2. What is PCR?
The polymerase chain reaction (PCR)
is a rapid, inexpensive, popular
molecular biology technique for
enzymatically replicating DNA
(without using a living organism such
as E.coli or yeast) & produces
microgram amounts of DNA from
minute quantities of template. This
technique allows a small amount of
the DNA molecule to be amplified
many times in an exponential
manner.
3. History
PCR technique was developed in
1983 by Kary Mullis. In 1993,
Mullis was awarded the Nobel
prize in Chemistry along with
Michael Smith for his work on
PCR.
Dr. Kary Mullis stated it “lets
you pick the piece of DNA you
are interested in & have as
much of it as you want”.
4. Steps
The PCR process consists of a series
of twenty to thirty-five cycles. Each
consists of three steps:
1. Denaturation of dsDNA
2. Annealing of the primers
3. Extension of the primers
5. The double-stranded DNA has to be heated
to 94-96 °C in order to separate the
strands. This is called denaturing; it breaks
apart the hydrogen bonds that connect the
two DNA strands. Prior to the first cycle,
the DNA is often denatured for an extended
time to ensure that both the template DNA
& the primers have completely separated &
are now single-strand only.
Time 1-2 minutes up to 5 minutes.
Also Taq–polymerase is activated by this
step.
Denaturing
6. After separating the DNA strands, the
temperature is lowered so the primers
can attach themselves to the single
DNA strands. This step is called
annealing.
The temperature of this stage depends
on the primers & usually 50 °C below
their melting temperature (45-60 °C).
A wrong temperature during the
annealing step can result in primers
not binding to the template DNA at all,
or binding at random.
Time 1-2 minutes.
Annealing
7. Extension
Finally, the DNA-polymerase has to fill in
the missing strands. It starts at the
annealed primer & works its way along
the DNA strand. This step is called
extension. The extension temperature
depends on the DNA-polymerase.
The time for this step depends both on the
DNA-polymerase itself & on the length of
the DNA fragment to be amplified.
As a rule-of-thumb, 1minute per 1 kbp.
8. Stages
The PCR process can be divided into three
stages:
1. Exponential amplification: At every cycle,
the amount of product is doubled
(assuming 100% reaction efficiency). This
reaction is very sensitive, only minute
quantities of DNA need to be present.
2. Leveling off stage: The reaction slows as the
DNA polymerase loses activity & as
consumption of reagents such as dNTPs &
primers causes them to become limiting.
3. Plateau: No more product accumulates due
to exhaustion of reagents & enzymes.
9. Components
• DNA template or cDNA which contains the region
of the DNA fragment to be amplified
• Two primers which determine the beginning & end
of the region to be amplified
• Taq polymerase (DNA polymerase) extends primers
& copies the region to be amplified
• Nucleotides from which the DNA-polymerase for
new DNA
• Buffers provides a suitable chemical environment for
the DNA-polymerase
• Mg²+ is required as co-factor for the thermostable
DNA polymerase
• Gelatin & Triton X-100 stabilise the DNA
polymerase
• dNTPs provide initial excess required for
incorporation into DNA
10. PRIMERS
These are short, artificial DNA strands not
more than fifty, usually 18-25 bp
nucleotides that are complementary to the
beginning & end of the DNA fragment to be
amplified.
Both primers which used in the reaction,
should have similar annealing temperatures
with a minimal degree of
self-complementarity in order to avoid the
formation of secondary structures & no
complementarity to each other so that the
primers dimers will not formed.
11. The PCR reaction is
carried out in a
machine that heats &
cools the reaction tubes
within it, to the precise
temperature required
for each step of the
reaction, is called
Thermal Cycler.
12. The PCR product
can be identified
by its size using
agarose gel
electrophoresis.
13. Applications
✓ Diagnosis & screening of genetic diseases
& cancer
✓ Rapid detection of slowly growing
microorganisms & viruses
✓ HLA typing in transplantation
✓Analysis of DNA in archival material
✓ DNA fingerprinting in forensic science
✓ Preparation of nucleic acid probes
✓ Clone screening, mapping & sub-cloning
✓ Paternity test
14. Disadvantages
▪ requires costly instruments
▪ adequate space with aircondition,
dehumidifier, laminar flow facilities
▪ costly & not all people can afford
to do test
▪ requires trained, experienced,
qualified manpower & technologists
▪ false positive & false negative
results may lower specificity &
sensitivity
▪ limited scope for diagnosis of
diseases
15. Practical Modification
These are some practical modifications
to PCR technique:
• Nested PCR
• Inverse PCR
• Reverse Transcriptase RT-PCR
• Asymetric PCR
• Quantitative PCR
• Real time PCR
• Touchdown PCR
• Colony PCR
• Allele-specific PCR
