An American biochemist named Kary B. Mullis invented PCR in 1983. Prior to the advent of PCR, the process of amplifying, or making copies of, recombinant DNA fragments was time-consuming and labor-intensive. The polymerase chain reaction (PCR), sometimes known as "molecular photocopying," is a simple and inexpensive process for amplifying, or copying, small pieces of DNA. Because molecular and genetic analyses require large amounts of DNA, studies of isolated fragments of DNA are nearly impossible without PCR amplification. To amplify the DNA segment in PCR, the template DNA is heated for its denaturation. Taq polymerase is an enzyme that creates two new strands of DNA using the previous strands as templates. This process causes the original DNA to be duplicated, with each new molecule comprising one old and one new strand of DNA. Then, for each of these strands, two new copies can be made, and so on. Denaturing and synthesizing new DNA is repeated 30 - 40 times, or 2n times, resulting in more than one billion exact duplicates of the original DNA segment. Thermal cycler is the equipment which takes few hours to complete the entire process. The change of temperature which allows DNA denaturation and synthesis is controlled in this thermocycler. Steps involved in it are:
Denaturation: A thermal cycler is used to heat the fluid in the tube to at least 94°C (201.2°F). Double stranded DNA is split into single stranded by the heat as the hydrogen bonds between them are broken (this is termed denaturation of double-stranded DNA).
Annealing: DNA primers and polymerase enzyme are cooled between 50 and 60 degrees Celsius allowing the attachment to the different strands of DNA that were separated by the heat (this is termed annealing of the primers). The individual split strands of DNA that emerged from the heating procedure will be extended by nucleotides A, T, G and C.
Extension: During this step the nucleotides are added to the primers to make the new strand complementary to the template DNA. Each of the single strand of the original template DNA molecule is used to create a new duplicate double-stranded DNA molecule. This step is performed at the temperature 72 degrees Celsius.
Application of PCR includes:
- Gene fragment amplification as a quick alternative to cloning.
- The tinkering with DNA fragments.
- The sensitive detection of pathogenic microorganisms, followed by precise genotyping if desired.
- Arachaeological specimen DNA analysis
- Finding mutations that are linked to inherited disorders, cancer transformation, or tissue typing.
- Genetic marker analysis for forensic applications, paternity testing, and the mapping of hereditary features.
- Amplification of DNA segments between interspersed-repeat elements that is species-specific.
- Gene expression research.
At Experiome, we provide hands-on training in PCR on the gradient BioRad thermal cycler.