Polymerase Chain Reaction (PCR)

The field of Molecular Biology would be forever changed one night while driving along the Pacific
Coast Highway of California in 1983. Kary Mullis, a biologist for Cetus Corporation, was attempting
to develop a method to monitor mutations in DNA when he instead happened upon something
even more remarkable. He had invented polymerase chain reaction (PCR), a simple and cost-
effective method of making copies of DNA sequences. Before PCR, copies of DNA sequences were
cloned inside of a living host organism. This was both time consuming and required a great deal of
technical proficiency. Now, the method was streamlined to last only 2-3 hours with much higher
accuracy and using only basic laboratory equipment. The applications of this technology range from
clinical research, forensic science, and even paternity testing.
What Goes Into PCR?
DNA Template A sample that contains the DNA sequence you
wish to copy
Primers Two primers (20-30 nucleotides in length) are
needed so that DNA polymerase can bind to the
DNA (one primer per strand) and begin making
new copies
Taq polymerase A DNA polymerase, which adds nucleotides to a
growing DNA molecule
Deoxynucleotide Triphosphates (dNTPs) These nucleotides are the Gs, Cs, As, and Ts
that make up DNA
Buffer Solution To provide a suitable chemical environment
*All reagents must be kept chilled on ice in order to prevent any unwanted reactions from occurring
What Lab Equipment Is Needed?
PCR uses special test tubes that hold a volume of 0.5 mL and
are designed to distribute heat evenly. This is important because
PCR cycles through steps of heating and cooling in order to
replicate the sample DNA. Also needed is a micropipette with
sterile tips, which must be changed after contact with each
solution to ensure there is no cross-contamination of DNA.
Finally, a thermal cycler (right) is needed for the necessary
heating and cooling of the PCR master mix as it progresses
through its three stages of denaturation, annealing, and
extension.


The Three Stages of PCR
Denaturation
During denaturation, the reaction tube is
heated to 94-95
o
C in a thermal cycler in
order to break the hydrogen bonds that
hold the double helix structure of DNA
together. Hydrogen bonds are weaker
than the ionic and covalent bonds that
hold together the rest of the DNA
molecule, which is why the rest of the
molecule doesnt come apart when
heated. This temperature is maintained
for anywhere between 20 and 30
seconds. This must be done so the
primers are able to bind each strand so
DNA polymerase can begin adding
nucleotides.
Annealing
For the next 20-40 seconds, the thermal cycler cools to approximately 45-50
o
C which allows the
primers to bind to the newly separated DNA. The temperature chosen for the annealing stage is
typically 5
o
C below the melting temperature of the primers being used. For a simple estimation, the
melting temperature of the primers can be estimated by adding 2
o
C for each A or T and 4
o
C for each
G or C. This is because there are 2 hydrogen bonds for each A-T pairing and 3 hydrogen bonds for
each C-G pairing. Since it takes more energy to break 3 bonds than it does to break 2 bonds, we
estimate 4
o
C for each G or C and 2
o
C for each A or T.
Extension
In this stage, the DNA polymerase extends the primers
to generate new strands of DNA which are
complementary to the DNA template added in the
beginning. The temperature at which this is done
depends on the polymerase. For Taq polymerase that
temperature is 70-72
o
C. The expression

where n
is the number of cycles of PCR that have been
completed allows us to calculate the number of copies
after n number of cycles. After only about 30 cycles,
billions of copies of the DNA sample have been made.
Applications
As was mentioned earlier, PCR has enabled further developments in other technologies such as
DNA sequencing, genetic fingerprinting, forensic analysis, and diagnosis of cancer, genetic
disorders, and infections. One of the reasons it has been so useful in these fields is its ability to
detect mutations in a genome. Due to the high specificity of PCR, its able to detect a viral infection
from a DNA sample before the onset of disease symptoms has even occurred. It can do this by
making copies of viral DNA so that doctors are able to identify the sequences in their patients
DNA.
Conclusion
PCR typically undergoes between 20 and 30 cycles of heating and cooling, each cycle defined as
being one progression through denaturation, annealing, and extension. This enables a significant
amplification of a provided DNA sample. The amount of DNA in the sample at the beginning
determines how many cycles of PCR you want to subject it to. The advances in PCR technology has
improved the efficiency as well as broadened the spectrum of applications in which this technology
can be used.