PCR Polymerase Chain Reaction - PowerPoint PPT Presentation

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PCR Polymerase Chain Reaction

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PCR Polymerase Chain Reaction PCR PCR Machine / Thermocycler PCR Components of PCR Template DNA primers dNTPs (dATP, dTTP, dCTP & dGTP) Taq DNA polymerase MgCl2 PCR ... – PowerPoint PPT presentation

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Title: PCR Polymerase Chain Reaction


1
PCRPolymerase Chain Reaction
2
PCR
- a method for amplifying (copying) small amount
of DNA in nearly any amount required, starting
with a small initial quantity. - an in vitro or
cell-free method for synthesizing DNA. - it was
invented in 1985 by Kary Mullis (received the
Nobel Prize for chemistry in 1993).
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4
PCR Machine / Thermocycler
5
PCR
  • Components of PCR
  • Template DNA
  • primers
  • dNTPs (dATP, dTTP, dCTP dGTP)
  • Taq DNA polymerase
  • MgCl2
  • PCR buffer, pH 8

6
PCR
  • Three major phases in PCR
  • Denaturing (94ºC)
  • Annealing (55ºC)
  • Extension (72ºC)
  • The total time to perform a standard PCR is
    approximately 4 hours.

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9
Factors influencing PCR
  • Quality of template DNA
  • Concentration of template DNA
  • Primers
  • Concentration of MgCl2
  • Annealing temperature

10
  • Quality of template DNA

- should be free of proteases that could degrade
the DNA polymerase. - template DNA with high
levels of proteins or salts should be diluted or
cleaned up to reduce inhibition of DNA polymerase
activity.
11
  • Concentration of template DNA

- highly concentrated template DNA may yield
nonspecific product or inhibit the reaction. - it
is rare that template DNA concentration is too
low.
12
  • Primers

- select primers with a random base distribution
and GC content similar to template DNA being
amplified. - avoid sequences with secondary
structure, especially at the 3 end. - check
primers for complementary and avoid primers with
3 overlaps to reduce primer-dimer artifacts. -
design so the base at the 3 end of the primer is
a G or C to enhance specificity.
13
  • Concentration of MgCl2

- MgCl2 concentration is very important. - excess
Mg2 promotes production of nonspecific product
and primer-dimer artifacts. - insufficient Mg2
reduces yield.
14
  • Annealing temperature

- annealing temperature depends on length and GC
content of primers (55ºC good for primers 20
nucleotides long 50). - Higher annealing
temperatures may be needed to increase primer
specificity.
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