Physical and chemical component principle and process of PCR its type and applications in aquaculture along with major advantages and disadvantage

1. Polymerase Chain
Reaction (PCR)
Dr. RITU SHARMA
Senior Technical Officer,
AAH&QTL-NFDB, Hyderabad 500052

2. A Brief History of PCR
1983: Dr. Kary Mullis developed PCR
1985: First publication of PCR by Cetus Corporation appears in
Science
1986: Purified Taq polymerase is first used in PCR
1988: PerkinElmer introduces the automated thermal cycler
1989: Science declares Taq polymerase “molecule of the year”
1993: Kary Mullis won Nobel Prize

3. Equipments
❖ PCR is a technique used in the lab
to make millions of copies of a
particular section of DNA
❖ Known as a Thermocycler, PCR
Machine or DNA Amplifier
❖ Rapid heating and cooling of the
samples
❖ These instruments contain
specialized 96-well thermal blocks
to hold the sample as it is
processed
❖ Different types of thermal cyclers
are also available for variant
methods of PCR

4. New automated PCR Old PCR

5. Principle
PCR uses the enzyme DNA polymerase that directs the
synthesis of DNA from deoxynucleotide substrates on a single-
stranded DNA template. DNA polymerase adds nucleotides to
the 3` end of a custom-designed oligonucleotide when it is
annealed to a longer template DNA. Thus, if a synthetic
oligonucleotide is annealed to a single-stranded template that
contains a region complementary to the oligonucleotide, DNA
polymerase can use the oligonucleotide as a primer and
elongate its 3` end to generate an extended region of double
stranded DNA.

6. PCR Steps
The PCR involves three
major cyclic reactions:
1. Denaturation
2. Annealing
3. Elongation

7. PCR Reagents
Nuclease Free Water
PCR Buffer/Cofactor
dNTPs
Primers
DNA Polymerase
DNA Template

8. PCR Reaction Chemistry
Water
❖ The medium for all other components
❖ Provides physiochemical environment

9. PCR Reaction Chemistry
PCR Buffer
❖ Stabilizes the DNA polymerase, DNA template,
and nucleotides
❖ Optimize conditions like salt concentration and pH
to enable Taq polymerase to work efficiently
❖ Having KCL to facilitate template primer binding
❖ Having MgCl2 Functions as a cofactor of Taq
DNA polymerase by providing magnesium ions

10. PCR Reaction Chemistry
dNTPs
❖ dNTP stands for deoxyribonucleotide
triphosphate
❖ There are four different dNTPs (dATP, dGTP,
dCTP, dTTP)
❖ The dNTPs serve as the building blocks for the
new strands being processed during PCR

11. PCR Reaction Chemistry
PRIMERS ❖ PCR primers are short fragments of single
stranded DNA (15-30 nucleotides in length)
that are complementary to DNA sequences
❖ The purpose of PCR primers is to provide
a “free” 3'-OH group to which the DNA
polymerase can add dNTPs
❖ They are initiators of polymerization
❖ Used in set: Forward and Reverse Primers

12. PCR Reaction Chemistry
Taq Polymerase
❖ Obtained from a bacterium Thermus aquaticus
❖ High temperature stability
❖ Performs the function of elongation of DNA
strands
❖ High rate of dNTP incorporation

13. PCR Reaction Chemistry
DNA Template
❖ The Target DNA sequence which need to be
amplified
❖ It is also called amplicon
❖ Should be free of polymerase inhibitors

14. PCR Reagents
Nuclease Free Water
PCR Buffer/Cofactor
dNTPs
Primers
DNA Polymerase
DNA Template
Ready to Start PCR Reaction

15. A Typical PCR Reaction
Setting Up PCR
Reactions
PCR Component Amount used
Nuclease free water 38.0 microliter
10X PCR Buffer 5.0 microliter
MgCl2 (50mM) 2.5 microliter
dNTPs (10mM each) 1.0 microliter
FWD Primer (20 pmol/microliter) 1.0 microliter
REV Primer (20 pmol/microliter) 1.0 microliter
DNA Polymerase 0.5 microliter
DNA Template 1.0 microliter
Total volume 50.0 microliter

16. Mastermix
PCR Component
Taq Polymerase
10X PCR Buffer/other
cofactors
MgCl2 (50mM)
dNTPs (10mM each)
Add primers NF-water and DNA template to the master
mix, and you are ready to start the PCR

17. Typical
Thermal Cycler
Conditions
Step
no
Steps name Temperature Time
1 Initial denaturation 94 to 98 °C 5 min
2 Denaturation 94°C 10-60 sec.
3 Primer annealing 50 to 60°C
generally 3-5
°C below
primer™
generally 20 to
60 sec.
4 Extension 70-75°C (72°C ) Amplicon
dependent
Go to step #2 and repeat the 40 more time 25 to 35 cycles
5 Final extension 72°C Amplicon
dependent
Rest 4°C Forever

18. Analyzing the
Amplified DNA
•After thermal cycling, tubes are taken out from the PCR machine
•Contents of tubes are loaded onto an agarose gel
•DNA is separated by size using an electric field
•PCR products are visible as different “bands”

19. Visualizing
Results
Bands can be compared
against each other and
to known size standards
to determine the
presence or absence of
a specific amplification
product
A typical PCR product on gel looks a like as
shown

20. Variants of
PCR
PCR have so many variance
use as per the target
sequence need to be amplify
or/and as per the type of
analysis required some
variants are field specific
Conventional PCR
RT-PCR
Quantitative PCR
Nested PCR
Multiplex PCR
Ligation mediated PCR
Colony PCR
Hot start PCR
Asymmetric PCR
LAMP
Among all the variance some are listed here

21. Advantages of PCR: Disadvantages of PCR:
● Specific and Sensitive
● Fast ( Can be done <1 days)
● Safe
● Usually not necessary to use
radioactive material
● Small amount of DNA is required per
test
● Detection of bacteria and viruses
● Setting up and Running requires high
technical
● Skills
● High equipment cost
● DNA contamination
● Taq polymerase is expensive
● Internal control
● False reactions

22. Applications of PCR
Molecular Identification:
➔ Classification of organisms
➔ Genotyping
➔ Mutation screening
➔ Drug discovery
➔ Genetic matching
➔ Detection of pathogens
➔ Molecular Archaeology
➔ Molecular Ecology
➔ DNA fingerprinting
DNA Sequencing
DNA fingerprinting
Genetic Engineering
Medical applications
Infectious disease
applications (detect bacteria or
viruses )
Forensic applications
Research applications
Bioremediation

23. Importance of Molecular
techniques in Aquaculture
and Fisheries
 The significant advances in molecular
biology during the last century greatly
influenced the development of genetic
research and application of molecular
methods in aquaculture and fisheries
 These methods provided substantial
opportunity for increased production
efficiency, better product quality and
improvement of animal health
 PCR can diagnose an array of viral,
bacterial and parasitic diseases of fish,
shellfish and crustaceans
 The advantages of the technique are
manifested in rapid obtaining of
results, high specificity and sensitivity

24. Thank you
Dr. RITU SHARMA
Senior Technical Officer, AAH&QTL-NFDB Hyderabad