Title: PCR Polymerase Chain Reaction
1PCRPolymerase Chain Reaction
2PCR
- 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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4PCR Machine / Thermocycler
5PCR
- Components of PCR
- Template DNA
- primers
- dNTPs (dATP, dTTP, dCTP dGTP)
- Taq DNA polymerase
- MgCl2
- PCR buffer, pH 8
6PCR
- 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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9Factors influencing PCR
- Quality of template DNA
- Concentration of template DNA
- Primers
- Concentration of MgCl2
- Annealing temperature
10- 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- 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- MgCl2 concentration is very important. - excess
Mg2 promotes production of nonspecific product
and primer-dimer artifacts. - insufficient Mg2
reduces yield.
14- 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.