Human Genetic Disease
Each year most of us are stricken with or at least exposed to infectious diseases
of all types. For example, diseases such as the flu or the common cold are
usually caused by viruses that enter our bodies and do their damage. There is,
however, another type of disease that lies hidden and often unrecognized in the
DNA of our own cells. These are human genetic diseases. While some are
detected at birth, others may not reveal themselves until late in adult life.
One such genetic disease is sickle cell anemia, a serious disease that is often
found in individuals who trace their ancestry to either the northern Mediterranean
or to Africa. In this disease, it is the hemoglobin molecule of red blood cells
which is affected. In humans, the hemoglobin molecule consists of four
polypeptide chains. The most common type of hemoglobin, known as
hemoglobin A, consists of two identical alpha chains and two identical beta
chains. In those individuals who have the mutation for sickle cell anemia, the
beta chain of the hemoglobin has acquired a mutation that results in the change
of a single amino acid from a glutamate to valine. This single amino acid
substitution causes the hemoglobin to form insoluble chains when the oxygen
concentration of the blood is low, resulting in the distortion of the cell and giving it
the characteristic sickle shape. This distorted shape gives rise to a number of
symptoms.
First, the sickled blood cells become hard and inflexible. As a consequence,
blood no longer flows freely through capillaries. This reduced blood flow causes
significant swelling and pain. Second, sickle-shaped red blood cells are
significantly less hardy than normal red blood cells. They are removed from
circulation prematurely which gives rise to both anemia and the production of a
yellow pigment (bilirubin), one of the by-products of the breakdown of
hemoglobin. If the liver, which is the site of elimination of the bilirubin, cannot
metabolize bilirubin fast enough, jaundice will occur.
While the sickle cell mutation in the beta chain of hemoglobin is required for the
sickle cell shape, environmental conditions also play a key roll in both the
appearance and severity of the disease. While sometimes there is no obvious
reason of the occurrence of an episode, it can be brought on by infection,
dehydration, high altitude, cold weather or even overexertion. At this time, there
is no cure for sickle cell anemia. Instead, antibiotics to suppress secondary
infections, bed rest, and pain killers are often prescribed for symptomatic relief.
This unit is organized into three parts: a pre-lab, a laboratory investigation and a
post-laboratory series of discussions and exercises. The Pre and Postlaboratory experiences will occur in the school science classroom, while the
laboratory exercise will take place at the University of South Carolina where the
experiments will be explained and guided by faculty and staff of the Department
of Biological Sciences. During the laboratory portion of the project, students will
construct their own agarose protein gels and assay protein samples for the
presence of the defective sickle cell globin protein, using known mutated and
normal globin proteins as control markers.
Pre-Lab Activities
Pre-Lab Materials
Video of doctor/patient discussion
Pre-Lab Engagement
(These activities need to be completed before attending the university.)
Before attending the university, students will participate in the introduction to the
Biologica program with their teacher. This program is designed to give the
students an initial introduction to genetic and inheritance. Students will also view
a video of a doctor and a patient discussing symptoms of Sickle Cell Anemia.
Students should take notes while watching the video to use in the pre-lab
discussion at the university.
Protein Electrophoresis: An Overview
(Solutions and sample preparation are given in "Laboratory Preparation")
The laboratory exercises are based on the fundamental principles addressed
during the Pre-lab exercises. More specifically, the students will take advantage
of the fact that a charge difference exists between normal and sickle cell
hemoglobin, and this charge difference can be visualized by subjecting the
proteins to agarose gel electrophoresis. Each student will receive three samples
of hemoglobin: 1. normal hemoglobin; 2. sickle cell hemoglobin; and 3. an
unknown "patient" sample that may have either normal, (homozygotic); sickle cell
(homozygotic); or a combination of normal and sickle cell (heterozygotic carrier)
hemoglobin. The samples of hemoglobin are loaded into the agarose gel and
subjected to a 100 volt electrical field. After this electrophoresis, the rate of
migration of the unknown sample will be compared to that of the two unknown
samples.
