Gel Electrophoresis

How DNA is processed for analysis

Introduction
Gel electrophoresis is a method used to separate macromolecule
fragments based on their size and charge. In this case we will be
looking at how gel electrophoresis can be used in DNA analysis.. The
end result would be various bands produced on a gel medium as shown
in Figure 1. Each band represents a different fragment of the DNA and
depending on whom or what it belongs to, it may or may not be present.
Hence it is an effective tool for comparing DNA samples to see if they
came from the same source. This is based on the fact that DNA
sequences are unique profiles and rarely do we ever get two sources
(especially in human context) having the same profile. Gel
electrophoresis is typically carried out with the use of: restriction
enzymes, agarose gel immersed in a buffer solution and an
electrophoresis chamber that contains the entire apparatus. (Figure 2).
Once the process is done, the bands can be visualized with the use of
ethidium bromide. The exact use of each component will be further
elaborated in the next few sections.


Figure 1. A gel electrophoresis sample
Source: "learning pod"
Figure 2. A gel electrophoresis chamber and power source
Source: Learn genetics, Utah

The Process
In order to carry out DNA gel electrophoresis, the following steps are typically taken:
1. The extracted DNA is treated with restriction enzymes.
2. The DNA is then loaded into the well of an agarose gel, along with a reference
sample of known fragments that have also been treated with the same restriction
enzymes.
3. The agarose gel is then placed in the gel electrophoresis chamber with the wells
closer to the negative terminal.
4. The chamber is then filled up with a buffer solution until the agarose gel is fully
immersed in it.
5. The chamber is switched on and set to the appropriate voltage.
6. Once an appropriate amount of time has passed, the chamber is switched off and the
agarose gel is removed and immersed in a solution of ethidium bromide.
How does it Work?
We will now breakdown how each individual step works.
1. Creating Fragments
In order to obtain the DNA fragments required for electrophoresis, the extracted
sample must first be treated with restriction enzymes. Restriction enzymes are
highly specific and cut the DNA macromolecules at specific markers to
produce fragments of varying lengths (Figure 3).

Since DNA from different sources have different markers where restriction
enzymes act upon, each DNA sample of would produce a unique set of
fragments. As shown in Figure 3, treatment with restriction enzyme produces
different set of fragments for each individual.

Figure 3. Restriction Enzymes Create Fragments

2. Loading The Samples
Agarose gel acts as a medium
for the DNA fragments to
move across. As shown in
Figure 4, the agarose gel is
prepared with agarose powder
and an electrophoresis buffer
that is heated up and casted
with a casting tray with a
comb to create the wells.
Each well can be loaded with
a separate sample that has
been treated with a restriction
enzyme. Typically a reference sample of known fragments is also added for
comparison.
3. Applying The Charge
In order for electrophoresis to work, a charge must
be applied so there will be an electrical gradient for
DNA to move across. A gel electrophoresis
chamber is specifically designed to hold an agrose
gel and apply a charge across it. As seen in Figure
5 the buffer solution contains ions which can
carry charges within the chamber, effectively
bridging the gap between the gel and terminal.
Once the chamber is turned on, the negatively
charged DNA fragments migrate towards the
positive terminal at varying rates and separate
accordingly.

4. Visualizing DNA Fragments
DNA fragments are barely visible under the agarose gel, hence analysis cannot
take place unless some sort of staining is used to visualize the fragments.
Ethidium bromide is a florescent dye that glows in a magenta color under UV
light and is typically used for such purposes. It intercalates with the bases
present on DNA, latching onto the nuclear bases present. Since ethidium
bromide is florescent, it is visible und
UV light. Figure 6 shows a
photographed gel electrophoresis
product immersed in ethidium
bromide and subsequently illuminated
*Fun fact:
Agarose gel is
made from
seaweed
extract!
*Warning:
Ethidium
Bromide is
carcinogenic
and should be
handled with
care.
Figure 4. Preparation of agarose gel
Source: Angelfire
Figure 5. The Chamber
Source "Gel Life Sciences
Figure 6. Ethidium Bromide Staining
Source "EHS Washington
with UV light. As shown, the DNA fragments show up as bands and the thicker
the band the thicker the concentration of that particular fragment. Ethidium
bromide diffuses within the gel, therefore a photograph should be taken as soon
as possible and used for analysis. Using the reference sample, the relative
distance traveled by the fragments can be compared to the known fragments in
the reference sample.

