Polymerase chain reaction (PCR) is a laboratory technique that allows scientists to

amplify a specific region of DNA in a test tube. PCR can produce millions or
billions of copies of a DNA segment from a very small amount of starting material,
such as a single cell or a drop of blood. PCR has many applications in biology,
medicine, and forensics, such as gene cloning, DNA sequencing, genetic testing,
disease diagnosis, and DNA fingerprinting.
The basic principle of PCR is to use a DNA polymerase enzyme to synthesize new
strands of DNA that are complementary to the target region. The DNA polymerase
needs a primer, a short piece of DNA that matches the beginning of the target
region, to start the synthesis. Two primers are used in each PCR reaction, one for
each strand of the target region. The primers are designed to flank the target
region, meaning that they bind to opposite ends of the region on opposite strands.
PCR is performed in a machine called a thermal cycler, which can rapidly change the
temperature of the reaction mixture. PCR consists of three main steps that are
repeated for a number of cycles, usually 25 to 40. The steps are:
- Denaturation: The reaction mixture is heated to a high temperature (usually 94 to
96 °C) to separate the two strands of the DNA template. This allows the primers to
access the target region.
- Annealing: The reaction mixture is cooled to a lower temperature (usually 50 to
65 °C) to allow the primers to bind to their complementary sequences on the
template strands. This provides the starting point for the DNA polymerase to extend
the new strands.
- Extension: The reaction mixture is heated to an optimal temperature (usually 72
°C) for the DNA polymerase to work. The DNA polymerase adds nucleotides to the 3'
end of the primers, following the rules of base pairing, and synthesizes new
strands of DNA that are identical to the target region.
After one cycle of PCR, each target region is copied once, resulting in two copies.
After two cycles, there are four copies. After three cycles, there are eight
copies, and so on. The number of copies increases exponentially with each cycle, as
the newly synthesized DNA segments also serve as templates for the next cycle. The
final product of PCR is a large amount of DNA that contains only the target region
and the primers.
The DNA polymerase used in PCR is usually derived from a heat-tolerant bacterium,
such as Thermus aquaticus, which lives in hot springs. This DNA polymerase, called
Taq polymerase, can withstand the high temperatures of the denaturation step
without losing its activity. This makes PCR more efficient and convenient, as the
DNA polymerase does not need to be added after each cycle.
PCR is a powerful and versatile technique that has revolutionized molecular
biology. PCR can be used to amplify any DNA region of interest, as long as the
sequence information is available to design the primers. PCR can also be modified
to introduce mutations, insertions, or deletions into the target region, or to
detect specific variants or pathogens in the DNA sample. PCR can also be combined
with other techniques, such as gel electrophoresis, DNA sequencing, or
hybridization, to analyze the amplified DNA in various ways. PCR has enabled many
discoveries and innovations in fields such as genetics, biotechnology, medicine,
and forensics..