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POLYMERASE CHAIN REACTION (PCR):
PCR, discovered by Kary Mullis in 1985, is an efficient and cost effective molecular tool to
copy or amplify small segments of DNA or RNA
...
Mullis for his invention of the polymerase chain reaction (PCR)
...
It combines principles of complementary
nucleic acid hybridisation with those of nucleic acid replication that are applied repeatedly
through numerous cycles
...
The thermocycler raises and lowers the temperature of the samples in
preprgrammed steps, allowing for denaturation and reannealing of samples with various
reagents
...
It is sensitive and not affected by the quality of DNA like other
techniques such as RFLP (Restriction fragment length polymorphism)
...
During replication, the DNA
molecule unwinds from double stranded DNA to single stranded, and the single stranded DNA
becoming a template for synthesis of a new, complementary strand
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In vivo replication
Place

where

Feature in PCR

DNA

polymerase attaches to DNA

RNA:DNA

DNA:DNA

strand
Separation of strands of

Helicase

Heat

DNA
Enzyme that elongates new
DNA strands
Primers are made up of

DNA polymerase

Taq polymerase

(Thermolabile)

(Thermostable)

RNA

DNA

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Steps of PCR:
•

Denaturation: PCR begins with the process of denaturation of a double stranded DNA sample
molecule
...


•

Annealing: In these two synthetic oligonucleotides which are complementary to the target
DNA segment are added to the DNA which has been denatured in the first step
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It is noted that the synthetic
oligonucleotides which are added to the target DNA should be in high concentration, so as to
ensure that they hybridize with the complementary sequences in the DNA sample
...


•

Extension: The annealed oligonucleotide along with dNTPs and Taq polymerase helps in the
chain elongation process
...
Taq polymerase can
withstand high temperature ranges, as in PCR it can remain active even after being heated
to95◦C and extends the primers which are at temperatures upto 72C
...
After this the temperature of the reaction
is lowered again and another cycle of synthesis of DNA duplex takes place as primer is still
present in excess in the mixture
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In PCR after each cycle, DNA doubles
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DENATURATION

EXTENSION

ANNEALING

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COMPONENTS OF PCR:
• Primers: A primer is a short synthetic oligonucleotide designed to have a
sequence which is the reverse complement of the target region of the
template DNA
...

➢ Inverted repeat sequences should be avoided so as to prevent the
formation of secondary structure in the primer, which may disrupt
the template hybridisation
...

• Taq DNA polymerase: Taq DNA polymerase is a thermostable DNA
polymerase enzyme which can survive high temperature ranges
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➢ Generally equimolar concentrations of dNTPs are used i
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➢ Tris-HCl buffer is generally used for PCR as it maintains the pH
between 8
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8 at room temperature and at 72C pH is 7
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➢ This reaction mixture of dNTPs and Buffer contains divalent cations
Mg2+ which forms a soluble complex with dNTPs which is essential for
the incorporation of dNTP
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TYPES OF PCR:
Need of different types of PCR:
•

To reduce contamination

•

In PCR we need to monitor in real time

•

To know how to perform PCR on unknown flanking sequence

•

To perform PCR on RNA sample

1
...
Amplification and simultaneous quantification of target DNA is
done in the same PCR machine using commercially available fluorescence detecting
thermocyclers
...


Working:

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Detector

Parent
DNA

DNA
polymerase

REAL - TIME PCR

Primer

dNTPs

•

Parent DNA is the DNA sample which is to be amplified
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•

DNA polymerase (heat resistant), generally Taq polymerase is used
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Sample preparation

PCR mixture is prepared

Either fluorophore or Taq man probe is added to the PCR mixture

Reaction started

Excitation of fluorescent dye by laser or tungsten lamp

Amplification proceeds

Fluorescent signal is captured after every cycle i
...
in real time

A graph is plotted by the software with cycle number vs
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➢ SYBER green is green fluorophore dye
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➢ It is very sensitive to double stranded DNA
...
When it binds with the minor groove
of ds-DNA, it becomes fluorescent
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Double stranded DNA in PCR binds to DNA binding dye (SYBR green) which leads to
Fluorescence

Intensity of fluorescence at each cycle increases as DNA product increases

Dye binds to all the PCR products including non specific PCR products
Advantages:
➢ Cheap and easy to use
...

