3. Introduction
Polymerase chain reaction (PCR) is a method
widely used to rapidly make millions to
billions of copies of a specific DNA sample,
allowing scientists to take a very small
sample of DNA and amplify it to a large
enough amount to study in detail.
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4. CONT…
It is fundamental to much of genetic
testing including analysis of ancient
samples of DNA and identification of
infectious agents.
Using PCR, copies of very small amounts
of DNA sequences are exponentially
amplified in a series of cycles of
temperature changes.
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5. CONT…
The majority of PCR methods rely on thermal
cycling.
Thermal cycling exposes reactants to repeated
cycles of heating and cooling to permit different
temperature-dependent reactions – specifically,
DNA melting and enzyme-driven DNA replication.
PCR employs two main reagents – primers (which
are short single strand DNA fragments known as
oligonucleotides that are a complementary
sequence to the target DNA region) and a DNA
polymerase.
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6. History
PCR was invented in 1983 by the
American biochemist Kary Mullis at
Cetus Corporation.
1966, Thomas Brock discovers Thermus
Aquaticus, a thermostable bacteria in the
hot springs of Yellowstone National Park
1985, Cetus Corp. Scientists isolate
Thermostable Taq Polymerase (from
T.Aquaticus), which revolutionized PCR
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8. DNA Template
DNA containing region to be sequenced
Size of target DNA to be amplified : up to 3 Kb
Buffer.
PCR is carried out in a buffer that provides a
suitable chemical environment for activity of
DNA polymerase. The buffer pH is usually
between 8.0 and 9.5
Mg2+
In PCR, MgCl2 is an essential cofactor that enhances the
activity of Taq DNA polymerase, which in turn increases
the amplification rate of DNA.
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9. Primer
2 sets of primers
Generally 20-30 nucleotides long
Synthetically produced
complimentary to the 3’ ends of target
DNA
not complimentary to each other
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10. Enzyme
Usually Taq Polymerase or anyone of the
natural or Recombinant thermostable
polymerases
Stable at T0 up to 950 C
High processivity
Taq Pol has 5’-3’ exo only, no
proofreading
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11. dNTP
dNTP stands for deoxyribose nucleotide
triphosphate employed in PCR to expand
the growing DNA strand. The function of
dNTPs in PCR is to expand the growing
DNA strand with the help of Taq DNA
polymerase. It binds with the
complementary DNA strand by hydrogen
bonds. The PCR is an in vitro technique of
DNA synthesis
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12. PCR Cycle
Comprised of 3 steps
Denaturation of DNA at 950C
Primer hybridization ( annealing) at 40-500C
DNA synthesis ( Primer extension) at 720C
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