GENERAL INSTRUCTIONS:
A. Preparation of a 1.3% agarose gel.
Students will dissolve a pre-weighed sample of agarose in the electrophoresis
buffer. The agarose will be contained in a 125 ml flask that will allow the
students to rapidly and safely bring the agarose/electrophoresis buffer to a full
boil in a microwave. Once the agarose is completely dissolved, the gel will be
cast as indicated in Figure 1.
Fig. 1
Flask
Hot agarose
Gel Casting Tray
It is important at this stage to emphasize to the students that the gel itself will act
as an obstacle course in which each negatively charged molecule will be pulled
by an electric current toward the positive pole. Since the proteins in this
experiment are the same size and the starting line is the same for each sample, it
is the charge of the individual hemoglobin molecules that will determine which
hemoglobin migrates faster.
B. Preparation of the gel electrophoresis box
The agarose gel will be formed in a casting tray. The tray needs to be sealed at
two ends. This is accomplished by inserting the tray sideways into the
electrophoresis chamber so that the two rubber gaskets touch the chamber walls.
Once the melted agarose has been poured into the gel tray a plastic comb will be
placed in the agarose. Slots are pre-cut into the tray to insure correct
positioning. Once the gel has set up (ca. 15 minutes), the tray is gently lifted
from the chamber and rotated so that the comb is closest to the negative (black)
electrode on the electrophoresis chamber. This is essential so that the
negatively charged proteins will migrate toward the positive (red) electrode.
Once the gel tray is properly positioned, the electrophoresis buffer is added. To
insure that the electrical field is even across the entire gel, the buffer must be
poured slowly into the electrophoresis chamber until it just covers the gel and
completely fills the wells. DO NOT ADD THE SAMPLES UNTIL AFTER THE
ELECTROPHORESIS BUFFER HAS BEEN ADDED!
C. Loading the samples
The comb is gently pulled straight up and out of the gel to create small, parallel
wells for samples. Each gel is sufficient for two groups to perform
electrophoresis and evaluate their known and unknown hemoglobins. They will
put 15 µl the patient sample, 15 µl of a normal hemoglobin and 15 µl of sickle cell
hemoglobin into three separate wells. In the fourth well, each team should place
a sample of bromphenol blue tracking dye so they can follow the movement of a
low molecular weight molecule through the gel. For purposes of comparison,
each working group should load their gels in the following order:
1.
2.
3.
4.
Normal hemoglobin
Sickle cell hemoglobin
Unknown patient hemoglobin
Bromphenol blue tracking dye
The unknowns will be different, but they all will be one of three possible protein
combinations: (a) Homozygous normal; (b) Homozygous sickle cell; and (c)
Heterozygous Normal and sickle cell.
D. Electrophoresis of the proteins
Each of the electrophoresis gel boxes is constructed so that the chamber is
covered during the actual electrophoretic run. You must be sure that the
appropriate color cable (red or black) attached to the lid corresponds to the
correct end of the box. After putting the lid on the box, connect the cables to the
appropriate terminals (red to red; black to black) of the power supply and turn it
on and set the voltage at 100 volts. Run the gels until the bromophenol blue dye
has migrated about 30 mm from the wells. Because the hemoglobin has a
distinct color of its own, the students will also be able to follow the migration of
each of their samples. Thus, it is necessary to run the gels only as long as it
takes to clearly separate the individual bands in each lane.
E. Analysis of the Results:
The students will be expected to identify which of the two known hemoglobins
(normal or sickle cell) migrates faster in the gel. These standards are then
compared to their unknown hemoglobin sample. Because each group will have
one of three possible combinations of hemoglobin, results will vary. Some gels
will display two bands representative of sickle cell trait (heterozygosity), while
others will show only one band corresponding to homozygosity of either the
sickle cell or the normal hemoglobin (Figure 2). As a part of the exercise,
students will be expected to write down both their results and their interpretation
of the results in either a laboratory notebook or on a separate piece of paper.
