(English captions by Andrea Matsumoto, University of Michigan.)
The polymerase chain reaction or PCR can target and amplify any specific nucleic acid from
complex biological samples.
The procedure can be used for diagnosis to determine whether a clinical sample contains
a nuclear sequence that is known to occur only in a specific pathogen.
Or the laboratory scientists may use PCR to amplify and color large quantities of a specific
gene for research.
To preform PCR you must already know the sequence of the nucleic acid you wish to amplify.
Then you define the boundaries of the target sequence by identifying short sequences at
each end on opposite strands.
Here, the boundaries of the target sequence are indicated by violet and green highlighting.
If you move from these sequence in the five prime to three prime direction, the direction
of normal DNA synthesis, the violet highlighting extends along one strand and the green highlighting
extends along the complementary strand.
It is difficult to show how PCR works using this double helix representation of DNA so
the diagram with be converted to more easily understood ladder image of the DNA.
In addition to the clinical sample, the PCR reaction requires three ingredients.
First, there must be a massive supply of each of the four nucleotides.
Second, the user must add a large supply of small synthetic primers that are designed
to hybridize to the bonding sequence of either end of the targeted DNA.
The primers are the ingredients that make the reaction specific since only DNA that
lies between these two primers will be synthesized in the PCR reaction.
Third, the reaction requires a DNA polymerase enzyme.
For PCR the polymerase is actually from a bacteria that normally grows in the sea around
hot geothermal vents on the ocean floor.
The bacterium is called Thermus Aquaticus and the polymerase is called Taq polymerase
for short.
This exotic enzyme is used because it is not inactivated by the high temperatures generated
in the PCR reaction.
All these elements are mixed together in appropriate proportions and placed in an instrument called
a thermocycler.
This instrument can be programed to change the temperature of the mixture through a series
of repetitive cycles.
The temperature of the reaction in this demonstration is presented in the lower right panel.
In the first round of PCR the temperature is raised to a point at which the DNA is melted
and the complementary strands separate from one another.
The temperature is then lowered to a level at which the complementary strands can re-associate.
However, since the primers are present in the mixture at huge numbers, they are most
likely to bind at the complementary sites when the strands re-associate.
As the temperature is lowered further, the polymerase finds the free prime ends of the
primers and the enzyme begins to add nucleotides to the end of the primer using the complementary
strand as a template.
The same process occurs when DNA replicates in normal cell division.
At the end of round one of PCR there will be two copies of the target sequence for every
one that was present in the clinical sample.
You can keep track of the amplification in the panel that will appear on the lower left.
The same process is repeated in the second round of PCR.
The theromcycler dramatically heats the sample to separate the complementary strands of DNA,
including those that have just been synthesized.
The temperature is lowered to allow primers to bind at their specific sites and to prime
synthesis of complementary strands by taq polymerase when the temperature is lowered
again.
In the third round the same cycling of the reaction temperature occurs with melting of
the strands, binding of primers when the temperature is lowered, and new strand synthesis when
the strands are primed for DNA polymerase to begin adding nucleotides.
At the end of round three there are now eight double strand copies of the target sequence
where there was originally only one.
The enlarging frame from the lower left will now show what happens with successive cycles
of PCR.
With each cycle the number of copies of the target sequence doubles so there will be sixteen
copies after four cycles, thirty-two copies after five cycles, and sixty-four copies after
six cycles.
By the time the thermocycler has completed forty cycles the primers and nucleotides will
likely be exhausted but there will theoretically be ten to the twelfth (10 ^ 12) copies.
The target sequence will have been amplified a trillion times.
This level of amplification produces enough of the specific DNA that it can now be visualized
by gel electrophoresis.
The large smear of DNA at the top of the gel represents the complex DNA that was present
in the clinical sample.
However, a new smaller band appears in samples taken from the later cycles of PCR.
For diagnostic laboratory purposes the amplified DNA can be detected and quantified by more
efficient and simpler methods than gel electrophoresis.
One of these methods is discussed in an accompanying program.
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