RECOMBINANT DNA TECHNIQUES: CLONING AND MANIPULATION OF DNA - A Guide to Methods in the Biomedical Sciences - page 54

to anneal by lowering the temperature. Third, the reaction is “extended”
by a polymerase; the temperature of this reaction is usually between
the other two. In its simplest form, PCR starts with two short oligonu-
cleotide primers, each of which is complementary to opposite strands of
the DNA. The DNA is heat denatured to separate the strands, and as the
temperature is lowered, the primers anneal to the DNA. In the presence
of DNA polymerase and each of the 4 dNTPs, copies of each strand
are produced. The sample is heated again, the temperature lowered
to allow annealing with the PCR primers, and the process is repeated.
The amplification process doubles the amount of DNA synthesized from
the two primers at each step, each of which takes a couple of minutes.
As a result, in an hour or two, 30 rounds of PCR can be carried out,
resulting in more than a billion copies of a specific region of DNA from
a single DNA template to be exact!). The DNA
needs not be abundant or pure. This is why DNA can be typed from a
single human hair or drop of blood. Small amounts of DNA have been
extracted from mummies, as well as from animals frozen in glaciers tens
of thousands of years ago. PCR allows this DNA to be compared to their
counterparts of today. And, as you might have guessed, DNA has been
extracted from insects preserved in amber and amplified by PCR, a pro-
cess that formed the basis of Michael Crichton's topic, “Jurassic Park”,
and the subsequent movies.
In its original incarnation, new DNA polymerase had to be added at
each step of PCR since the temperature used to separate the DNA
strands (commonly 94°C, which is near boiling) also inactivated the
enzyme. This problem was solved when a DNA polymerase that was
stable at high temperatures was isolated from the bacterium Thermus
aquaticus (Taq polymerase) which thrives in hot springs. Today, a
number of genetically engineered heat stable DNA polymerases are
available for use in PCR, and the PCR reaction is carried out in pro-
grammable instruments that rapidly heat and cool samples for each
round of amplification.
PCR has been adapted to a number of uses in biomedical sciences.
In some cases, nested PCR primers are used, particularly when the
template DNA is in low abundance. Nested primers are primers in which
a first pair of oligonucleotides is used to amplify DNA for a number of
rounds, and then a second pair of primers (located within the bounds
of the first PCR product) is used for further amplification. This not only
allows for the amplification of small amounts of DNA, but sometimes
significantly improves specificity.
A small sampling representing the most common variations of PCR
are outline below.