PCR principle, Steps, and Method

1. Polymerase Chain Reaction
(PCR)
Presented by:
Amany ELshamy

2. Outlines
• Introduction
• What is a PCR?
• Principle
• Steps
• Material
• Equipment
• Application
• Advantages
• Limitation
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3. Introduction
• The PCR technique was invented
in the 1983s by Kary B. Mullis.
• In 1993, Kary B. Mullis and
Professor Michael Smith awarded
the Nobel prize in the chemistry.
• In 1985, PerkinElmer introduced
the first automated PCR
machine.
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4. What is a PCR?
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5. What is a PCR?
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• A technique used to make numerous copies
of a specific segment of DNA quickly and
accurately.
• PCR is an in vitro DNA replication!! (in a test
tube at the laboratory not in the cell).
• PCR targets and amplifies a specific region
of a DNA strand.

6. Principle
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Amplification of the target DNA/RNA region

7. Principle
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8. Steps
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PCR step 1
Denaturation
PCR step 2
Annealing
PCR step 3
Extension
Temperature: 90°C - 95°C
Time 60 - 120 sec
Temperature: 50°C - 65°C
Time: 30 - 60 sec
Temperature: 72°C
Time: 45 Sec

9. Denaturation step
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• The dsDNA is denatured into ssDNA by disrupting
the hydrogen bonds between complementary
bases.

10. Annealing step
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• Annealing step: Allow annealing of the
primers to the ssDNA template.

11. Steps
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12. Extension/Elongation step
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• Elongation step: Taq polymerase adds dNTP's
from 5' to 3', reading the template from 3' to 5'
side.

13. Steps
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14. Steps
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15. Material
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PCR
Buffer
Taq
Polymerase
Sample
Primers
Enhancer
dNTPs
MgCl2

16. Equipment
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17. Equipment
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18. Applications of PCR
Medicine
Research
Forensic
Agriculture
Biotechnology
Genetic
Engineering
Genome
sequencing
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19. Advantages
• Determine pathogens that are difficult to isolate
(ex; viruses).
• Simple to use for Rapid diagnosis.
• Valuable screening tool.
• High sensitivity.
• Identify mutations, genetic diseases and
alteration of gene expression.
• Cost-effective compared to the other molecular
tools.
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20. Limitation
• Short size range of amplification Products
(100bp- 500bp).
• Chances of Contamination.
• Possibility of false negative/positive Results.
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21. Video
• https://www.youtube.com/watch?v=iQsu3Kz9
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• https://www.youtube.com/watch?v=a5jmdh9
AnS4
• https://www.youtube.com/watch?v=Nl6eLez3
CNI
• https://www.youtube.com/watch?v=VD5qEVT
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