Mystery of the Crooked Cell
By Donald A. DeRosa and B. Leslie Wolfe
Adapted by The Institute for Genomic Research
This document contains information and handouts needed to prepare
the MdBioLab activity, Mystery of the Crooked Cell, an investigation into
the molecular basis of sickle cell anemia.
Table of Contents
T EAC HER
M AT ERIALS
Background Information
1
Pre-Lab Activity Information
2
Characterization of Sickle Cell Anemia Event
Flow Chart
4
Laboratory Explanation
5
Laboratory Preparation
9
Extension Activities
ST UD ENT
ACT IVIT Y
12
HANDOUTS
Patient Description
Station A, B, C & D Guide Sheets
Laboratory Protocol
Observation & Data Sheet
A Closer Look at the Cause of Sickle Cell Anemia
Documents 1, 2 & 3
AND
LABO RATO RY
PRO TOCO L
TEACHER MATERIALS
Background
Sickle cell anemia is a genetic disease that affects the hemoglobin molecule in red blood cells.
Normal red blood cells are round like doughnuts, and they move through small blood vessels in
the body to deliver oxygen. Diseased red blood cells become hard, sticky and shaped like sickles
used to cut wheat. Then these hard and pointed red cells go through the small blood vessels, they
clog the blood flow and break apart. This can cause pain, damage and a low red blood cell count
or anemia.
Each person has two copies of the gene for hemoglobin. Normal hemoglobin is referred to as
Hemoglobin A. The letters AA are used to indicate that both hemoglobin genes are normal. The
gene that causes sickle cell anemia is referred to as Hemoglobin S. There are three possible
combinations of the genes for hemoglobin:
AA
AS
SS
Individual is homozygous for the hemoglobin A gene. So both copies of hemoglobin
code for normal hemoglobin and the person does not have the disease.
Individual is heterozygous. One copy of hemoglobin codes for normal hemoglobin
and the other copy of the gene codes for sickled hemoglobin. This person does not
have the disease and will not develop it later in life.
Individual is homozygous for the sickled hemoglobin S gene. So both copies of
hemoglobin code for diseased hemoglobin. This person suffers from sickled cell
anemia.
The irregularly shaped blood cells lead to a cascade of symptoms. The sickle-shaped blood cells die
prematurely resulting in anemia and the production of excess bilirubin (a yellow pigment resulting
from the breakdown of hemoglobin). Jaundice often results when the liver cannot metabolize
bilirubin fast enough.
Infection, dehydration, overexertion, high altitude, chills, or cold weather can bring on a sickling
episode, or crisis. Sometimes there is no apparent precipitating factor. People with sickle cell
disease are susceptible to fevers and infection.
There is no cure for sickle cell anemia. Hydration, bed rest, painkillers, and antibiotics are often
prescribed. Recent research has focused on re-expressing the fetal hemoglobin gene. After birth, the
gene for fetal hemoglobin turns off while the gene for adult hemoglobin becomes activated. If the
gene for fetal hemoglobin could be turned on again, it may compensate for the diseased hemoglobin
and provide relief for people with sickle cell anemia.
Sickle trait may serve as a protective mechanism against malaria. Malaria is a deadly disease found
in countries along the equator. People with sickle cell trait are protected from malaria while those
with normal hemoglobin are susceptible to it. Over the years, people with sickle trait migrated to
other continents. Sickle cell disease is seen predominantly in the African descendant populations
but is also seen in people of other ethnic groups. These ethnic groups include individuals from
parts of the Middle East, Central India, and countries bordering the Mediterranean Sea, especially
Italy and Greece.
This lesson is organized into two parts: A pre-lab and a laboratory investigation. During the prelab, students visit learning stations and acquire clues about a mystery disease (sickle cell anemia).
Each station challenges the students to explore different aspects of sickle cell anemia. Working in
groups, students manipulate models and gather data to construct an explanation of how sickle cell
anemia affects the patient at the molecular level. Following the pre-lab, students enter the
Mystery of the Crooked Cell: Background
1
laboratory where they apply the concepts acquired in the pre-lab to test a fictional patient for the
presence of sickle cell hemoglobin using gel electrophoresis.
Mystery of the Crooked Cell: Background
2
Pre-Lab Activities
The purpose of the pre-lab is to explore the connection of hemoglobin to symptoms exhibited in sickle cell
anemia. It provides students with the opportunity to construct ideas and concepts about the mechanism
of the disease based on their prior experience.
The objectives of the pre-lab are as follows:
· Observe prepared normal and sickle cell slides.
· Manipulate models of blood cells to gather data and make inferences about sickle cell anemia.
· Analyze an inheritance pattern using a pedigree.
· Work cooperatively to explain the symptoms exhibited in sickle cell anemia.
· Construct an explanation of the disease mechanism.
