Polymerase chain reaction. Who would have thought a bacterium hanging out in a hot spring in Yellowstone National Park would spark a revolutionary new laboratory technique? The polymerase chain reaction, now widely used in research laboratories and doctor's offices, relies on the
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Who would have thought a bacterium hanging out in a hot spring
in Yellowstone National Park would spark a revolutionary new
laboratory technique? The polymerase chain reaction, now widely
used in research laboratories and doctor's offices, relies on the
ability of DNA-copying enzymes to remain stable at high
temperatures. The polymerase from Thermus aquaticus (Taq),
a bacterium from Yellowstone can produce millions of copies of
a single DNA segment in a matter of hours. In nature, most organisms
copy their DNA in the same way. PCR mimics the natural process,
only it does it in a test tube. When any cell divides, enzymes called
polymerases make a copy of all the DNA in each chromosome.
The first step in this process is to "unzip" the two DNA chains of
the double helix. As the two strands separate, DNA polymerase
makes a copy using each strand as a template.
To copy DNA, polymerase requires two other components:
1. a supply of the four nucleotide bases
2. a primer.
DNA polymerases, whether from humans, bacteria, or
viruses, cannot copy a chain of DNA without a short
sequence of nucleotides to "prime" the process, or get it
started. So the cell has another enzyme called a primase
that actually makes the first few nucleotides of the copy.
This stretch of DNA is called a primer. Once the primer
is made, the polymerase can take over making the rest of
the new chain.
The three parts of the polymerase chain reaction are
carried out in the same vial, but at different temperatures.
The first part of the process separates the two DNA
chains in the double helix. This is done simply by
heating the vial to 95 oC for 30 seconds.
double-stranded DNA single-stranded DNA
The primers cannot bind to the DNA strands at such a
high temperature, so the vial is cooled to 55 oC. At
this temperature, the primers bind or "anneal" to the
appropriate location in the DNA strands.
This takes about 20 seconds.
single-stranded DNA + primer annealed DNA
The final step of the reaction is to make a complete
copy of the templates. Since the Taq polymerase works
best at ca. 75 oC, the temperature of the vial is raised.
The Taq polymerase begins adding nucleotides to the
primer and eventually makes a complementary copy
of the section of the template that lies between the
primers. This completes one PCR cycle.
primer-annealed DNA primer-extended DNA
with Taq polymerase and
dATP, dTTP, dGTP, dCTP
At the end of a cycle, each piece of DNA in the vial
has been duplicated. Since the cycle can be repeated
30 or more times and each newly synthesized DNA
piece can act as a new template, one can obtain 230 or
ca. 1 million copies of a single piece of DNA.
Note that the region of the DNA between the two
primers will be amplified. The flanking sequences
not. The entire process takes about three hours.
The figure on the left shows the series of steps
in a single cycle. The exponential growth of
the double helical segment between the two
primers is illustrated above.
Taq Polymerase In Complex With Tp7, An Inhibitory Fab