PCR is a technique that amplifies a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample in the presence of primers and a DNA polymerase. During each cycle, the DNA strands are separated by heating, then primers allow the polymerase to selectively copy the target sequence. This results in exponential amplification of the target DNA. PCR is used in a wide range of applications including pathogen detection, genetic testing, and forensic analysis due to its ability to rapidly produce large amounts of a specific DNA sequence from a small sample.

1. Brief Introduction to PCR
2 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 1
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida
Abhijit Debnath
Asst. Professor
NIET, Pharmacy Institute
Unit: 2
Subject Name: Biotechnology
(BP605T)
Course Details
(B. Pharm 6th Sem)

2. Brief Introduction
to PCR
2 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 2
 What Is PCR
 Principle of PCR
 Denaturation
 Annealing
 Extension
 Cycling
 Products of Extension
 Overall Principle of PCR
 Basic Requirements for PCR Reaction
 Instrumentation
 Advantages of PCR
 Applications of PCR
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida

3. •PCR is a technique that takes specific sequence of DNA of small amount and
amplifies it to be used for further testing.
• In vitro technique.
• 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.
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WHAT IS PCR? (CO2.2)

4. 2 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 4
WHAT IS PCR? (CO2.2)

5. 2 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 5
WHAT IS PCR? (CO2.2)

6.  Purpose:To amplify a lot of double-stranded DNA molecules (fragments) with
same (identical) size and sequence by enzymatic method and cycling condition.
 Condition: 1. Denaturation of ds DNA template
2. Annealing of primers
…………… …3. Extension of ds DNA molecules
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PRINCIPLE OF PCR (CO2.2)

7. single stranded
92C
3’
• Temperature: 92-94C
• Double stranded DNA melts DNA
5’
3’ 5’
+
5’
3’
5’ 3’
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DENATURATION (CO2.2)

8.  Temperature: ~50-70C (dependant on the melting temperature
of the expected duplex)
 Primers bind to their complementary sequences
5’
3’
5’ 3’
Forward primer Reverse primer
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ANNEALING (CO2.2)

9.  Temperature: ~72C
 Time: 0.5-3min
 DNA polymerase binds to the annealed primers and extends DNA
at the 3’ end of the chain
Taq
5’
3’
Taq
5’
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EXTENSION (CO2.2)

10. 2 July 2021 Abhijit Debnath BP605T and Biotech Unit-2 10
CYCLING (CO2.2)

11. 3’
5’
3’ 5’
3’
5’
3’ 5’
Taq
Taq
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PRODUCTS OF EXTENSION (CO2.2)

12. • DNA – 1 copy
• Known sequence Sequence of interest Known sequence
• PCR
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OVERALL PRINCIPLE OF PCR (CO2.2)

13.  Magnesium chloride: .5-2.5mM
 Buffer: pH 8.3-8.8
 dNTPs: 20-200µM
 Primers: 0.1-0.5µM
 DNA Polymerase: 1-2.5 units
 Target DNA: 1 µg
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OVERALL PRINCIPLE OF PCR (CO2.2)

14.  DNA sequence of target region must be known.
 Primers - typically 20-30 bases in size. These can be readily
produced by commercial companies. Can also be prepared
using a DNA synthesizer.
 Thermo-stable DNA polymerase - eg Taq polymerase
which is not inactivated by heating to 95C.
 DNA thermal cycler - machine which can be programmed to
carry out heating and cooling of samples over a number of
cycles.
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BASIC REQUIREMENTS FOR PCR REACTION (CO2.2)

15. 2 July 2021 Abhijit Debnath BP605T and Biotech Unit-2 15
INSTRUMENTATION (CO2.2)

16.  Specificity
 Efficiency
 Fidelity
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THREE ASPECTS OF PCR (CO2.2)

17. •
If no product ( of correct size ) produced:
 Check DNA quality
 Reduce annealing temperature
 Increase magnesium concentration
 Add dimethylsulphoxide ( DMSO ) to assay ( at around 10% )
 Use different thermostable enzyme
 Throw out primers - make new stocks
If extra spurious product bands present:
 1 Increase annealing temperature
 2 Reduce magnesium concentration
 3 Reduce number of cycles
 4 Try different enzyme
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Things to try if PCR does not work (CO2.2)

18.  Initial denaturation : 95C for 5 mins 30
 Thermo-cycle file - cycles of 95C for 30
 Denaturation : secs 55C for 30 secs
 Annealing : 72C for 45 secs 72C
 Extension :for 5 mins
 Final extension
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EXAMPLE A PCR PROGRAMME (CO2.2)

19.  Small amount of DNA is required per test
 Result obtained more quickly - usually within 1 day for PCR
 Usually not necessary to use radioactive material (32P) for
PCR.
 PCR is much more precise in determining the sizes of alleles -
essential for some disorders.
 PCR can be used to detect point mutations.
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ADVANTAGES OF PCR (CO2.2)

20.  Neisseria gonorrhea
 Chlamydia trachomatis
 HIV-1
 Factor V Leiden
 Forensic testing and many others
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APPLICATIONS OF PCR (CO2.2)

21. Molecular Identification Sequencing Genetic Engineering
Molecular Archaeology Bioinformatics Site-directed mutagenesis
Molecular Epidemiology Genomic Cloning Gene Expression Studies
Molecular Ecology
DNA fingerprinting
Classification of organisms
Human Genome Project
Genotyping
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens
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APPLICATIONS OF PCR (CO2.2)

22.  PCR is not only vital in the clinical laboratory by amplifying small amounts of DNA for
STD detection, but it is also important for genetic predisposing for defects such as
Factor V Leiden.
 The PCR technology can also be employed in law enforcement, genetic testing of animal
stocks and vegetable hybrids, and drug screening along with many more areas.
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CONCLUSION (CO2.2)

23. Introduction:
• Basic principle.
• Highly specific genetic manipulation of genetic material.
• Gene transfer between unrelated species.
• Applications in health, agriculture, environment, industry.
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THREE ASPECTS OF PCR (CO2.2)

24. Antibiotics:
• Artificially prepared antibiotics also available.
• They denature harmful living pathogens.
• pencillin in 1928.
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THREE ASPECTS OF PCR (CO2.2)