Fig.2
Because each of the working groups has a different unknown, it is useful to have
a representative of each group bring their gel to the front of the class. The deep
color of the hemoglobin makes it possible to visualize the banding patterns by
simply placing the gels on an overhead projector. As a part of the discussion of
the class results, the instructor can ask the students to identify which of the
unknown patients have common hemoglobin beta genes. Another question
might be how can there be two bands in some lanes while only one in others?
Human Genetic Disorders:
Laboratory Preparation
Materials
Hemoglobin A
Hemoglobin S
Glycine
Tris-base
Glycerol
Bromophenol blue
Agarose
Sample Preparation
A) Hemoglobin Samples
1) Prepare 500 ml of 1.5M Tris, pH = 9.2.
Weigh out 90.86 g of Tris-base and place in a 1 L beaker
Add approximately 400 ml of dH2O to the beaker
r & add dH2O to 500 ml final volume
2) Dissolve HgA and HgS in 5 ml of 1.5M Tris, pH = 9.2.
Hemoglobin is purchased in 100 mg aliquots. Add 5 ml of 1.5M Tris, pH = 9.2
directly to the bottle the hemoglobin was shipped in. Gently swirl to mix.
Remove five -1 ml aliquots and freeze at -20 C.
3) Prepare 2X Sample Dye by mixing the following:
Glycerol - 8 ml
1.5M Tris, pH 9.2 - 4 ml
Deionized H2O 28 ml
Bromophenol Blue 0.01 g
Store at 4 degrees C.
4) Prepare Hemoglobin Sample Mixes:
HgA
HgA/S
1.5 M Tris, pH 9.2 270 µl
230 µl
2x Sample Dye
500 µl
500 µl
HgA
230 µl
115 ul
HgS
--115 ul
HgS
270 µl
500 µl
---230 µl
This recipe will make approximately 50 aliquots of each type.
5) Aliquot Hemoglobin samples:
Label microcentrifuge tubes “N”, "S" and “U”.
Into tubes labeled “N” add 18 µl of Hg A mix.
Into tubes labeled "S" add 18 ul of HgS
Into tubes labeled “U” add 18 µl of either HgA, HgA/S, HgS mix.
Each student will load 15 ul of Sample “N”, 15 ul of Sample "S" and 15 ul of
Sample “U” into a well. Sample “N” is the normal control, sample "S" is the sickle
cell control, and Sample “U” is the unknown hemoglobin of the patient they are
testing. Patient samples should be distributed amongst the students so that all
results (normal-HgA, carrier- HgA/S, and sickle-HgS) will be represented.
B) Hemoglobin Electrophoresis Buffer
1) 10X Stock
1 liter
2 liters
Tris
160 g
320 g
Glycine 72 g
144 g
Add approximately 700 ml (or 1400 ml if preparing 2 liters) of dH20.
Add a magnetic stir bar and mix on stir-plate until dissolved.
Pour into a 1 liter (or 2 liters) graduated cylinder and add dH2O until the final
volume is 1 liter (or 2 liters)
Pour the electrophoresis buffer into a storage bottle and label.
2) 1X Working Solution: For each electrophoresis apparatus to be used:
Add 50 ml of 10x Stock to an empty 500 ml cylinder.
Add dH2O to 500 ml “fill line”.
Cover top and mix very well.
Label student bottles “Hemoglobin Electrophoresis Buffer”.
Fill student bottles with 500 ml of 1X Working Solution.
Laboratory Station Set-up (for groups of four students)
• 125 ml flask containing 650 mg agarose
• gel electrophoresis box with casting trays
• 1 comb
• hemoglobin samples in buffer
• yellow pipette tips
• 500 ml of electrophoresis buffer and a 100ml graduated cylinder
• practice loading dye
• laboratory protocol
• 4 data/observation sheets
Human Genetic Disorders:
Laboratory Protocol
Part I: Preparation of the gel
At your station, you will find a 125 ml flask containing 650 mg of dry agarose.
This is the correct amount of agarose to make 50 ml of a 1.3% agarose solution.
