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 Post-laboratory 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. 1Flask
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. Normal hemoglobin
2. Sickle cell hemoglobin
3. Unknown patient hemoglobin
4. 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
Add magnetic stir bar and mix on a stir plate until dissolved
Adjust the pH to 9.2 using HCl
Pour into a 500 ml graduated cylinder & add dH2O to 500 ml final volume
Pour into a 500 ml bottle, label, and use as needed.
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 -20C.
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 HgS
1.5 M Tris, pH 9.2 270 µl 230 µl 270 µl
2x Sample Dye 500 µl 500 µl 500 µl
HgA 230 µl 115 ul ----
HgS --- 115 ul 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
Waitfor 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 mmlayer 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!
Waitfor 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.