Dangerous Ideas and Forbidden Knowledge, Spring 2005
Lab 3
Part 2: In Search of the
Sickle Cell Gene
OBJECTIVE:

Visualize our Southern Blot and interpret the results
General Background:
Last week in lab we carefully loaded digested (cut) genomic DNA from three controls,
and three unknowns (individuals) into gels for electrophoresis. The DNA electrophoresis sorted
the DNA fragments based on their size. We then transferred these sorted fragments onto a more
durable membrane using a technique known as a Southern Blot. Today in lab we will probe this
blot, looking for the sickle cell gene!
We’re able to find this specific gene
fragment among all the thousands of fragments in
your gel by taking advantage of the
complementary nature of the DNA molecule. As
we discovered last week “A” always pairs with
“T”, and “C” with “G”. DNA is normally found in
its double-stranded form but in the case of our
membrane, one of the solutions you soaked your
gel in separated the two strands. Thus you now
have single-stranded DNA on your membrane.
We will know take this membrane and
soak it in a solution containing a probe. A probe
is a short piece of single stranded DNA of a
specific known sequence to which a dye has been
attached. (Thus in the diagram above, our probe has the sequence TCCGATA and is labeled with
a dye, indicated by the letter F.) In our case, the probe we will use is specific to the sickle cell
gene. Because of the complementary nature of the DNA molecule, this probe can only bind to
the sickle cell sequence. (Just as the probe shown above can only bind to the corresponding
sequence: AGGCTAT.) It will bind, carrying a blue dye with it, and allowing us to find the
sickle cell gene!
Our next challenge will be to determine if these three individuals, our unknowns, have
two good copies of this gene, a normal and a mutant copy, or two mutant copies. We are able to
do this because one of the restriction enzymes (molecular scissors) we used can cut the normal
hemoglobin gene, but not the mutant sickle version. Thus the normal version of this gene will be
present as a slightly smaller fragment, and will have traveled farther in our gel.
Procedure:
1. Carefully identify your membrane from last week and place it in a staining tray, trying to
avoid getting fingerprints on the membrane. Note that I marked the positions of your wells
using a blue ink pen. This is the side of the membrane that has the DNA on it; it should be
facing up in your staining dish.
2. Pour enough of the Blue-Blot staining solution into your tray to cover the surface of your
membrane. Allow the membrane to soak at room temperature 10-15 minutes.
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Dangerous Ideas and Forbidden Knowledge, Spring 2005
3. Pour the staining solution off into the waste beaker provided, and refill your staining dish with
water for rinsing. Shake gently for ~ 1 minute and pour this water down the sink.
4. Repeat the rinse step 3 to 4 times or until the bands are clearly visible. Record your results in
the space provided below.
Results: Answer the following questions. Note that you should turn in this sheet.
1. In the space below, draw a diagram of your results, indicating both where you loaded each
sample, and the relative positions of the bands on your membrane.
2. Based on these results, identify the mother, father, and child as affected with the disease,
carriers of the disease, or “normal” (wild-type).
Mother: ____________________
Child: ______________________
Father: _____________________
3. Carriers of the sickle cell disease will display two bands on the southern blot. In your own
words, explain why two bands are visible.
4. Consider two carriers of the sickle disease. If these two have a child together, what is the
probability that their child will have the disease? Explain how you determined this
probability.
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