WAIT for the instructor to demonstrate the use of the equipment. Then follow the
procedure below.
Step 1:
Use the 100 ml graduated cylinder to measure 50 ml of electrophoresis buffer
Pour 50 ml of buffer into the flask containing the agarose powder.
Step 2:
Carefully swirl the mixture to suspend the agarose.
Step 3:
Give the flask with the agarose-buffer solution to an instructor who will put it into
a microwave for about two minutes to melt the agarose. The instructor will notify
you when the mix has cooled and is ready to pour.
StopHere/StopHere/StopHere
Wait for the instructor to demonstrate pouring the gel and cleaning the flask.
Step 4:
Place the gel tray into the gel box so that the rubber gaskets seal it against the
wall of the gel box. This will prevent the liquid agarose from running out of the
gel tray while it is setting up.
Step 5:
Put the box on a level surface.
Step 6:
When your group is called come to the instructor to retrieve your flask containing
the melted agarose.
.
CAUTION!!
The flask is hot. Hold the flask by the top. DO NOT grab it
by the base.
Step 7:
Pour the agarose into the center of the casting tray.
Step 8:
Insert a comb into the slots at the end of the casting tray. This comb will create
the wells into which you will place your hemoglobin samples once the gel has
had a chance to harden.
Step 9:
Rinse the flask five times with running tap water.
Hang the flask on the drying rack above the sink.
Step 10:
Wait for the gel to harden. It will become opaque and firm to the touch in about
15 minutes.
StopHere/StopHere/StopHere
Wait for the instructor to demonstrate the removal of the comb.
Step 11:
Remove the comb from the tray. The depressions left in the gel are referred to as
"wells".
Step 12:
Carefully remove the gel tray from the electrophoresis box, rotate it and place it
back in the box with the wells close to the black (negative) electrode. Your gel is
now ready for addition of the electrophoresis running buffer and the loading of
your sample proteins.
Step 13:
Slowly pour the contents of the bottle labeled "Electrophoresis Buffer" into the
electrophoresis box. Fill the electrophoresis box until the gels are covered with a
2 - 3 mm layer of buffer and the wells are filled with buffer. You can pour the
buffer directly from the bottle.
StopHere/StopHere/StopHere
Wait for the instructor to demonstrate how to use micropipettes and load
samples.
Part II: Loading the gel
Be sure to practice loading gels before you load your samples in the wells.
Technique for loading gels:
Fig. X Correct way to insert the pipette into well
Fig. X Incorrect way to insert the pipette into well
Step 1:
Your instructor will demonstrate for you how to correctly use a micropipette. The
micropipettes are very precise instruments that will allow you to measure
accurately very small volumes. BE CAREFUL! These instruments are quite
expensive and dropping them, even on a counter top, will damage them.
Use a micropipette and a new tip to put 15 µl of normal (N) hemoglobin into a
well. Keep a record on your data sheet of the well into which you put sample N.
Step 2:
Use a micropipette and a new tip to put 15 µl of sickle cell (S) hemoglobin into
the next well. Keep a record on your data sheet of the well into which you put
sample S.
Step 3.
Use a micropipette and a new tip to put 15 µl of your unknown (U) patient's
hemoglobin into the next (3rd) well. Keep a record on your data sheet of the well
into which you put sample U.
Step 4.
Use a micropipette and a new tip to put 10 µl of the Bromphenol blue tracking
dye into the next (4th) well. Keep a record on your data sheet of the well into
which you put the dye.
Step 5.
Since two working groups can use one gel having 10 lanes, the above procedure
is now repeated by the second group using lanes 7 through 10 as though they
were 1 through 4.
Caution: AFTER LOADING YOUR GEL, GENTLY CLOSE THE LID OF
THE ELECTROPHORESIS BOX TO AVOID EXPOSURE TO HIGH VOLTAGE.
StopHere/StopHere/StopHere
*** DO NOT TURN ON THE POWER SUPPLY!***
Wait for an instructor to assist you.
