Polymerase Chain Reaction
(PCR)
• PCR produces billions of copies of a specific
piece of DNA from trace amounts of starting
material. (i.e. blood, skin cells, bone)
• Allows scientists to isolate pure quantities of
specific DNA sequences
• 230 = over 1 billion copies of a specific DNA
fragment; large enough quantity to be analyzed
• Used in:
– Criminal Investigations to identify suspects
– Sequencing the Human genome
How does DNA from the Crime
Scene help in the lab?
• 46 Chromosomes code for 30,000 to
50,000 genes; only 5% of your DNA
– Exons = DNA that is coded or expressed into
proteins
• Noncoding DNA has more diversity; since
this DNA rarely leaves the DNA to head to
ribosomes
– Introns = DNA that is rarely expressed
– Increased number of mutations
• Target DNA – the DNA that needs to be copied
• Primers – short pieces of DNA that are designed
to attach to each end of the DNA fragment that
will be replicated
• Taq polymerase – enzyme that reads the DNA
– Comes from the bacteria Thermus aquaticus
– Lives in the hot springs in Yellowstone; doesn’t fall
apart in high temperatures
• dNTPs – 4 nucleotides with the 4 different bases
that are needed to replicate DNA
• Buffer – gives the best environment for the
enzymes to work
• Mg Ions – needed by DNA polymerase to make
DNA copies
• PCR machine is known as thermal cylcer
• Machine changes to three different
temperature changes during one cycle
• Average number of cycles per run is 30 to
40
Temperature at 94ºC
• Denaturing
temperature
– The target DNA
falls apart
– The H bonds
holding the nitrogen
bases together
break
– 2 individual strands
of DNA are now
present instead of a
double helix.
Temperature at 65ºC
• Annealing
Temperature
– Primers attach to the
ends of the Target
DNA that needs to be
copied
– Annealing =
attachment of the
primers
– Attach to
complimentary bases
of target DNA
Temperature at 72ºC
• Extension
Temperature
– Provides best temp
for Taq polymerase
to begin reading the
DNA
– Taq polymerase will
synthesize a second
strand of
complimentary DNA
– Taq polymerase
always read target
DNA from 3’ to 5’
end
Repeat 30 times
• The three temperature changes
represents one cycle
– Denature
– Anneal
– Extend
• Repeat 30 times 230 = over 1 billion copies
of the Target DNA
• Once DNA is amplified (copied), it is
visible on a gel
One Cycle has all three different
temperatures.
Another Animation
PCR Animation