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Overview
PCR : is a molecular biology technique to amplify a
short region of a DNA molecule by million fold or
more
PCR can be used to clone a given DNA sequence in
vitro without the use of living cells during the
cloning process
Developers: Kary Mullis, who received a Nobel
Prize in 1993 for this work, invented the method in
1983
The Nobel Prize in Chemistry 1993 was awarded "for contributions to the
developments of methods within DNA-based chemistry
Principles of PCR
PCR uses synthetic oligonucleotides
complementary to known sequences to prime
enzymatic amplification of the intervening
segment of DNA in the test tube
Carried out by the DNA polymerase I enzyme
(normally from Thermus aquaticus)
This organism lives in hot springs, and many of
its enzymes, including the Taq polymerase, are
thermostable
The Procedure
Most PCR methods typically amplify DNA
fragments from few hundred bp up to ~10 kb
A basic PCR set up requires several components
and reagents in a reaction volume of 10200l in
small reaction tubes (0.20.5 ml volumes)
The reaction is set up in a thin walled PCR tube
permit favorable thermal conductivity to allow for
rapid thermal equilibration in a thermal cycler
PCR Cycle
Initial denaturation
Denaturation
Annealing
Extension
Final extension
Hold
Denaturation (Td):
The first step: thermal denaturation of the DNA sample
at 94-96oC for 30 s-1 min
Double-stranded DNA template denature at a
temperature that is determined in part by their G + C
content
The higher the proportion of G + C, the higher the
temperature required to separate DNA strands
The longer the DNA molecules, the greater the time
required at the chosen denaturation temperature to
separate the two strands completely
Number of Cycles
The number of cycles required for amplification
depends on the number of copies of template DNA
present at the beginning of the reaction
The reaction proceeds until one of the components
becomes limiting
At least 25 cycles are required to achieve acceptable
levels of amplification of single-copy target sequences
Usually is between 25-40 cycles
PCR Cycle
25-40
cycles
Summary
1.
2.
3.
4.
Principles
Procedure
7 Components of PCR
PCR Cycle
End of Lecture 9
Thank You