Intro to Polymerase Chain Reaction (PCR)

This short animation introduces the polymerase chain reaction (PCR) procedure. This resource was developed by Yaw Adu-Sarkodie of the Kwame Nkrumah University of Science and Technology and Cary Engleberg of the University of Michigan. It is part of a larger learning module about laboratory methods for clinical microbiology. The full learning module, editable animation, and video transcript are available at http://open.umich.edu/education/med/oernetwork/med/microbiology/clinical-microbio-lab/2009. Copyright 2009-2010, Kwame Nkrumah University of Science and Technology and Cary Engleberg. This is licensed under a Creative Commons Attribution Noncommercial 3.0 License http://creativecommons.org/licenses/by-nc/3.0/.

(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.