PCR (Polymerase Chain Reaction)

PCR (Polymerase Chain Reaction)
3. Taq polymerase or another DNA
The polymerase chain reaction (PCR) is “a
biochemical technology in molecular
polymerase with a temperature
biology to amplify a single or a few copies
optimum at around 70 °C.
of a piece of DNA across several orders of
4. Deoxynucleoside triphosphates
magnitude, generating thousands to millions
(dNTPs, sometimes called
of copies of a particular DNA sequence.”
"deoxynucleotide triphosphates";
nucleotides containing triphosphate
After amplification, the DNA produced by
groups), the building-blocks from
PCR can be used in many different
which the DNA polymerase
laboratory procedures. PCR is also valuable
synthesizes a new DNA strand.
in a number of newly emerging laboratory
5. Buffer solution, providing a suitable
and clinical techniques, including DNA
chemical environment.
fingerprinting, detection of bacteria or
Taq polymerase was found in a bacterium,
viruses (particularly AIDS), diagnosis of
Thermus aquaticus, which was first
genetic disorders and forensic science.
discovered in the Lower Geyser Basin of
Yellowstone National Park.
PCR was developed by Kary Mullis in 1983.
At that time, he was working in Emeryville,
PCR has 6 steps.
California for Cetus Corporation, one of the
1. Initialization step: This step consists
first biotechnology companies. He was
awarded the Nobel Prize in Chemistry in
of heating the reaction to a
temperature of 94–96 °C.
2. Denaturation step: It causes DNA
The following are components and reagents
melting of the DNA template by
for a basic PCR set up.
disrupting the hydrogen bonds
between complementary bases,
1. DNA template that contains the
yielding single-stranded DNA
DNA region (target) to be amplified.
molecules. It consists of heating the
2. Two primers that are complementary
to the 3' (three prime) ends of each
reaction to 94–98 °C for 20–30
of the sense and anti-sense strand of
the DNA target.
PCR (Polymerase Chain Reaction)
3. Annealing step: The reaction
temperature is lowered to 50–65 °C
for 20–40 seconds allowing
annealing of the primers to the
single-stranded DNA template. The
polymerase binds to the primertemplate hybrid and begins DNA
4. Extension/elongation step: The
temperature at this step depends on
the DNA polymerase used; Taq
Fig 1. Diagram of PCR
polymerase has its optimum activity
temperature at 72°C. Under optimum
Now PCR is so essential in the
conditions at each extension step, the
biotechnological field that the
amount of DNA target is doubled,
biotechnological industry cannot
leading to exponential (geometric)
exist without PCR.
amplification of the specific DNA
5. Final elongation: This single step is
occasionally performed at a
temperature of 70–74 °C for 5–15
minutes after the last PCR cycle to
ensure that any remaining singlestranded DNA is fully extended.
6. Final hold: This step at 4–15 °C for
an indefinite time may be employed
Fig 2. Thermocycler of PCR
for short-term storage of the reaction.
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