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PCR
Polymerase Chain Reaction
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 main steps of PCR reaction
❑Denaturation (90-95°C)
❑Annealing (50-65°C)
❑Extension (72°C)
Initial denaturation
Denaturation
Annealing
Extension
Final extension
Holding / storage
How is it
done ?
Autopipettes (1-10µl, 10-
100µl & 100-1000µl)
0.2ml- 1.5ml
microcentrifuge tubes, tips,
PCR stands etc.
Gloves
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.
Components of PCR
1. Taq polymerase & other thermostable polymerases
2. DNA template
3. Primers
4. dNTPs
5. MgCl2
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.
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.
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.
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
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
DNA template
Primer bind to template
Taq polymerase extend primers
PCR episode1.pdf
PCR episode1.pdf
PCR episode1.pdf
PCR episode1.pdf
PCR episode1.pdf
PCR episode1.pdf
PCR episode1.pdf

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PCR episode1.pdf

  • 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)
  • 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.
  • 15.
  • 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
  • 21. Primer bind to template