Electrophoresis: How scientists
observe fragments of DNA
Electrophoresis Introduction
How do scientists look at DNA?
As genetic analysis has become more precise, the average person may get the
impression that modern geneticists have powerful microscopes in their labs that zoom
in on the DNA molecule and reveal information. Fans of “Star Trek” television reruns
will envision devices that are waved over tissue and produce meaningful DNA data. In
reality, DNA analysis techniques reveal information by using a more indirect method of
looking at the molecule. Electrophoresis is the central technique in this analysis.
Electrophoresis means to carry with electricity. The technique allows scientists to
detect sequence variation among specific DNA segments based on how fast these
segments are carried by electricity. There are two key facts that allow this technique to
work.
1) Nucleotide sequence differences in one DNA segment compared to another can
result in size differences in these segments.
2) Longer segments of DNA will be carried through the gel more slowly than shorter
segments. Speed comparisons bring to mind a race, so let’s think about
electrophoresis using a racing analogy.
Electrophoresis gels: the race track
You need a track if you’re going to race and the
electrophoresis gel is the molecular race track. The
track is a gel matrix that is similar to a slab of gelatin.
The electrophoresis gel is made by adding a powdered
substance such as agarose (Fig. 1) to water, bringing
the water to a boil, and then allowing the solution to
cool. Agarose will form a gel matrix as it cools,
conforming to the shape of the container or mold. The
matrix will have spaces that are filled with the water.
The size of these spaces depends upon the
concentration and physical properties of agarose.
Change the concentration or switch to a different
substance (acrylamide for example) and you alter the
size of the holes or spaces in the matrix. Therefore, a
matrix made from agarose and water is the race track
used to compare the speed of DNA from different
samples.
Fig. 1. Agarose powder used to make an
electrophoresis gel.
Fig. 2. Measuring the correct amount of agarose
Current is the key
Several differences distinguish the electrophoresis
gel (our DNA race track) and a slab of gelatin you
would eat for desert. The water used to make the
gel has electrolytes in it to conduct current. This
water plus electrolytes solution is called an
electrophoresis buffer. An agarose gel is made of
complex carbohydrates extracted from seaweed
and is not sweet to eat. The matrix creates the
right size spaces for sieving DNA and holds it's
shape even if it heats up when electric current is
run through it. Finally, the electrophoresis gel is
placed in an electrophoresis apparatus or gel box
that is designed to hold the gel in place and
conduct a current through it.
Preparing a gel
The first step in gel preparation is to weigh out
the appropriate amount of agarose (Fig. 2). This
amount depends on the size of the gel and the
size of the DNA segments being separated.
Smaller segments will require separation with a
more dense electrophoresis gel. The agarose is
poured into the water solution with the
electrolyte (Figs. 3,4). Usually the electrolyte
solution such as Tris borate EDTA is made as a
concentrated stock and them diluted for each
gel. The agarose and electrolyte solution is then
heated and stirred. This works well using a
stirring hot plate found in most molecular
genetics labs. One can also heat the gel solution
in a microwave. As the mixture heats, the
agarose goes into solution, forming a clear
liquid. Now the gel is ready to be poured.
Fig.3. Preparing the TBE
electrophoresis buffer.
Fig.4.Mixing the agarose powder and
electrophoresis buffer on a stirring hotplate
Pouring a gel
Pouring an electrophoresis gel is the final step in preparing the molecular
race track. The mold the gel is poured into is a plexiglass tray that is missing
the two ends.
Tape or removable
ends are added
prior to pouring to
hold the hot liquid
in place (Fig 5).
Fig. 5. Preparing the gel mold by
taping the ends.
Pouring a gel
Pouring an electrophoresis gel is the final step in preparing the molecular
race track. The mold the gel is poured into is a plexiglass tray that is missing
the two ends.
A plexiglass comb is also added (Fig. 6).
The teeth on this comb will stick into the
cooling gel but not extend to the bottom
of the mold. As the gel cools, each tooth
in the comb forms a well. This is where
the DNA sample will be placed. If many
samples need to be compared in our
electrophoresis race, combs with more
teeth are used or more rows of combs
are placed in the gel. When the gel
cools, it solidifies to a semi-solid matrix
that holds it’s shape.
Fig. 6. Placing combs in the gel mold
to establish the sample wells.
Pouring a gel
Pouring an electrophoresis gel is the final step in preparing the molecular
race track. The mold the gel is poured into is a plexiglass tray that is missing
the two ends.
When the gel cools, it
solidifies to a semi-solid
matrix that holds it’s shape.
