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PCR Technology
PCR is the cornerstone of current forensic DNA analysis as well as many other biological disciplines,
such as drug discovery and disease diagnosis. PCR was invented in the mid-1980s by Dr. Kary Mullis,
who was awarded the Nobel Prize in Chemistry in 1993 for this revolutionary breakthrough. PCR is
like a biological photocopier. This sensitive technique is capable of producing millions, even billions
of copies of DNA from just a few input strands (templates) of DNA. Once copied a billion-fold, this
small amount of original DNA can now be visualized using gel electrophoresis in cases where, prior
to this process, it was undetectable.
Ingredients of PCR:
Template DNA: the input DNA to be copied (DNA
from the crime scene).
This DNA is obtained by extracting it from
a cell’s nucleus and separating it away from
protein and other cellular components.
Primers (forward and reverse): bookends used to
define the region of junk DNA to be copied
Primers are short pieces of DNA synthesized
in a laboratory that bind to DNA adjacent to
the polymorphic or variable region. Each
section of DNA used for amplification requires
two primers, a forward and reverse, to
define the start and stop point for the reaction.
dNTPs: the building blocks of DNA
PCR products are made from the same building
blocks as DNA. dNTPs (deoxynucleotide
triphosphates) is a collective term for any or
all bases.
PCR Buffer: the solution in which PCR occurs
A solution containing precise concentrations
of salts and Magnesium that facilitate an ideal
environment for PCR to occur.
Taq Polymerase: the enzyme responsible for making
the copies of DNA
This enzyme was isolated from a bacterium
(Thermus aquaticus) originally found in hot
springs. In order to survive in this hostile
environment, this bacterium evolved a specialized
enzyme to replicate DNA at temperatures
that would normally prevent enzymatic
activity. This heat-stable enzyme is crucial
to the PCR technique, as it assembles the raw
components into the desired PCR products
under the higher temperatures needed for
the reaction.
Three Stages of PCR:
PCR is conducted in commercially available instruments
called thermal cyclers. These instruments
heat and cool the reaction tubes that contain
the reaction components. Three steps are
repeated approximately 30 times (called cycles),
which results in the replication of billions of
copies of the targeted DNA sequence.
1. Denaturation: Temperature greater than
90°C. The hydrogen bonds of the doublestranded
DNA ladder are broken, allowing
the two strands to separate.
2. Annealing: Temperatures between 50°C to
65°C. The primers sit down on the DNA,
outside the targeted region to be amplified,
binding by reforming hydrogen bonds to the
specific complementary sequences flanking
the target region, thus defining the region
to be copied.
3. Extension (Elongation): Temperature of
approximately 72°C. Taq polymerase is activated
and incorporates the free dNTPs into
the area between the primers.
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The Mystery of Lyle and Louise