain reaction (RT-PCR) is a variation of PCR that can amplify RNA instead of DNA. RNA is a molecule that carries genetic information from DNA to proteins in many organisms. However, RNA is less stable and more prone to degradation than DNA, so it is often difficult to study RNA directly. RT-PCR solves this problem by converting RNA into complementary DNA (cDNA) using an enzyme called reverse transcriptase. Reverse transcriptase can make a DNA strand that is complementary to an RNA strand, following the rules of base pairing. The cDNA can then be used as a template for PCR amplification, using the same principles as described above. RT-PCR is useful for studying gene expression, identifying RNA viruses, and detecting mutations in RNA.

In this article, we will explain the objectives, principle, enzymes, types, steps, uses, advantages, and limitations of RT-PCR in detail. We will also compare RT-PCR with other methods of RNA analysis and provide some examples of RT-PCR applications in different fields of biology and medicine.