Polymerase Chain Reaction

NAME - HArsH yAdAV
ClAss - 12 A
TH
AdM NO - 471
TO - GUNJAN NIGAM
Polymerage
TOPIC -
chain reaction
pOlyMErAsE CHAiN
The polymerase chain reaction (PCR) is a

method widely used to make millions to
billions of copies of a
specific DNA sample rapidly, allowing
scientists to amplify a very small sample
of DNA (or a part of it) sufficiently to
enable detailed study.
PCR was invented in 1983 by
American biochemist Kary Mullis at Cetus
Corporation. Mullis and
biochemist Michael Smith, who had
developed other essential ways of
manipulating DNA, were jointly awarded
the Nobel Prize in Chemistry in 1993.
# priNCipAl Of (pCr)
The PCR technique is based on the enzymatic

replication of DNA. In PCR, a short segment
of DNA is amplified using primer mediated
enzymes. DNA Polymerase synthesises new
strands of DNA complementary to the template
DNA. The DNA polymerase can add a nucleotide
to the pre-existing 3’-OH group only. Therefore,
a primer is required. Thus, more nucleotides are
added to the 3’ prime end of the DNA
polymerase.
#Components of PCR
Components Of PCR constitutes the following:
1. DNA Template– The DNA
2. of interest from the sample.
3. DNA Polymerase– Taq Polymerase is used.
It is thermostable and does not denature at
very high temperatures.
4. Oligonucleotide Primers- These are the
short stretches of single-stranded DNA
complementary to the 3’ ends of sense and
anti-sense strands.
5. Deoxyribonucleotide triphosphate– These
provide energy for polymerization and are
the building blocks for the synthesis of DNA.
These are single units of bases.
6. Buffer System– Magnesium and
Potassium provide optimum conditions for
DNA denaturation and renaturation. It is
also important for fidelity, polymerase
activity, and stability.
#Types of PCR
PCR is of the following types:
Real-time PCR
In this type, the DNA amplification is detected in
real-time with the help of a fluorescent
reporter. The signal strength of the fluorescent
reporter is directly proportional to the number
of amplified DNA molecules.
Nested PCR
This was designed to improve sensitivity and
specificity. They reduce the non-specific binding
of products due to the amplification of
unexpected primer binding sites.
Multiplex PCR
This is used for the amplification of multiple
targets in a single PCR experiment. It amplifies
many different DNA sequences simultaneously.
Quantitative PCR
It uses the DNA amplification linearity to detect,
characterize and quantify a known sequence in
a sample.
Arbitrary Primed PCR
It is a DNA fingerprinting technique based on
PCR. It uses primers the DNA sequence of which
is chosen arbitrarily.
#Applications of PCR
The following are the applications of PCR :
Medicine
• Testing of genetic disease mutations.
• Monitoring the gene in gene therapy.
• Detecting disease-causing genes in the
parents.
Forensic Science
• Used as a tool in genetic fingerprinting.
• Identifying the criminal from millions of
people.
• Paternity tests
Research and Genetics
• Compare the genome of two organisms in
genomic studies.
• In the phylogenetic analysis of DNA from
any source such as fossils.
• Analysis of gene expression.
• Gene Mapping
#PCR Steps
The PCR involves three major cyclic reactions:
Denaturation
Denaturation occurs when the reaction mixture
is heated to 94℃ for about 0.5 to 2 minutes. This
breaks the hydrogen bonds between the two
strands of DNA and converts it into a single-
stranded DNA.
The single strands now act as a template for the
production of new strands of DNA. The
temperature should be provided for a longer
time to ensure the separation of the two
strands.
Annealing
The reaction temperature is lowered to 54-60℃
for around 20-40 seconds. Here, the primers
bind to their complementary sequences on the
template DNA.
Primers are single-strand sequences of DNA or
RNA around 20 to 30 bases in length.
They serve as the starting point for the
synthesis of DNA.
The two separated strands run in the opposite
direction and consequently there are two
primers- a forward primer and a reverse
primer.
Elongation
At this step, the temperature is raised to 72-
80℃. The bases are added to the 3’ end of the
primer by the Taq polymerase enzyme.
This elongates the DNA in the 5’ to 3’ direction.
The DNA polymerase adds about 1000bp/minute
under optimum conditions.
Taq Polymerase can tolerate very high
temperatures. It attaches to the primer and
adds DNA bases to the single strand. As a
result, a double-stranded DNA molecule is
obtained.
These three steps are repeated 20-40 times in
order to obtain a number of sequences of DNA
of interest in a very short time period.

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NAME - HArsH yAdAV

The polymerase chain reaction (PCR) is a

The PCR technique is based on the enzymatic

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