Biotechnology

1. Polymerase Chain Reaction (PCR)
Sushil Kumar
Deptt. Of Zoology
Govt. P.G. College, Bisalpur
(U.P.)-262201

2. What is Polymerase Chain Reaction?
PCR is an exponentially progressing
synthesis of the defined target DNA
sequences in vitro.
It is called “polymerase” because the
only enzyme used in this reaction is DNA
polymerase.
It is called “Chain Reaction” because
the products of the first reaction
become substrates of the following one,
and so on. . . . . . . .

3.  PCR was invented by Kary Mullis in 1983 while
working in Biotechnology company Cetus Corporation
of Emeryville, California as a DNA chemist.
 Nobel Prize in Chemistry along with Michael
Smith -1993“ (Mutagenesis at a Specific Position in a
DNA Sequence“)
Born
December 28, 1944
Lenoir, North Carolina,
United States
Fields Molecular biology
Education
Georgia Institute of
Technology University of
California, Berkeley, PhD
Doctoral advisor Lee Wittenauer
Known for
Invention of polymerase
chain reaction
Notable awards
Nobel Prize in Chemistry
and Japan Prize (both in
1993)

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

5. Typical PCR Mix
In a thin wall Eppendorf tube assemble
the following
PCR components Amount
Template DNA (5-200 ng)
1 mM dNTPs (200 uM final)
10 X PCR buffer
25 mM MgCl2 (1.5 mM final)
20 uM forward primer (20 pmoles final)
20 uM reverse primer (20 pmoles final)
5 units/uL Taq DNA polymerase (1.5 units)
Water
Final Volume
variable
10 uL
5 uL
3 uL
1 uL
1 uL
0.3 uL
Variable
50 uL

6. The Reaction
THERMOCYCLER
PCR tube
(Eppendorf tube)

7. Taq DNA polymerase
 Derived from Thermus aquaticus
 Thermostable DNA polymerase
 Ideal temperature 72 0C
 Other Thermostable DNA Polymerase enzymes
1) Pflu DNA Polymerase (isolated from Pyrococcus furiosus)
2) Vent polymerase (isolated from Thermococcus litoralis)

8. Properties of DNA polymearse
3’ 5’
5’ 3’
 Needs a pre-existing DNA to duplicate
– Cannot assemble a new strand from
components
– Called template DNA
 Can only extend an existing piece of DNA
– Called primers

9. Properties of DNA polymearse
 DNA polymerase needs Mg++ as cofactor
 Each DNA polymerase works best under
optimal temperature, pH and salt
concentration
 PCR buffer provides optimal pH and salt
condition

10. Properties of DNA polymearse
 DNA strands are anti-parallel
– One strand goes in 5’  3’
– The complementary strand is opposite
 DNA polymerase always moves in one
direction (from 5’  3’)
3’ 5’
5’ 3’

11. Properties of DNA polymearse
 DNA polymerase incorporates the four
nucleotides (A, T, G, C) to the growing
chain
 dNTP follow standard base pairing rule
3’ 5’
5’ 3’
dCTP
dTTP
dCTP
dGTPdATP
dGTP
dCTP
dTTP
dATP
dGTP
dCTP
dTTP
dATP
dATP
dGTPdCTP dTTPdATP dGTP
dATP
dGTP
dTTP
dATP
dCTP
dTTP

12. Properties of DNA polymearse
 The newly generated DNA strands
serve as template DNA for the next
cycle
 PCR is very sensitive
 Widely used

13. Setting up a PCR Reaction
 Add template DNA and primers
3’ 5’
5’ 3’
dCTP
dTTP
dCTP
dGTPdATP
dGTP
dCTP
dTTP
dATP
dGTP
dCTP
dTTP
dATP
dATP
dGTPdCTP dTTPdATP dGTP
dATP
dGTP
dTTP
dATP
dCTP
dTTP
 Add dNTPs
 Add DNA polymerase

14. Thermal Cycling
 A PCR machine controls temperature
 Typical PCR go through three steps
– Denaturation
– Annealing
– Extension

15. Denature (heat to 95oC)
Lower temperature to
56oC Anneal with primers
Increase temperature to
72oC DNA polymerase +
dNTPs

17. 0
500000
1000000
1500000
2000000
2500000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
DNAcopies
Cycle number
DNA copies vs Cycle number

18. Applications of PCR
 Classification
of organisms
 Genotyping
 Molecular
archaeology
 Mutagenesis
 Mutation
detection
 Sequencing
 Cancer research
 Detection of
pathogens
 DNA fingerprinting
 Drug discovery
 Genetic matching
 Genetic engineering
 Pre-natal diagnosis

19. Detection of Unknown Mutations
Molecular Identification:

20. Classification of
Organisms
1) Relating to each other
2) Similarities
3) Differences
* Fossils
* Trace amounts
* Small organisms
! DNA !
Molecular Identification:
Insufficient data

23. Detection Of Pathogens
Molecular Identification:

24. Conclusion
The speed and ease of use, sensitivity,
specificity and robustness of PCR has
revolutionised molecular biology and made PCR
the most widely used and powerful technique
with great spectrum of research and diagnostic
applications.

25. THANK YOU