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The
PCR depends on the ability to alternately
denature (melt) double-stranded DNA molecules
and renature (anneal) complementary single
strands in a controlled fashion. As in the
membrane-hybridization assay described earlier,
the presence of noncomplementary strands in a
mixture has little effect on the base pairing of
complementary single DNA strands or
complementary regions of strands. The second
requirement for PCR is the ability to synthesize
oligonucleotides at least 18–20 nucleotides long
with a defined sequence. Such synthetic
nucleotides can be readily produced with
automated instruments based on the standard
reaction scheme.
A typical PCR
procedure begins by heat-denaturation of a DNA
sample into single strands. Next, two synthetic
oligonucleotides complementary to the 3' ends of
the target DNA segment of interest are added in
great excess to the denatured DNA, and the
temperature is lowered to 50–60 C. These
specific oligonucleotides, which are at a very
high concentration, will hybridize with their
complementary sequences in the DNA sample,
whereas the long strands of the sample DNA
remain apart because of their low concentration.
The hybridized oligonucleotides then serve as
primers for DNA chain synthesis in the presence
of deoxynucleotides (dNTPs) and a
temperature-resistant DNA polymerase such as
that from Thermus aquaticus (a bacterium that
lives in hot springs).
This enzyme, called Taq polymerase, can remain
active even after being heated to 95 C and can
extend the primers at temperatures up to 72 C.
When synthesis is complete, the whole mixture is
then heated to 95 C to melt the newly formed DNA
duplexes. After the temperature is lowered
again, another cycle of synthesis takes place
because excess primer is still present. Repeated
cycles of melting (heating) and synthesis
(cooling) quickly amplify the sequence of
interest. At each cycle, the number of copies of
the sequence between the primer sites is
doubled; therefore, the desired sequence
increases exponentially—about a million-fold
after 20 cycles—whereas all other sequences in
the original DNA sample remain unamplified.
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