Step 6.
Turn on the power supply and adjust the voltage up or down until the unit reads
100 volts. You gel is now running!
_____________________________________________________________
AFTER LUNCH!
Wait for an instructor.
PART 4: Ending electrophoresis and recovering
the gel
Step 1:
Turn off the power switch on the power supply.
Step 2:
Disconnect the cables from the power supply.
Step 3:
Open the lid of the electrophoresis box.
Step 4:
Gently lift the gel tray out of the box.
Step 5:
Hold the clear plastic bag provided at your lab bench and label the top of the bag
with your initials.
Step 6:
Gently slide the gel off the tray and into the plastic bag. Seal the bag.
Part V: Analysis of the gel
1. Observe the banding patterns on your gel and record the results on your data
sheet.
2. What is your conclusion? Which of the two known hemoglobins ran the
fastest?
What was the composition of your unknown sample?
What does this tell you about the probability that your patient will have sickle cell
anemia, be a carrier (sickle cell trait) or be totally free of the disease?
Based on what you know about how genes are inherited, what is the chance that
a child with two heterozygous parents will have the same genotype as your
patient?
How do you know this?
Write your answer on your data sheet.
Name______________________________ Group Number: _______
Human Genetic Disorders:
Data/Observation sheet*
I. Preparation of the Agarose Gel:
1. The agarose gel plays what role in this experiment?
2. The comb does what in this experiment?
3. What would have happened to the rate of electrophoresis if you used 0.065 g
of agarose instead of 0.65 g? What effect would that have on your experiment?
II. Preparation of the Gel Electrophoresis Box:
1. Why did you not put the wells of the gel at the positive (red) pole?
2. What role does the electrophoresis buffer play?
III. Loading the Samples:
1. What was the most difficult problem you encountered when loading the
samples into the wells?
2. Predict what will happen to the hemoglobin molecules after the gel is loaded
and the electric current is applied. Why?
3. Diagram the final results as they would appear on the gel if the patient (sample
U) has sickle cell anemia.
4. Diagram the expected results if the patient (sample U) has sickle cell trait.
(1.) Use the diagram below to record where you loaded each sample.
LANE #:
SAMPLE TYPE
1.
____________________________ Work Group 1
2.
____________________________
3.
____________________________
4.
____________________________
7.
____________________________ Work Group 2
8.
____________________________
9.
____________________________
10.
____________________________
(2.) Record the results of the electrophoresis (sketch the results).
Lane#
1
2
3
4
5/6(empty)
7
8
9
10
Conclusions:
1. What was the purpose of sample N and S?
2. Based on your results, what have you learned about the hemoglobin from your
patient (sample U)?
3. Assuming that sample U came from a person you were testing for sickle cell
anemia, what would be your conclusion?
4. What are the chances that two parents of your patient's genotype will have a
child with sickle cell anemia?
Group 1:
_______________________________________________
Group 2:
5. Assuming that your patient does not know or understand how the test works,
how might you explain your results to them?
Post-laboratory Exercises:

Discussion
- Review gels from previous day at USC
This is an opportunity to take the student gels and look at some length at each
set of results using an overhead projector or light box if the class is small
enough. Not only should the students reinforce their conclusions by doing the
appropriate Mendelian genetics, but they should also give serious consideration
to any inconsistent or missing data. It is during this period that the students
should begin to understand that the data is real, not the "proposed" correct
answer. If the two are in conflict, this is the perfect opportunity for the teacher to
reinforce the basic concept that data is data. If inconsistent, it needs to be
reconciled. That is a fundamental premise of science. Usually this means that
there was an error in the experimental protocol. Perhaps lanes were loaded
backwards or mislabeled, or perhaps a well never got filled due to a pipetting
error. None the less, the students should be asked, "If the data is consistently in
conflict with the hypothesis, what must be wrong?" (e. g., the hypothesis has an
error that must be fixed.)
-
Go over questions, ask questions and in any other way try to assess if the
students have understood the principles of the experiment and
experimentation itself.