Disadvantages:
➢ Non specific in activity
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•

Taq Man Probes: It recognises specific DNA sequence of interest and probe binds to the
strand
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This method depends on a DNA based probe with a fluorescent reporter at one end and
quencher at opposite end
...

In Taq Man, when the reporter and the quencher are connected to each other, the quencher
reduces the fluorescent signal of the reporter dye as it absorbs the energy via a mechanism
known as fluorescence resonance energy transfer (FRET)
...

Structure of a Taq Man probe:
F

Q

Quencher (Q) is inhibitor of fluorophore (F) as it absorbs light emitted by fluorophore as it
absorbs light emitted by fluorophore
...
The fluorophore is
thereby released from its molecular attachment to the quencher and fluoresces
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•

It is used to carry out RT-PCR i
...
Real time PCR with many genes
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•

It is quite expensive
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Application of Real Time PCR:

•

It is used for the calculation of starting template concentration
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•

Measuring m-RNA expression
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•

It has ability to process multiple samples simultaneously
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2
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Principle: In this, reverse transcription and PCR amplification can be performed as a twostep process in a single tube or even with two separate reactions
...
The primer used for cDNA synthesis can be either nonsequence specific primers (a mixture of random hexamers or oligo-dT primers) or sequence
specific primers
...


•

Oligo-dT primers are the complementary sequences to the poly-A tail of mRNA molecules and
allow the synthesis of cDNA only from mRNA molecules
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•

They are designed to mRNA molecules of interest which makes reverse transcription a target
specific process
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But in the process of reverse transcription, cDNA is synthesized from mRNA using
Reverse transcriptase enzyme
...
e
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Reverse transcriptase enzyme:
Reverse transcriptase is an RNA- dependent DNA polymerase enzyme discovered, by Satoshi
Mizutani and David Baltimore in 1970, in many retroviruses such as HIV and Avian
myeloblastosis virus (AMV)
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It possesses
three enzymatic activities:
•

RNA-dependent DNA polymerase: It synthesizes a DNA strand complementary to the RNA
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•

DNA-dependent DNA polymerase: It completes double stranded DNA synthesis
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But this high error rate of viral reverse transcriptase enzyme provides
selective advantage for their survival in the host system
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Reverse
transcriptase use an RNA template and a short primer complementary to 3’end of RNA to direct
the synthesis of first strand of cDNA, which can be used directly as a template for the PCR
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ONE STEP RT-PCR:

mRNA
Reverse transcription

First strand cDNA

PCR cycle 1

PCR cycle 2

AMPLIFICATION

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ii
...


•

Many clinically important viruses have their genomes composed of RNA, RT-PCR is quite
useful for detecting such viruses like:

➢ Dengue virus
➢ Severe acute respiratory syndrome (SARS)
➢ Human meta-pneumonia virus
➢ Recently it has been used for the detection of Novel Corona Virus in humans
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Multiplex PCR:
This technology was first used by Chamberlain et al for diagnosis of Duchenne muscular
dystrophy
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While designing amplification primers for multiplex PCR, several factors must be considered
such as:
•

Primer length: As the technique involves the use of a large numbers of primers, hence it is
required that the designed primer should be of accurate length
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•

Melting temperature (Tm): Primers which have similar Tm are used
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The sequences which have high GC (Guanine-Cytosine) content, primer with high Tm
(75◦C – 80◦C) are preferred
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•

Avoiding primer dimer formation: It should be checked that designed primer should not
form primer dimer in the reaction mixture as it leads to reduced yield and non specific
amplification
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•

Regions with repetitive sequences, called germline, SNPs and regions with high homology
should be avoided as it may affect the efficiency of PCR amplification and create amplification
bias
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False negatives are often revealed in the multiplex PCR
because each amplicon provides an internal control for other amplified fragments
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•

Indication of template quality: The quality of template may be dteremined more effectively
in multiplex PCR
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Nested PCR:
In this PCR type, two sets of primers are used against same target and two successive PCR
reactions occur for the same
...

