POLYMERASE
CHAIN REACTION
(PCR)
Prepared by: M. Mohsin Ali Dynamo
Introduction
 You might have often strolled by clinics during your lounging
out, and seen a small statement “PCR Lab” written on flex
signs and boards. PCR is a technique to amplify the genetic
information. In simpler terms, PCR is a method used to
obtain large amounts of DNA in a laboratory test tube—large
enough that analysis can be performed easily.
 PCR was developed in 1983 by Kary Banks Mullis, an
American Biochemist who won the Nobel Prize in Chemistry
for it. You might be interested to note that while he got a
Nobel prize in Chemistry, the PCR application is used mostly
in biochemical analysis studied in biology.
 PCR is entirely in vitro, i.e. it is performed in a completely
artificial environment. PCR analysis is carried out in a PCR
machine, formally called a thermocycler. We will get on to
discuss the physics behind a thermocycler, but later.
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What you should
know about PCR
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Specificity: PCR is very specific.
The starting material for PCR
amplification does not need to be
purified DNA [DNA Purification is
the extraction of DNA from cell
debris, other nuclei acids and
soluble proteins]. Only minute
amounts of DNA are needed to
carry out PCR analysis, and this
DNA can be in a partially degraded
state as well.
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Purpose: You might already know a bit
about gene fingerprinting and genomic
libraries. In a genomic library, there are
clones of cells, each with a particular gene.
To find a gene, we simply use a radioactive
or a fluorescent tracer and off we go.
Now, PCR can be used to amplify a gene
prior to cloning. As a result, that gene
becomes our most abundant DNA
fragment, and it is easy for a tracer to spot
it and the clone containing that gene.
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Note: Traditional cloning is still used whenever a large quantity of
gene or protein product is needed.
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Primers:
These are
sequences of 20 bases
complementary to bases on either
side of the target DNA. In PCR, we
use one forward and one reverse
primer. Primers are used because
DNA polymerase (an enzyme
discussed below) cannot start a
nucleotide chain; it can only add
nucleotides to an already growing
chain.
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Taq Polymerase:
This is type of DNA polymerase
enzyme, extracted from the
thermophilic bacterium Thermus
aquaticus. This bacterium inhabits
hot springs, and the DNA
polymerase extracted from it is
thermostable (it can exist in its
natural state even at high
temperature. Other enzymes get
denatured at high temperature).
Ordinary DNA polymerase III
denatures above 37°C, so it is not
practical or efficient to use. See
figure to visualize Thermus aquaticus.
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Denaturation:
The DNA is briefly heated at 94-96◦C
to separate its strands.
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Primer Annealing:
Annealing means: “Hardening
something by heat treatment.” In
this step, the DNA is cooled to 5060◦C to allow the primers to bind by
Hydrogen bonding to the ends of the
target sequence, one primer on each
strand. (Since DNA polymerase adds
nucleotides from the 5’ to the 3’ end,
therefore, technically, a forward
primer and a reverse primer are
used).
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Primer
Extension:
At about
72◦C, DNA Taq Polymerase adds
nucleotides to the 3’ end of the
primer using longer DNA strands as
templates.
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The first cycle produces
variable-length strands.
After primer annealing in
the second cycle of PCR,
constant-length strands
are produced.
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The doubling of the
targeted DNA
sequence takes a
mere 5 minutes.
After about 20
cycles, all product
DNA molecules will
consist of targeted
sequences.
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To amplify molecules or sequences of
RNA, it is initially converted to DNA using
the enzyme reverse transcriptase. Then
the converted DNA is amplified using the
standard PCR procedure. This is called RT
PCR.
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PCR has been used to amplify DNA from:
1. 40,000 years old frozen wooly
mammoth.
2. Blood, tissue or semen for forensic
analysis.
3. Single embryonic cell for rapid
antenatal (before birth) diagnosis of
genetic disorders.
4. Viral genes from cells infected with HIV.
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