POLYMERASE CHAIN
REACTION
(PCR)
Introduction
Polymerase chain reaction (PCR) is an advance
molecular laboratory technique for exponential
amplif ic ation of selected DNA sequence in vitro
within a short, specified time
It is also termed as “molecular photocopying/In
vitro cloning”
It was invented by Kary B. Mullis in 1983
Principle
It is based on the principle of enzymatic
replication of the nucleic acids
3-Steps
Denaturation
When DNA double helical strand is heated at high temperature (96⁰C)
→ hydrogen bonds between the two chains are broken and the two
strands separate → unwinding / melting
Annealing
Primers get bound to specif ic complementary sequences on the
flanking region of ssDNA at specific temperature (annealing-i.e. 55ºC)
Extension
At 72⁰C - optimum temp for enzyme - Taq polymerase extends the 3´
end of primers by adding dNTPs to create new daughter strands
Equipment Required
Reagents Required
PCR/Taq Buffer
I t prov id e s the suitable c he mic al
environment and maintains the pH
The cations from the buffer also promote
primer annealing
MgCl2
I t ac ts as a po te ntial c o - fac to r and
enhances the activity of Taq polymerase
which in turn increases the DNA
amplification
dNTPs
Building block for PCR reaction
It expands the growing chain of DNA
along with polymerase
Primers
Cont.
These short fragments of single strand
b i n d t o f la n k i n g r e g i o n o f D N A
complimentary to it
Provide free 3´-OH for addition of dNTPs
Taq Polymerase
A thermostable DNA enzyme derived
from bacterium T. aquaticus
Its heat stability makes it suitable for
PC R . Thi s e nz yme synt he si ze s ne w
strands of DNA by using existing strands
as DNA template
NF Water: It maintains the f inal volume and
prevents the reaction from the attacks of
nucleases
For 25 µl Reaction Mixture
All reagents are kept at -20⁰C
1. 1X Taq Buffer 2.5µl storage
2. MgCl2 1.5 µl
Taken out from -20⁰C a few
3. dNTPs 0.5 µl
4. Forward Primer 1 µl minutes before making the
5. Reverse Primer 1 µl reaction mixture
6. Taq polymerase 0.5 Thaw at room temperature
µl Vortex before mixing
7. DNA template 1 µl Vortex after addition of each
8. NF Water 17 µl
ingredient – for even mixing
Finally centrifuge at 4000g for 30
sec (brief spin-to remove bubbles)
PCR Protocol
1 million copies of a DNA fragment are produced in 30 cycles
It takes 2-3 hours to complete the amplification
Results are checked by performing Gel Electrophoresis and seen
on Gel Documentation System
Clinical Applications
Detection of SNPs
Detection of VNTRs i.e., DNA finger printing
Paternity disputes
Criminal evidence
Detection of viral load e.g., HCV, HIV, and HBV
Prenatal diagnosis of genetic diseases (Sickle cell anemia, CF,
PKU)
Cloning genes of interest
Advantages
Small amount of DNA is required per test
Results obtained more quickly – usually within
one day for PCR
PCR is more precise in assessment of alleles –
specific genetic disorders
PCR can be used to detect point mutations
Types of PCR
Conventional PCR (Qualitative PCR)
RT-PCR (Reverse transcriptase PCR)
ARMS and Tetra - ARMS PCR
qPCR (real time / quantitative PCR)
RT- qPCR (Reverse transcriptase – real time
/ quantitative PCR)
Multiplex PCR
Nested PCR
Conventional PCR
Qualitative
Allows only detection of amplif ie d
DNA
RT-PCR (Reverse transcriptase PCR)
Qualitative
RNA templates are used → converted into
cDNA by reverse transcriptase enzyme
and then amplified
ARMS PCR AND TETRA - ARMS PCR:
AMPLIFICATION REFRACTORY MUTATION SYSTEM PCR
ALLELE SPECIFIC PCR
Qualitative
Both amplif ie s only a specif ic allele having expected
single nucleotide polymorphism
ARMS PCR – uses a two primers (one forward and one
reverse)
Tetra ARMS – uses two sets of primers (two forward
and two reverse)
qPCR (real time / quantitative PCR)
Quantitative
Allows detection and quantification of amplified DNA
Fluorescent dye is used that binds with the amplif ied
DNA