The comb can be removed
from the solid gel and the
empty wells will serve as the
starting line for the DNA
samples (Fig. 7).
Fig. 7. Taking the combs out of the
solidified gel. Note the wells.
Pouring a gel
Pouring an electrophoresis gel is the final step in preparing the molecular
race track. The mold the gel is poured into is a plexiglass tray that is missing
the two ends.
The tape is removed so
current will flow through
the gel once it is in the
gel box (Fig. 8).
Fig. 8. Removing the tape from the
ends.
Loading a gel established the starting line
DNA samples will be run
in the electrophoresis
box and the gel can be
placed in the box with
electrophoresis
buffer
prior to loading the
sample wells (Fig. 9).
Fig. 9. Placing the gel into the gel box.
Loading a gel established the starting line
Fig. 10. A tracking dye is mixed with
each DNA sample.
DNA samples are brought to
the starting line of the
electrophoresis gel with the
use of a pipette. The pipette
can dispense a measured
amount of DNA sample into
each sample well. Since the
DNA in solution lacks color, a
dense tracking dye solution
is often mixed in to visualize
the gel loading process (Fig.
10).
Loading a gel established the starting line
It is easy to see that the gel
loading step must be done
with care to avoid mixing of
samples (Fig. 11).
Fig. 11. Using a pipette to load the
DNA samples.
Loading a gel established the starting line
Fig. 12. The tracking dye aids in loading the
samples.
When the DNA solution plus
tracking dye are loaded, the
well will hold the colored
mix in place until all the
samples are loaded (Fig. 12).
Now it is time for the race to
determine which lane has
DNA samples with longer
fragments and which lane
has DNA samples with
shorter fragments.
Conducting a fair race
Fig. 14. Current is run through the gel by
connecting it to a power supply.
Electrophoresis uses electricity as the force to
move the DNA molecules through the gel. The
electrophoresis apparatus is designed to run a fair
race by conducting the current evenly across the
gel (Fig. 14). A wire is found along the bottom of
the apparatus at both ends. Electrons supplied by
the electrophoresis buffer will flow to the positive
pole. The phosphates making up the DNA
molecule's backbone create a negative charge so
DNA will move toward the positive pole once the
circuit is complete. As long as the current is
flowing, DNA molecules will be subjected to the
same force (the voltage) which carries them
toward the positive end of the gel. This will cause
the DNA fragments to move in a straight lane
rather than wander toward the positive end.
Therefore, each well in the electrophoresis gel
establishes a lane for the DNA sample
The agarose obstacle course
Fig. 15. DNA fragments move through the gel by
weaving in and out of the gel pours.
The DNA race occurring in an electrophoresis gel is
fair but it is an obstacle course. This is because the
DNA molecules are forced to travel through the
matrix of buffer filled spaces or pores that is
created when the agarose gel is formed. The
agarose gel provides a three dimensional lane. A
random mix of small and large spaces will be found
in any given part of the lane. If a DNA molecule
cannot fit through a pore it moves up, or down to
find a pore big enough to squeeze through. The
molecule does not take a straight path to the
positive end of the gel but instead wiggles through
(Fig. 15). Shorter DNA segments find more pores
that they can wiggle through, longer DNA
segments need to do more squeezing and up or
down moving. For this reason, shorter DNA
segments move through their lane at a faster rate
than longer DNA segments. That is why the gel
electrophoresis method separates DNA molecules
based on their size.
The race in action
Molecular geneticists are like racing
fans. They need to observe the action to
gain information about the DNA. The
tracking dye provides some information.
In addition to helping the geneticist load
their DNA sample in the well, the
tracking dye will also move in response
to electric current and as it move it
provides an indicator of how the DNA is
moving. In Fig. 16, the tracking dye looks
dark purple as it just begins to leave the
wells.
Fig. 16. The tracking dye moves along with the
DNA sample.
The race in action
Fig. 17. The two dyes used to make the tracking
dye separate as the dye moves through the gel.
Molecular geneticists are like racing
fans. They need to observe the action to
gain information about the DNA. The
tracking dye provides some information.
In addition to helping the geneticist load
their DNA sample in the well, the
tracking dye will also move in response
to electric current and as it move it
provides an indicator of how the DNA is
moving. In Fig. 17, the dye is now seen
to consist of a mixture of a faster moving
purple dye and a slower moving blue
dye. By observing how the tracking dye
moves, the geneticist can determine that
current is moving through the gel.
However, in room light, the DNA is not
visible.
