The Polymerase Chain Reaction
In 1993, Kary Mullis received the Nobel Prize for
inventing the Polymerase Chain Reaction, also known as PCR. This
procedure for making billions of copies of DNA has revolutionized
modern biology and has become a standard technique for anyone studying
DNA. With PCR, specific DNA sequences from very small amounts of
DNA sample can be amplified for testing and analysis.
The procedure makes use of the physical properties
of DNA. At elevated temperatures (around 95oC), the two strands
of the DNA helix unwind and separate. The separated strands then
serve as templates for forming new DNA molecules. In a double-stranded
DNA molecule, the base adenine (A) pairs with thymine (T) and guanine
(G) pairs with cytosine (C). In a solution containing the single
strands and individual bases, complementary bases would attach to
the bases found on the single strands.
There are five essential components of a PCR reaction:
DNA template–DNA
that has been released from the source, e.g. buccal swabs, and purified.
Primers–small pieces
of single-stranded DNA that attach to complementary target sequences
on the DNA molecules.
Bases–the free bases
(A,T,G,C) that are added to the primer, extending the complementary
sequence to form double-stranded DNA
DNA polymerase–an
enzyme that facilitates the extension of the DNA molecule
Buffer–the solution
that maintains the favorable conditions in which PCR takes place
All of the above ingredients form the PCR mix, which
is then placed in a thermal cycler. The thermal cycler heats and
cools the PCR mix as follows:
95oC DNA strands
separate (or denature).
60oC Primers attach to the single
strands.
72oC DNA polymerase extends
the primers using the free bases.
Each cycle is repeated 25-35 times. After the
first cycle, two copies are produced of each DNA template. The PCR
products exponentially multiply through the 30 or so cycles, until
there are about one billion copies of each target sequence. Since
the DNA sample typically contains more than one copy of the DNA
template, billions of copies of the target sequence are produced.
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