Taylor Peltier

3/23/15

The Process of Agarose Gel Electrophoresis

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and
proteins) and their fragments, based on their size and charge. Gel electrophoresis is typically
used in a molecular biology lab to determine differences in genetic material. The basic process of
gel electrophoresis involves nucleic acid molecules that are separated by applying an electric
field to move negatively charged molecules through a matrix of agarose or other gel substances

Main Components
There are six main components of a successful gel electrophoresis experiment. These include the
gel itself which is made from agarose, TBE Buffer, dye and genetic material and the physical
equipment used to run an electrical current across the gel (including the electrophoresis chamber
and a power supply.) There are many variations of gel electrophoresis, but this paper focuses on
standard agarose specific gel electrophoresis.
Agarose and TBE Buffer
Agarose is a powdery substance derived from natural polysaccharide polymers extracted from
seaweed. The agarose gel is easily made using a TBE Buffer Liquid in combination with an
agarose powder. Often, the agarose powder is used in a ratio of 1.5% - 2% to TBE Buffer. The
combination of these two ingredients creates a clear liquid that sets to form a 3D, solidified,
rectangular gel.
Agarose gels are easily cast and handled compared to other matrices, because the gel setting is a
physical rather than chemical change. Agarose is also a safer substitute to use versus many other
types of gel substance, which can be poisonous to humans. Agarose also proves beneficial
because samples can be easily recovered. After an experiment is finished, the resulting gel can be
stored in a plastic bag in a refrigerator for further analysis or reuse.
Dye
Used in conjunction with the agarose and TBE Buffer is a fluorescent dye that allows genetic
material to be viewed under UV light after the electrical current is sent through the gel. The
fluorescent dye used most frequently during agarose electrophoresis experiments is called
Green&Glo, which is a non-mutagenic, environmentally safe substance.
Genetic Material
Genetic material is eventually added to the agarose, TBE Buffer and fluorescent dye finished gel.
The genetic material is dispensed via pipetting into wells within the gel. Genetic material can
range from double stranded DNA to RNA and proteins of interest. This will be explained more in
depth in the applications section of this paper.

Loading Buffer
A loading buffer must be added to the gel in order for the electric current to travel across the well
plate. The loading buffer can be the same material used to make the agarose gel to save
economically during the experiment. TBE Buffer is most commonly used.
Electrophoresis Chamber & Power Supply
The apparatus used in gel electrophoresis consists of an electrophoresis chamber and power
supply. The electrophoresis chambers holds the DNA containing gel and TBE buffer. A power
source is used to run the electrical current through the apparatus. A simplified image of the entire
system is shown below.
Figure 1. Simplified Image of a Gel Electrophoresis Chamber and Agarose Gel

Image from http://www.sciencebuddies.org/

Experimental Process & Analysis

Once all of the main components are explained, the methodology behind gel electrophoresis is
fairly simple. Nucleic acid samples are loaded into the gel (via well plates and pipetteing), and
then are exposed to an electric current. Fundamental biology of DNA and nucleic acids allow for
the separation and movement the fragments across the gel. DNA is negatively charged due to all
of the phosphate groups found in the backbone of DNA (see Figure 3.) Thus, DNA will move
towards the positive electrode on the opposite end of the gel. As the pieces of DNA move
through the gel, they will meet with resistance within the agarose gel. This resistance provides
the mechanism for gel electrophoresis.

Figure 2. Basic structure of DNA groups (notice the phosphate backbone.)

www.scienceaid.co.uk

Larger pieces of DNA will have more difficulty moving through the gel than smaller fragments.
Thus, larger fragments will move slower than smaller fragments. This allows separation of all
different sizes of DNA fragments and analysis of the subsequent image. When looking at a
finished gel under UV you can analyze your results. A series of bands will appear under the UV
lighting. By examining the different distances of how far fragments traveled, it becomes possible
to decipher how large nucleic acid fragments are. If the fragment has travelled a larger distance
than other bands, it can be deduced that the fragment is smaller and met with less resistance in
the gel.
As seen below, there are different fragments that have travelled different distances across the gel.
(NOTE: Notice where the negative and positive electrodes are in relation to each other)
Figure 3. Example of a Finished Gel Post Electric Current Treatment

Image taken from www.teachengineering.org

Applications

Gel electrophoresis can be used for a variety of purposes. Two examples include to get a DNA
fingerprint for forensic purposes and to check a PCR reaction. Both of these procedures are done
in a laboratory setting. The basic gel electrophoresis mechanisms are used across these two
different purposes.
Forensic Purposes
Identifying individuals from a crime scene has improved the way court cases are handled and
often verdicts are decided. Forensic gel electrophoresis requires a crime scene sample and a
suspect sample to compare. By amplifying these two samples, scientists can compare them in a
gel.
Gel electrophoresis allows forensic scientists to check whether or not certain DNA sequences
have been copied or not (depending on the individual.) If the sequence of interest has been
copied, a single band will appear in the gel. If the size of the DNA fragment that is being
searched for is known, scientists can match the bands up with bands from the size of the DNA
fragment that was copied in the first place. This is how individuals can be identified. The DNA
of each person is unique and therefore the patterns of separation created using the gel are unique.
PCR Product Reaction
PCR stands for Polymerase Chain Reaction. PCR is a machine that can amplify certain DNA
strands of interest post DNA extraction protocols. Unlike the forensic gel electrophoresis, PCR
can amplify any gene of interest. This can include processes such as sexing animal feces,
identifying tissue species samples and a plethora of other tasks. Gel electrophoresis utilizing
PCR product reaction is versatile and incredibly important in genetic studies.
Using gel electrophoresis is a standard method for analyzing these different objectives. Ideally,
electrophoresis yields a single strong band of correct size, as determined by comparison with size
markers run on the same gel. In sexing processes, often a male will appear on a gel with two
bands. This results from having an X and Y chromosome. The Y chromosome is much smaller
than the X chromosome. Therefore, in gel electrophoresis, the Y chromosome travels farther than
the X chromosome. This can be observed in the gel as two separate bands. In a female sample,
there will be one, very bright band visible. This is because females have two X chromosomes of
the same size. The two X chromosomes travel the same distance on the gel.