hello everyone in our course. now you are watching Level TWO Episode 1 in the practical molecular biology course from A to Z. in this video, we will discover: -introduction to PCR -Main steps in PCR

1. PCR
Polymerase Chain Reaction

2. What is PCR ?
• Polymerase chain reaction is an in vitro
technique that enables replication and
amplification of a DNA sequence to billions
fold amplitude

3. 3 main steps of PCR reaction
❑Denaturation (90-95°C)
❑Annealing (50-65°C)
❑Extension (72°C)

4. Initial denaturation

5. Denaturation

6. Annealing

7. Extension

8. Final extension

9. Holding / storage

10. How is it
done ?

11. Autopipettes (1-10µl, 10-
100µl & 100-1000µl)
0.2ml- 1.5ml
microcentrifuge tubes, tips,
PCR stands etc.
Gloves

12. Thermal cycler
• a device has a thermal block with holes where
tubes holding the reaction mixtures can be
inserted.
• The cycler then raises and lowers the temperature
of the block in discrete, pre-programmed steps.

13. Components of PCR
1. Taq polymerase & other thermostable polymerases
2. DNA template
3. Primers
4. dNTPs
5. MgCl2

14. 1.Taq polymerase
• Thermostable enzyme
• Thomas D. Brock isolated Thermus aquaticus, a new
species of thermophilic bacterium found in the Lower
Geyser Basin of Yellowstone National Park
• if inhibitors (e.g., low purity of template DNA) are
present in the reaction mix, higher amounts of Taq DNA
Polymerase (2-3U) may be required to obtain a better
yield of amplified products.

16. 2.Template DNA
• DNA should be pure as even a trace amount of
phenol, EDTA, Proteinase K ,etc. used during
DNA isolation strongly inhibit Taq DNA
Polymerase action.
• ethanol precipitation of DNA and washing with
70% ethanol to DNA pellet is usually effective
in removing these contaminants from the DNA
sample.

17. 3. Primer
• Primers are short synthetic oligonucleotide used in many molecular
techniques from PCR to DNA sequencing
• Primers designed to have a sequence reverse complement targeted
template.
• Some tips should be kept in mind while designing the primers.

18. 4.dNTPs
• The concentration of each dNTP in the final reaction mixture is
usually 200µM and the concentrations of each dNTPs (dATP, dCTP,
dGTP, dTTP), should be equal

19. 5.MgCl2
• Mg2+ metal ions act as essential cofactor for the DNA polymerase in
PCR.
• Faciltate the nucleophilic attack and bond formation and
polymerization.
• Mg2+ ion enter into the protein for joining and creating forces making
the polymerase stronger and capable to join dNTPs but at the
expense of specificity.
• Recommended range of MgCl2 concentration is 1-4 mM in the
standard reaction mixture

20. DNA template

21. Primer bind to template

22. Taq polymerase extend primers