Why Does It Work?
The principles of DNA gel electrophoresis are based on the following facts:
1. DNA is negatively charged due to the phosphate groups present.
2. Fragments of DNA have different sizes, weights and configurations.
3. DNA Fragments of different size, weight and configuration travel with different rates
across a medium.
When a charge is applied across the agarose gel, the negatively charged DNA will migrate
towards the positive terminal; the buffer solution contains ions that carry charges across the
agarose gel. The agarose gel in this case acts as a sieve, slowing down the movement of DNA.
The larger and heavier the piece, the slower it moves across the gel, in addition, if the fragment
has a conformation with a lot of loose ends it will move at a slower rate compared to a
fragment that is tightly conformed. Hence over time, the fragments will start to separate, much
like in paper chromatography, with the lighter fragments ahead and the heavier, bulkier
fragments lagging behind. This separation forms clear distinct bands when immersed in ethidium
bromide, since the gel medium is consistent, fragments of the same size and configuration would
travel the same distance. Hence a simple comparison can be made within the gel to know if the
samples contain the same fragment, keeping in mind that restriction enzymes are highly specific
cut at the same position every time.
Using Figure 7 as an example, if the crime scene sample as well as known samples from
suspects were treated with the same restriction enzyme; a simple gel electrophoreses would
reveal that suspect 2s DNA matches the one at the crime scene since it contains all the same
fragments with the same concentrations.
*Think about it:
What happens
if we place the
gel in the
opposite way?
Figure 7. Analyzing Results
Source "Gene Ed
A Few Considerations
When running comparison tests such as this one it is important to consider factors that would
affect the consistency of the test. In order to ensure each test is repeatable and that the same
sample would produce the same result as the last one, there are a few factors that have to be
considered. These factors affect the rate at which DNA fragments move across the gel medium
other than its conformation and size.

1. Voltage
At lower voltages, the rate of migration is proportional to the applied voltage.
However at higher voltages, the migration speed of higher weight fragments increase
differentially, hence decreasing the reliability of the results. Therefore the optimum
voltage should be around 5-8V for DNA more than 2kb in size

2. Concentration of Ethidium bromide
Ethidium bromide may be added in the beginning with the samples and loaded into
the wells. It inculcates with the DNA fragments hence changing its length, charge as
well as conformation. Thus its presence early could drastically affect the results of the
electrophoresis.

3. Gel concentration
The concentration of agarose in the gel determines how porous the gel will be. A
lower concentration results in more pores and better fluidity, allowing the fragments
to migrate with more ease at a higher rate.
The varying conditions that may affect the distance travelled of a sample is the reason a
reference sample is used. Even though same samples may migrate different distances on separate
tests, the relative distance would remain the same. In other words the distance migrated relative
to the reference sample should be constant with varying conditions.
Applications
DNA gel electrophoreses has many applications that span a variety of scientific sectors. The two
main uses are in research and forensic inquiries. Research applications are in simple terms,
finding out what genes a certain organism possess. Certain genes contain restriction enzyme sites
and may be longer than DNA segments that do not contain the gene; hence simple gel
electrophoresis can be used to determine if a gene is present. This is used especially in the field
of bacterial research where plasmid DNA is studied. Forensic inquiry is similar to the example
shown in Figure 7, in which a suspects DNA is matched to the one at a crime scene. Gel
electrophoresis may even be used for paternal testing and many other forensic processes.