The race in action
Fig. 18. The ethidium bromide stained DNA can
be observed running through the gel with a UV
light source.
Because DNA lacks color, another type
of dye molecule that binds specifically to
DNA is added to the electrophoresis
buffer or to the gel. A commonly used
dye is ethidium bromide. This dye has a
structure that allows it to bind to the
DNA helix and stay there. (note:
Ethidium bromide’s DNA binding
abilities make it a mutagen. Molecular
geneticists wear gloves to prevent this
dye from binding to the DNA in their
own cells.) Ethidium bromide has an
orange color in visible light but it’s real
power for detection comes in the
ultraviolet range of wavelengths. In UV
light, the dye fluoresces brightly.
Therefore to see the DNA, the gel is
placed on a UV light source (Fig. 18)
The race in action
Ethidium bromide will emit a strong fluorescent
signal, but detection of DNA depends on one
additional factor; copies of the DNA segment. A
single molecule of DNA cannot emit a strong
enough fluorescent signal to be seen or detected.
Instead, the molecular geneticist attempts to have
hundreds or thousands of copies of the DNA
segments they are trying to detect and load them
in the gel well together. If the segments are the
same length, they will move at about the same
rate through the gel and form a band of DNA. The
molecules making up this band collectively bind
enough of the ethidium bromide to emit a
detectable fluorescent signal. Therefore, when we
view an electrophoresis gel that has been run and
stained, we can observe bands of DNA. This is
called the DNA fragment banding pattern. A band
contains many copies of the same length molecule.
Fig. 18. The ethidium bromide stained DNA can
be observed running through the gel with a UV
light source.
A photo finish
A gel run can last hours so a molecular geneticist
only views the finish of the DNA fragment race.
The gel is run for a set period of time and then the
stained gel is photographed. The timing of this
photo is important to properly compare the lanes
of DNA segments. If the race is ended too early,
the smaller segments may not have enough time
to get ahead of the larger segments (Fig. 19). .
Fig. 19. This gel has not run long enough to
resolve two fragments
If the race is ended too late, the smaller segments
may have already reached the end of the gel and
run off into the gel box. However, if the gel run is
ended at the correct time, DNA samples with
shorter fragments can be pictured forming bands
that have moved farther through the gel than
samples of longer fragments (Fig. 20).
Fig. 20. Running the gel longer resolves the DNA
sample into two bands.
A photo finish
When a DNA fragment banding pattern
is different between samples, genetic
differences are inferred to exist between
the sources of each sample. The
geneticist will describe this DNA
fragment pattern as polymorphic (many
forms). If the fragment banding is the
same among samples they are
monomorphic
and
no
genetic
differences are inferred from this
analysis. So, whether a DNA race ends in
a tie or whether fragments move at
different speeds, gel electrophoresis
reveals genetic information.
Fragment patterns and molecular genotypes.
One application of gel electrophoresis in DNA analysis is that it can reveal
an individual’s genotype at a specific genetic locus. In this case, the DNA
segments loaded into a sample well are copies of the DNA from one
chromosomal region or locus from a single individual. In most
applications, the DNA segment copies are made with the use of the PCR
technique. In other applications (i.e. Southern blotting), these segments
are detected among thousands of different length DNA segments that
have been run through the gel. Molecular genotypes can be inferred from
the fragment banding pattern observed if one keeps in mind what is
happening
at
the
molecular
level.
Fig. 21. The genotype of an individual at a
specific locus can be inferred from a fragment
banding pattern.
We will describe the molecular basis of a genotype using a diploid
organism as an example. In any tissue source from which we obtain DNA,
this organism will have two copies of every chromosome and thus two
copies of each gene on those chromosomes. If copies are made of the
DNA at a specific locus, half of these copies in our sample will originate
from the locus of one chromosome and the other half from the
homologous chromosome. We can use electrophoresis to observe these
DNA segments and determine if the organism was homozygous or
heterozygous with respect to the DNA at that locus. Homozygous
individuals will have the same DNA sequence at this locus in both
chromosomes. Heterozygous individuals will have two different versions
of this DNA. If the DNA sequence differences that occur result in a longer
DNA segment from one chromosome (Fig. 21, lane 1) and a shorter
segment from the other (Fig 21, lane 2), then we can visualize this
difference in the electrophoresis banding pattern observed in the gel. If
an individual is homozygous at the locus being analyzed, both
chromosomes have the same DNA (Fig 21 lanes 1 and 2). If an individual is
heterozygous at the locus, the DNA segment copies from one
chromosome will be different than the DNA segment copies from the
homologous chromosome (Fig. 21, lane 3). We will therefore see more
DNA bands in our gel from a heterozygote.
