Name:_________________________________
DIAGNOSING DISEASE USING GEL ELECTROPHORESIS
GEL ELECTROPHORESIS & A REAL-WORLD APPLICATION
Gel electrophoresis is a technique that allows us to separate
certain macro-molecules, like fragments of DNA, based on their size.
The sample DNA is placed in a gel, which is submerged in a salt
solution and subjected to an electric current. The phosphate group in
DNA is negatively charged (Figure 1), so fragments travel towards the
positive node of the electric current (positive electrode). The shortest
fragments travel the farthest and fastest, whereas the larger fragments
will move slowly through the matrix of the gel.
There are many uses for this technique, including paternity
testing and in the study of diseases and mutations. Huntington’s
disease is a brain disorder that impairs a person’s ability to move,
speak, and think. It is an autosomal dominant disease, meaning that it
does not occur on the sex chromosomes, and that you only need 1 copy
of a defective allele to develop Huntington’s disease. Defective alleles
have a larger number of repeats of the nucleotide sequence CytosineAdenine-Guanine (Figure 1). These kinds of differences in allele sizes
can be visualized using gel electrophoresis.
Figure 1. One side of a fragment of DNA
showing the nucleotides Cytosine, Adenine,
and Guanine (top to bottom). The negatively
charged phosphate groups are circled in grey.
LAB ACTIVITY – DIAGNOSING CULLENOSIS
In this lab, you will use gel electrophoresis to determine which
of your patients has the disease Cullenosis. Symptoms include an iron-deficiency, elongated canines, pale skin,
and a sensitivity to sunlight. Cullenosis is also an autosomal dominant disease, but it is caused by an abnormally
small allele. Rather than use DNA, we will use dyes. Dyes contain charged molecules of different sizes, and
thus can behave in a similar way as would fragments of DNA.
PROCEDURE
MAKING AN AGAROSE GEL
MATERIALS: 1 gram of Agarose powder, 100 mL of buffer solution,
Erlenmeyer flask for mixing, electrophoresis chamber (Tupperware), gel comb, tape,
plastic knife, towel / oven mitt, disposable gloves, extra buffer solution
1. Hang your gel comb across your electrophoresis chamber (Tupperware) 1 – 2 inches from one side (you
may need to use tape). The ends of the teeth should be close to, but not touching, the bottom or the sides
of the chamber. Make sure the tips of the teeth are as straight as possible. While 1 – 2 people do this,
other team members can begin Step 2.
2. Measure out the appropriate amount of Agarose powder and buffer solution. Add both to your flask and
gently swirl a few times. This will make a 1% agarose gel.
3. Microwave the flask for up to 2 minutes, stopping to swirl the beaker every 20 seconds so that it does
not boil over. CAUTION: BEAKER & SOLUTION WILL BE HOT!
4. Once you can no longer see any small pieces of clear gel floating in your flask, return to your station.
Swirl the flask gently with the bottom beneath running water for 15 – 20 seconds before pouring the
solution in to your chamber. Make sure your comb has not moved. Wash and scrub the flask with soap.
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5. Let the liquid cool and solidify in to a gel (25 – 30 minutes).
6. Once the gel has solidified, put on your gloves and gently pull the comb straight up and out of the gel.
7. Use a plastic knife to cut the gel in to a rectangle (see Figure 2 below). Cut a little gel from the top, and
more off from the bottom. Do not cut the sides of the gel. Be sure not to cut through any of the wells.
8. Throw extra scraps of gel in to the trash, and fill your chamber with extra buffer solution so that it is just
deep enough to cover the top of the gel and fill the wells. Cover your chamber if you aren’t going to
immediately run the gel.
Wells
Agarose gel
Chamber
containing
buffer solution
Negative electrode
(black alligator
clip & wire)
Positive electrode
(red alligator clip
& wire)
Power source
Figure 2. Gel electrophoresis chamber set up.
LOADING & RUNNING THE GEL
MATERIALS: micropipette, micropipette tips, samples of patient DNA (dye mixture), seven 9 V batteries,
stainless steel wire & alligator clips for electrodes, disposable gloves
Make sure you remember which patient’s sample was put in which well.
Write the patient number next to the appropriate well on the figure below.
1. While wearing your gloves, set your micropipette to 10 microliters.
2. Insert the end of the micropipette in to a plastic tip. Flick your patient’s DNA to mix it thoroughly.
Depress the plunger to the first stop and suck up the patient’s DNA sample. Gently insert the plastic tip
in to the appropriate well in the gel, and slowly depress the plunger to the second stop to load the
sample in to the well. Discard this plastic tip in to the designated container by pressing the eject button.
3. Repeat Step 2 for each of your patients in a new well, letting each member of your team have a turn.
4. Link your 9 V batteries together in an assembly. Connect positive node of one battery to the negative
node of the other (See Figure 2). This is your power source.
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5. String a wire through each of the holes in the side of the Tupperware, making sure the section of wire
in the Tupperware spans its width, is resting in the buffer solution, and is on or near the bottom of the
Tupperware. Don’t let the wire touch the gel.
6. Attach the black alligator clip to the wire closest to the wells, and then to the negative node of your
power source. Attach the red alligator clip to the other wire, and then to the positive node of your
power source. Your set up should look pretty much like what you see in Figure 2.
7. Place the lid on your electrophoresis chamber. Let your gel run for 30 – 45 minutes.
8. Disconnect the clips from the power source and take the top off your chamber. Place the chamber over
a white sheet of paper so you can easily see all of the fragments of DNA.
COLLECTING THE DATA & DIAGNOSING PATIENTS
On the figure below…
1. Label which patient was associated with each well, and use colors to draw where you saw bands of dye.
2. Cullenosis is caused by the smallest allele (smallest fragment). Across all of your patients, which
fragment of DNA is the smallest? Circle this fragment. Patient(s) with this fragment have Cullenosis.
3. Rank the colored fragments from largest to smallest
______________ > ______________ > ______________ .
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