•

The first set of primer is used for annealing the sequence upstream from the second set of the
primers
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•

The second set of primer is nested with respect to the first set of the primer
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The traditional approach to nested PCR to perform a number of PCR cycles using first set of
primers and then open the reaction vessel and add the second, nested set of primers to run the
second PCR cycle
...
To address this issue, single-tube nested PCR (STN PCR) reactions have
been developed wherein both sets of primers are added to the initial reaction vessel and an
extended PCR is performed
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•

Less chances of contamination
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•

Detection of Herpes virus and entero virus in CSF
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tuberculosis in sputum sample
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Inverse PCR:
Inverse PCR was describes by Ochman et
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This involves series of DNA digestions
and self ligation process resulting in known sequences at other end of the unknown sequence
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In this primers are arranged in reverse direction of usual orientation
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It is mostly used for the
identification of sequences flanking transposable elements and in the process of mutagenesis
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•

It cannot be used in the routine genetic diagnostic labs
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The cost of the overall process is quite high compared to conventional PCR
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•

It is useful in identification of unknown mutations such as gene rearrangements, gene fusion,
and oncogenic gene arrangement on a chromosome
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•

The most important application of the inverse PCR is in the site-directed mutagenesis
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Colony PCR:
This technique is specifically used at the time we perform cloning and we need to grow the
host cells in agar plates (as if we are growing libraries in the agar plates) and we obtain a
bacterial colony from it, for the amplification of that colony, we use this technique Colony
PCR
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Principle:
The bacterial colony which has been cloned (i
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the bacterial colony containing the plasmid)
can directly be amplified using two sets of primers
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The vector specific flanking primers: They are used for the amplification of the plasmid DNA
other than the inserted DNA
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Additionally, by adding one initial heating step to the PCR, the plasmid DNA comes
out from the bacterial cell and amplified in the reaction
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Uses of colony PCR:

•

It is used to get information about the presence or absence of the insert only
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•

To get information about the orientation f the insert
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•

Higher accuracy and specificity
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•

Laborious steps are not required
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It is time effective technique
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•

The sequence information cannot be obtained by the colony PCR
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•

The chance of false positive results is high
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•

It is used for the assay of number of colonies simultaneously and there is no need of storing
large number of transformed clones for long period
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...
In this the DNA
polymerase has some residual activities, which results in primer dimer formation and non
specific primer annealing
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In this technique we withheld the key components of PCR reaction like MgCl2 and DNA
polymerase from the reaction mixture until specific temperature is reached
...
Then
it is subjected to PCR and when the temperature reaches about 65◦C to 70◦C, we manually add
the MgCl2 and DNA polymerase to the reaction tube
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The limitation of this technique is that while we are manually adding MgCl 2 and DNA
polymerase, the chances of contamination are quite high
...

•

Physical barrier hostart PCR: In this physical barrier hotsart PCR, DNA polymerase and
MgCl2 are separated from the main reaction mixture by the layer of wax
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So this technique does not involve any manual working leading to less chances of
contamination
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•

Hotsart PCR using monoclonal antibody coupled with DNA polymerase: This is the best
method of hotsart PCR
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APPLICATIONS:
•

In clinical diagnosis: As PCR is highly specific and sensitive in nature, it is highly useful in
the diagnosis of various diseases in humans including viral diseases, genetic diseases, bacterial
diseases etc
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We can diagnose the normal, carrier or diseased stage of
sickle cell anaemia using PCR
...

•

Basic biomedical research: It is very useful technique in biomedical research where it is used
for the amplification of specific DNA for downstream applications such as cloning and
expression of recombinant proteins
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•

Evolutionary studies: DNA extracted from fossils can be amplified using PCR
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Forensic sciences: PCR is helpful in investigations related to crime purposes
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