SUPERVISED BY- PRESENTED BY-

PROF. S. P. VYAS SUDIPTA NANDI

M.PHARM 1ST SEMESTER
ROLL NO- Y19254029

Department of Pharmaceutical Sciences

Dr. Harisingh Gour Vishwavidyalaya
(A Central University)
Sagar, M.P.
 TOPICS TO BE DEALT WITH
 WHAT IS PCR
 HISTORY AND PERSPECTIVE OF PCR
 COMPONENTS OF PCR
 PRINCIPLES INVOLVED
 TECHNIQUE OF PCR
 INSTRUMENTATION
 ADVANTAGES
 APPLICATIONS
 VARIANTS OF PCR
 FUTURE PROSPECTIVES
 REFERENCES
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 WHAT IS PCR

 PCR is laboratory or in vitro cell-free

amplification technique
 Uses specific sequence of DNA of small
amount and amplify to synthesize multiple
identical copies of DNA segment of interest.

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From its birth, PCR changed the molecular biology world.

• Dr. Kary Mullis developed PCR

1983

• First publication of PCR by Cetus Corporation appears in Science

1985

• Purified Taq polymerase was first used in PCR

1986

• PerkinElmer introduced the automated thermal cycler

1988

• Taq polymerase declared as “Molecule of the year”

1989

• Dr. Kary Mullis shares Nobel Prize in Chemistry for conceiving PCR
1993 technology

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 COMPONENTS OF PCR
• Essential requirements include :
 Target DNA : 0.1- 10 kbp in length; upto 40 kbp.
 Two DNA primers (synthetic oligonucleotide of 17-30 nucleotides length; 0.1-
0.5μM)
 A thermostable DNA polymerase (e.g. Taq polymerase; 1- 2.5 units)
 Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP; 20- 200 μM)
 Buffer : pH 8.3- 8.8
 Bivalent cations (Mg2+, Mn2+)

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 PRINCIPLE INVOLVED

1. Separation of DNA double-stranded template

2. Hybridization of strand with primer

3. Extension of new DNA strands by a DNA polymerase

and deoxynucleoside triphosphates (dNTPs)

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 TECHNIQUE OF PCR
• PCR consists of a series of 20- 40 repeated temperature changes called
thermal cycles, with each cycle commonly consists of 2-3 discrete
temperature steps as below:

DENATURATION
ANNEALING
of ds DNA
of primers
template

SYNTHESIS of ds
DNA molecule

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 TECHNIQUE OF PCR
 DENATURATION :
Samples are heated to 94-96°C for 30 sec
to 1 minute to denature the two strands
of of ds target DNA.

 RENATURATION OR ANNEALING :
Next the temperature is slowly
cooled to 55-65°C for 30 sec or
more, the primers base pair with the
complementary regions flanking
target DNA.

 EXTENSION OR SYNTHESIS :
Finally the temperature is raised to 75-
80°C, allows the Taq polymerase to bind
at each primed site and extend a new
DNA strand.

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AT A GLIMPSE….

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 INSTRUMENTATION
 Quantitative PCR instrument is a machine that amplifies and detects DNA.
 It combines the functions of
 Thermal cycler
 fluorimeter
 ADVANTAGES
 Sensitivity
 Speed
 Easy and simple
 Cost effective
 flexibility
 Usually not necessary to use radioactive
materials
 More precise in determining size of alleles

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 APPLICATIONS

Molecular Identification Sequencing Genetic Engineering

 Molecular Archaeology  Bioinformatics  Site-directed
 Molecular Epidemiology  Genomic Cloning mutagenesis
 Molecular Ecology  Human Genome  Gene Expression
 DNA fingerprinting Project (HGP) Studies
 Classification of
organisms PRENATAL DIAGNOSIS
 Genotyping FORENSIC LABORATORY
 Mutation screening
 Drug discovery
 Genetic matching
 Detection of pathogens
 Diagnostic purpose

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 Inverse PCR
 In-situ PCR
 Anchored PCR
 Reverse Transcription PCR  Asymmetric PCR
(RT-PCR)  Assembly PCR
 Real-time PCR  Hot-start PCR
 Miniprime PCR  Touchdown PCR
 Multiplex PCR  Solid phase PCR
 Variable Number of
Tandem Repeats (VNTR)  Intersequence Specific PCR
(ISSR)
 Quantitative real time PCR
(Q- RT PCR)  Suicide PCR
 Nested PCR  Colony PCR
 Long-range PCR  Digital PCR
 Single cell PCR  Ligation mediated PCR
 High-fidelity PCR
 Cold PCR

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 FUTURE PROSPECTIVES
 Preimplantation diagnosis
 PCR and Virology
 PCR and Bacteriology
 Cancer therapy

 CONTINUOUS FLOW PCR (The future of PCR) –

Most interesting and innovative way to increase speed of
PCR.
• Advantage :
 Reduce time needed for reaction
 Thermal equilibrium can be reached easily

 In an recent approach (February, 2019) Bio-Rad released

first FDA-cleared droplet digital PCR system and BCR-ABL
fusion test for monitoring chronic myeloid leukemia
treatment response.

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 Satyanarayana U, Chakrapani U. “Biochemistry”, Polymerase Chain
Reaction, Books & allied (P) Ltd.,2013, 4th edition;594-596.
 Vyas S. P, Dixit V. K, “Pharmaceutical Biotechnology”, CBS Publishers,
New Delhi,1998, 1st edition;353
 Innis Michael A., Gelfand David h., Sninsky John j., “PCR
APPLICATIONS- PROTOCOLS FOR FUNCTIONAL GENOMICS”,
Academic press,1999;33-263.
 Mullis Kary B, Ferre Francois, Gibbs Richard A. “PCR – The Polymerase
Chain Reaction”, Springer Science+Business Media, New York, 1994;165-
182.

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“….innovation is also on its face often marked with a bit of insanity.
If no one mentions loudly that one things you’re out of your mind,
then, you probably not being innovative.”
- Kary B. Mullis

THANK
YOU