Fragment patterns and molecular genotypes.
Fig. 21. The genotype of an individual at a
specific locus can be inferred from a fragment
banding pattern.
The molecular genotypes just described are
illustrated in the animation. Sample A or
sample B would be from individuals that are
homozygous and sample A & B would be
from a heterozygous individual. In this case,
one version of this locus has two smaller
segments of DNA and the other version has
one longer segment. Therefore, the
heterozygous sample contains all three DNA
segments. One segment is observed in one of
the homozygous types and the other two
DNA segments observed in the other
homozygous type. The DNA sequence
differences that result in the DNA segment
lengths are described in another lesson.
Alternative gels
Fig. 21. The genotype of an individual at a
specific locus can be inferred from a fragment
banding pattern.
The basics of DNA analysis described in this lesson can be applied to “modern”
DNA genetic analysis that is becoming more common-place: DNA sequencing
and high throughput DNA analysis. In DNA sequencing, the exact nucleotide
order of a segment of DNA is determined. Here, an electrophoresis gel system is
used that can separate fragments that differ in length by a single nucleotide.
This is done by employing three technical alterations to the electrophoresis.
First, acrylamide is used rather than agarose. This creates a more dense gel
matrix that has a higher resolving power to separate DNA segments. Second, a
longer gel is run. This is synonymous with a longer race that provides more
spread between the fastest and the slowest fragments. Finally, the DNA
segments are often labeled with a fluorescent dye and allowed to run past a
detector and off the gel. The detector then sends this information to a
computer. This allows the geneticist to worry less about timing a single photo
finish of a gel and more information can be obtained from each gel. Therefore,
the gel electrophoresis methods can be modified, depending on the analysis
needs of the geneticist.
When genetic analysis needs to be applied to many regions of the
chromosomes on hundreds of individuals, the time and expense of pouring gels
can become a limitation. Therefore, capillary electrophoresis systems have been
developed. In this system a lane in a gel is substituted with a capillary tube
which is the matrix. DNA forced into the capillary tube is labeled with some
detectable molecule and moves through the tube in the same manner as has
been described with gel electrophoresis. The advantage of the capillary system
is that electrophoresis runs can be done in less time compared to gels. The tube
may also be used again and again. The capillary system can also be integrated
into an automated system that uses robotics to pipette arrays of samples into
the tubes. Consequently, the capillary system has some advantages over
traditional slab gel electrophoresis when a high through-put of samples is
required.
RNA and Protein electrophoresis
The electrophoresis of RNA and proteins has a longer history than DNA
electrophoresis. This is because these molecules are shorter in length than DNA
in a cell and techniques did not need to be developed that either made a
discrete segment of DNA (PCR) or detected that segment (Southern Blotting).
RNA and proteins have other properties, however, that influence the
electrophoresis methods used to separate them. One property is the formation
of a secondary structure. Both molecules are single chains of subunits and these
subunits can interact, forming loops, hairpins or sheets. If the molecule retains
this secondary structure in the gel, it's mobility will not be directly related to it's
length. This can be remedied by treating the molecules physically or chemically
to eliminate secondary structure. RNA can be coated with formaldahyde or
dimethyl formamide while protein can be treated with a detergent called
sodium dodecyl sulfate (SDS). The treated molecules do not retain their
secondary structure and thus move through an electrophoresis gel as linear
chains. An additional issue with proteins is that the amino acid subunits will
each have their chemical properties. Some amino acids have a negative charge,
some a positive charge and some no charge at a given pH. The amino acid
charges will determine the overall charge of a protein and thus how it moves in
an electrophoresis gel. Treating proteins with SDS tends to create a uniform
negative charge on the molecule. Proteins treated with SDS will thus have a
negative charge, no secondary structure and an SDS gel will separate proteins
based on their length. Thus the electrophoresis method has wide applications
in molecular genetics but requires modifications based on the molecule of
interest and the question being addressed.
Fig. 21. The genotype of an individual at a
specific locus can be inferred from a fragment
banding pattern.
Summary
Electrophoresis is the method used by molecular
geneticists to observe DNA, RNA and proteins and
compare their relative lengths. This observation is
indirect. A single segment of DNA and the dozens,
hundreds or thousands of nucleotides that make
it are too small to see. However, careful
deployment of this technique and thoughtful
interpretation of the fragment banding pattern
can reveal the smallest differences in DNA
sequences that account for the genetic variation
among living things.