The amount of f luorescent labelled DNA is measured at
the end of each cycle and displayed on the screen. As
the DNA is amplif ie d, the amount of f lu orescence
detected also increases proportionately → DNA is
“quantified” in “real time”
RT- qPCR (Reverse transcriptase – real time /
quantitative PCR)
Quantitative
RNA templates are used → converted into
cDNA by reverse transcriptase enzyme and
then amplified
Fluorescent dye is used that binds with the
amplified DNA
Multiplex PCR
Can be Qualitative or Quantitative
Allows amplif ic ation of several different DNA sequences simultaneously by
using multiple sets of primers in a single reaction mixture
Nested PCR
It is a modif ic ation of conventional PCR with increased
sensitivity and specificity
Can be Qualitative or Quantitative
Involves two sets of primers and two successive PCR
reactions
The products of the f ir st round of amplif ic ation are
subjected to a second round of amplif ic ation using the
second set of primers
GEL ELECTROPHORESIS
Gel electrophoresis is a widely used technique in molecular
biology to separate DNA, RNA, or proteins based on their size
and charge
Principle:
The principle of gel electrophoresis is based on the movement
of charged molecules in an electric field. DNA and RNA
molecules are negatively charged due to their phosphate
backbone and will move towards the positive electrode (anode)
when an electric current is applied.
Cont.
The gel, usually made of agarose or polyacrylamide, acts as a
molecular sieve
Smaller molecules migrate faster and farther through the pores
of the gel, while larger molecules move more slowly
DNA is visualized by an intercalating dye, ethidium bromide
Procedure
Gel Preparation: Agarose powder is dissolved in a buffer solution
and heated, upon cooling to 50ºC, EtBr is added. The molten
agarose is poured into a casting tray with a comb to create wells
and allowed to solidify
Sample Preparation: DNA samples are mixed with a loading dye,
which adds color and density to the samples, making them easier
to load and track
Loading the Gel: Once solidified, the gel is placed in an
electrophoresis chamber filled with running buffer. DNA samples
and a DNA ladder (molecular weight marker) are carefully loaded
into the wells
Cont.
Running the Gel: An electric current is applied across the gel.
DNA fragments, being negatively charged, migrate towards the
positive electrode
Visualization: DNA bands are then visualized under UV light
MAKING 1% AGAROSE GEL
Agarose solution at room
40 ml 1XTBE 0.4 gm Agarose Mix temperature
buffer powder
Add 4µl of
Heat in Mix by swirling Clear Agarose Ethidium
microwave motion of hand solution bromide and Pour in Gel
mix casting tray and
let set
Ethidium Bromide
It intercalates between the nitrogenous
bases of DNA and f luoresces under UV
light
EtBr is an irritant to the skin, eyes, mouth,
and up p e r re sp i rato ry t rac t and
potentially carcinogenic
EtBr can be absorbed through skin, so it
is important to avoid any direct contact
with the chemical
Handling should be performed while
wearing lab protection
DNA Loading Dye
DNA loading dye contains a
colored dye and a density
agent
The density agent serves to
enhance the density of the
DNA sample allowing the
DNA to sink into the bottom
of the well
Purified
GEL ELECTROPHORESIS
DNA
Loading
dye
GEL ELECTROPHORESIS IMAGE
1 KB DNA LADDER
A 1 kb DNA ladder is a
molecular size marker
It contains multiple DNA
fragments of known lengths,
typically ranging from 250 base
pairs (bp) to 10,000 bp
Used as a reference to compare
and estimate the sizes of
unknown DNA samples.
Clinical Applications
Genetic Disease Diagnosis: Detects mutations,
deletions, or insertions in DNA (e.g., sickle cell anemia,
thalassemia)
DNA Fingerprinting: Used in forensic medicine and
paternity testing
Analysis of PCR products: To confirm the amplification
of target DNA sequences in PCR experiments
