olymerase chain reaction (PCR) is a method widely used in molecular biology to make several copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.

2.  PCR is an in vitro technique for the amplification of DNA.
 PCR is use to create millions or billions of copies of DNA
through repeated cycles of denaturing, annealing, and
extension/elongation.
 Where the DNA strands are used as templates to build
two new strands of DNA.
 PCR is a molecular biochemical technology.
 It is called “polymerase” because the only enzyme used in
this reaction is DNA polymerase.
 It is called “chain” because the products of the first
reaction become substrates of the following one, and so on.

3. 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

4. 1993: Kary Mullis won Nobel Prize

7. 1. Template DNA:
 Contains the sequence to be
amplified.
 Different sources including human
blood, saliva, skin or hair and cells
from other smaller organisms.
 Generally, PCR requires 1-1000 ng
(104-107 molecules) of DNA
template.
Template DNA

8. 2. Primer:
 short oligonucleotides (synthetic short DNA
strand)
 20-30 bases in size
 Specificity of PCR depend on primer.
3. dNTPs:
 Deoxynucleotide Triphosphates (Nucleotides)
 DNA building block.
 In general, 20-200μM (50μM of each is ideal)
of each dNTP should be included in the
reaction.
 Excess dNTPs inhibits polymerase activity

9. 4. DNA polymerase:
 Thermostable enzyme that synthesizes copies of DNA.
 Key of PCR
 Polymerases are responsible for binding free nucleotides complementary to the template DNA.
 Polymerases obtain from organism such as bacteria (including Taq and Pfu (Pyrococcus furiosus.)
Pyrococcus furiosus:
• Extremophilic species of Archaea.
• Thermal marine sediments
• hyperthermophile

10. Taq DNA polymerase:
 Isolated from Thermus aquaticus is the first isolated and best
known enzyme.
 Activity: 5’ – 3’ polymerase activity, but lacks 3’ – 5’ exonuclease
activity
 Works at high temperature.
 Stability: Half life of <5 min at 100 C, but retains activity up to 40
min at 95°C.
 Fidelity low
 heat-loving bacterium that is naturally found in hot springs,
Structure of Thermus aquaticus

11. Hot springs with algae and bacteria in Yellowstone
National Park
Microscopic view Thermus aquaticus

12. Enzyme Source Optimum
temperature
Fidelity Proofreading
rTth T. thermophilus 75-80 Low None
Pfu Pyrococcus furiosus 72-78 High Yes
Pwo P. woesei 60-65 High Yes
Deep Vent Pyrococcus strain GB-D 70-80 High Yes
“When greater fidelity is required, other thermostable enzymes may have
significant advantages.”

13. 5. Cofactors:
Magnesium (Mg2+):
 Cofactor of the enzyme.(DNA polymerase.)
 Mg2+ concentration should be maintained at
around 0.5-5.0mM.
 Can be changed to optimize PCR
amplification.
 Increasing Magnesium concentration results
in higher product yield.

14. 6. Buffer solution
 Maintains pH & ionic strength of the
reaction solution suitable for the
activity of the enzyme.
 pH range: 8.3-8.9
Buffer solution

16. 6 Following steps,
1. Initial Denaturation (Melting) : 94ºC for 2 minutes
2. Denaturation (Melting) : 94ºC for 1 minutes
3. Annealing : 55ºC for 30 seconds
4. Extension (Synthesis) : 72ºC for 1 minute
5. Final Extension: 72ºC for 6 minutes
6. Storage: 4ºC forever

17.  Temperature: 94C, For 1 mintutes
 Double stranded DNA melts - single stranded DNA.

18.  Temperature: 55ºC for 30 seconds ( Time vary depend on the nature of primer)
 In this steps, mixture now cooled.
 Primers bind to their complementary sequences

19.  Temperature:72ºC for 1 minute
 DNA polymerase binds to the annealed primers and extends DNA at the 3’ end of the chain
 DNA polymerase duplicats DNA

22. Equipments
Thermal Cycler:
 also 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.

23. Logaritmic multiplication

24. Logaritmic multiplication

25. Logaritmic multiplication

26. Logaritmic multiplication

27. Logaritmic multiplication

28. Logaritmic multiplication

29. Logaritmic multiplication

31. Advantages of PCR:
 Most specific, sensitive
 Fast ( Can be done <1 days)
 Safe
 Usually not necessary to use radioactive material
 Small amount of DNA is required per test
 Reduction of DNA for sequencing
 Detection of bacteria and viruses
 Setting up and Running requires high technical
skills .
 High equipment cost.
 High Test cost.
 DNA contamination.
 Taq polymerase is expensive
 Internal control
 False reactions
Disadvantages of PCR:

32.  Molecular Identification:
1. Classification of organisms
2. Genotyping
3. Pre-natal diagnosis
4. Mutation screening
5. Drug discovery
6. Genetic matching
7. Detection of pathogens
8. Molecular Archaeology
9. Molecular Ecology
10. DNA fingerprinting
 DNA Sequencing
 DNA fingerprinting
 Genetic Engineering
 Medical applications
 Infectious disease applications (detect bacteria or viruses )
 Forensic applications
 Research applications
 Bioremediation

33. Restrictions of PCR
 Contamination of reagents or lab results in false
 positive results
 Failure due to a mistake in the protocol
 Different materials/parts of the sample can inhibit the PRC process

34. Thank you….