Polymerase Chain Reaction - Xeroxing DNA
National Center for Human Genome Research, National Institutes
of Health. "New Tools for Tomorrow's Health Research." Bethesda, MD:
Department of Health and Human Services, 1992.
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. No problem
for Thermus aquaticus, the sultry bacterium from Yellowstone that now
helps scientists 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. The PCR
mimics this 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.
The four nucleotide bases, the building blocks of every piece of DNA,
are represented by the letters A, C, G, and T, which stand for their
chemical names: adenine, cytosine, guanine, and thymine. The A on one
strand always pairs with the T on the other, whereas C always pairs
with G. The two strands are said to be complementary to each other.
To copy DNA, polymerase requires two other components: a supply of the
four nucleotide bases and something called 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.
A PCR vial contains all the
necessary components for DNA duplication: a piece of DNA, large
quantities of the four nucleotides, large quantities of the primer
sequence, and DNA polymerase. The polymerase is the Taq polymerase,
named for Thermus aquaticus, from which it was isolated.
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 90-95 degrees centigrade (about 165
degrees Fahrenheit) for 30 seconds.
But the primers cannot bind to the DNA strands at such a high
temperature, so the vial is cooled to 55 decrees C (about 100 degrees
F). At this temperature, the primers bind or "anneal" to the ends of
the DNA strands. This takes about 20 seconds.
The final step of the reaction is to make a complete copy of the
templates. Since the Taq polymerase works best at around 75 degrees C
(the temperature of the hot springs where the bacterium was
discovered), the temperature of the vial is raised.
The Taq polymerase begins adding nucleotides to the primer and
eventually makes a complementary copy of the template. If the
template contains an A nucleotide, the enzyme adds on a T nucleotide
to the primer. If the template contains a G, it adds a C to the new
chain, and so on to the end of the DNA strand. This completes one PCR cycle.
The three steps in the polymerase chain reaction - the separation of
the strands, annealing the primer to the template, and the synthesis
of new strands - take less than two minutes. Each is carried out in
the same vial. At the end of a cycle, each piece of DNA in the vial
has been duplicated.
But the cycle can be repeated 30 or more times. Each newly
synthesized DNA piece can act as a new template, so after 30 cycles, 1
billion copies of a single piece of DNA can be produced! Taking into
account the time it takes to change the temperature of the reaction
vial, 1 million copies can be ready in about three hours.
PCR is valuable to
researchers because it allows them to multiply unique regions of DNA
so they can be detected in large genomes. Researchers in the Human
Genome Project are using PCR
to look for markers in cloned DNA segments and to order DNA fragments
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