Polymerase chain reaction (PCR) is a technique developed by Kary Mullis that amplifies a specific sequence of DNA to generate multiple copies of that sequence. PCR involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Each cycle doubles the number of target sequences. Key components of PCR include primers that define the target sequence, DNA polymerase, dNTPs, and buffer solution. PCR has many applications in research, forensics, medicine, and more by allowing scientists to easily make millions of copies of a specific DNA sequence.

1. Polymerase chain reaction
By
Dr. Priti D.Diwan
Assistant Professor
Department of Zoology
J.D.Patil Sangludkar Mahavidyalay
Daryapur

2. PCR is a technique that
takes a specific sequence
of DNA of small amounts
and amplifies it to be
used for further testing
What is PCR?
PCR- Polymerase Chain Reaction

3. Kerry Mullis's PCR Idea
“lets you pick the piece of
DNA you’re interested in
and have as much of it as
you want”.

4. The history of the Polymerase Chain
Reaction
Cooperative teamwork of many researchers. A list
of some of the events before, during, and after its
development:
On April 25, 1953 James D. Watson and Francis Crick published "a
radically different structure" for DNA thereby founding the field of
molecular genetics
Starting in the mid 1950s, Arthur Kornberg began to study the
mechanism of DNA replication By 1957 he has identified the first DNA
polymerase

5. In 1969 Thomas D. Brock reported the isolation of a new species of bacterium
from a hot spring in Yellowstone National Park. Thermus aquaticus (Taq),
Taq polymerase isolated by Chien et al. in 1976
became a standard source of enzymes able to withstand higher temperatures
than those from E. Coli
In 1970 Klenow reported a modified version of DNA Polymerase I from E. coli.
Treatment with a protease removed the 'forward' nuclease activity of this
enzyme. The overall activity of the resulting Klenow fragment is therefore biased
towards the synthesis of DNA, rather than its degradation

6. PCR Targets
.
The targets in PCR are the sequences of DNA
on each end of region of interest,
which can be a complete gene or a small sequence

7. The “Reaction” Components
 Target DNA - contains the sequence to be
amplified
 Pair of Primers - oligonucleotides that define the
sequence to be amplified.
 dNTPs - deoxynucleotidetriphosphates: DNA
building blocks.
 Thermostable DNA Polymerase - enzyme that
catalyzes the reaction
 Mg++ ions - cofactor of the enzyme
 Buffer solution – maintains pH and ionic strength
of the reaction solution suitable for the activity of
the enzyme

8. PCR tube THERMOCYCLER
The Reaction

10. PCR Cycles Review
 Denaturalization: 94°- 95°C
 Primer Annealing: 55°- 65°C
 Extension of DNA: 72°
 Number of Cycles: 25-40

11. PCR Denaturing
Denaturation is the first
step in PCR, in which
the DNA strands are
separated by heating to
95°C.

12. PCR Cycles

13. PCR Primers
Primers range from 15 to 30 nucleotides, are
single-stranded, and are used for the
complementary building blocks of the target
sequence.

14. PCR Primers
A primer for each target sequence on the
end of your DNA is needed. This allows
both strands to be copied simultaneously
in both directions.

15. PCR Annealing
Annealing is the process of allowing two
sequences of DNA to form hydrogen bonds.
The annealing of the target sequences and
primers is done by cooling the DNA to 55°C.

16. PCR Cycles

17. PCR Taq DNA Polymerase
Taq stands for Thermus aquaticus, which is
a microbe found in 176°F hot springs in
Yellow Stone National Forest.

18. PCR Taq DNA Polymerase
Taq produces an enzyme called DNA
polymerase, that amplifies the DNA from the
primers by the polymerase chain reaction, in
the presence of Mg.

19. PCR Cycles

25. RT-PCR

26. Synthesis of Gene

28. LA-PCR Long accurate PCR

29. Nested Primer

30. Real Time Quantitative PCR

31. Applications of PCR
• Mutation screening
• Drug discovery
• Classification of organisms
• Genotyping
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies
• Genetic matching
• Detection of pathogens
• Pre-natal diagnosis
• DNA fingerprinting
• Gene therapy
Basic Research Applied Research