PCR Polymerase Chain Reaction

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PCRPolymerase Chain Reaction
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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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PCR Machine / Thermocycler
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PCR

• Components of PCR
• Template DNA
• primers
• dNTPs (dATP, dTTP, dCTP dGTP)
• Taq DNA polymerase
• MgCl2
• PCR buffer, pH 8

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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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Factors influencing PCR

• Quality of template DNA
• Concentration of template DNA
• Primers
• Concentration of MgCl2
• Annealing temperature

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• 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.
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• 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.
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• 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.
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• Concentration of MgCl2

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