Pre-lab Materials
¨
¨
¨
¨
¨
¨
Four microscopes with 1000x capability
Prepared slides of sickle cell blood and normal blood. Prepared slides are available from several
biological supply companies.
One model of a red blood cell with normal hemoglobin and one model of a red blood cell with
diseased hemoglobin. We make models of red blood cells from snap lock beads and balloons.
One capillary model (Y connector) with several models of round and sickled red blood cells. We
use Model Magictm to make the cells.
Newsprint
Markers
Pre-lab Engagement (10-15 minutes)
Organize the students in groups of four. Have each team read a description of the patient who came to
Dr. Herrick, a Chicago physician, in 1904 (Patient Description File). The essential question is. “What is
the mechanism of the disease?” Instruct the students to make observations and gather clues about the
condition described in the patient scenario. Ask students to identify and underline any clues in the
description that may help them determine the effect of the disease on the patient.
When they are finished, invite a student from each team to write two clues on the board. Discuss the
clues as a class. Ask for clarification or expansion of ideas where appropriate. Encourage the students to
think freely and make connections based on the evidence given in the patient description as well as on
their own experience. The discussion usually leads to many good ideas about the mechanism of the
disease. However the students soon determine that they need to explore the disease in greater depth in
order to substantiate their ideas and gain a deeper understanding of the disease.
Pre-lab Exploration (40-50 minutes)
To assist the students in their investigation, set up the four stations described below. Each station is
comprised of manipulatives that in some way model or illustrate concepts relating to the mechanism of
the disease. Give each team descriptions of the stations (Station Sheets) that include directions that
encourage exploration. Urge the students to gather observations that may yield insights to the
mechanism of the disease. It is helpful to assign the roles of “reader” and “recorder” at each station to
facilitate cooperation among team members. Rotate each team through stations A, B, C and D, allowing
about ten to fifteen minuets per station.
Mystery of the Crooked Cell: Pre-lab Activities
3
Set up each station as follows:
Station A:
Station B:
Station C:
Station D:
Four microscopes with prepared slides of sickle cell blood and normal blood
Y connectors, clay models of normal and sickled red blood cells
One balloon model of a cell with normal hemoglobin and one of a cell with sickle cell
hemoglobin in red blood cells.
Pedigree and pedigree symbol key (Station D sheet).
Pre-Lab Explanation (20-30 minutes)
Collect and display the data and ideas collected by the class by putting four pieces of newsprint around
the room and labeling them A, B, C, and D respectively. Have each team write their observations for each
station on the newsprint, using a different colored marker for each team. If another group already writes
an idea or observation, students need not repeat it. In this manner, all the observations are recorded and
each group is required to read the observations of the other groups.
At this junction we find it helpful to ask the students to consider the information the class has collected at
the stations and to reflect individually in writing on the essential question, “What is the mechanism of the
disease?” Individual reflection gives each student time to collect and organize their thoughts in
preparation for group discussion.
After individual reflection, ask the members of each team to regroup and synthesize an explanation for
the mechanism of the disease. Next ask teams to present their explanations to the entire class. Encourage
students to be creative in their presentations by giving them the option to present verbally, in writing,
with diagrams or concept maps, or by using role-play. Students often generate many ideas and
interesting topics for discussion. Encourage the students to debate their ideas and consider them in light
of the observations they made. The discussion frequently becomes lively with considerable studentstudent dialogue. Challenge and elaborate on students’ ideas to lead them to discover the following
points:
1.
2.
3.
4.
The blood cells are irregularly shaped.
The irregular shape of the red blood cells interferes with ability to flow through the blood
pathways.
The hemoglobin units connect to each other when oxygen concentrations in the blood are
low resulting in abnormally shaped blood cells.
The condition is inherited.
Refer to the stations to assist the students’ discovery of the above points. Demonstrate the cause of
sickling using the models form station C. The blockage created by the sickled cells is illustrated in station
B while the prepared blood slides at station A indicate anemia and irregularly shaped red blood cells.
The family history suggests the possibility that the condition is inherited. At this point, the students are
usually curious about the name of the disease. Let them generate their own name for the condition based
on their understanding of it and emphasize that their name is just as valid as the name given by Dr.
Herrick. He based the name on his observations of sickle shaped cells and the decrease in the number of
red blood cells or anemia.
Mystery of the Crooked Cell: Pre-lab Activities
4
Characterization of Sickle Cell Anemia Event Flow Chart
Ask the students to make a concept map depicting what they learned today about sickle cell anemia such
as the one shown below.
Transmitted through
SICKLE CELL
Is characterized
INHERITANCE
ABNORMAL
HEMOGLOBIN
Which Reacts to
LOW OXYGEN
Causing the
Hemoglobin
to form
Which contributes to
Which contributes to
CHAINS
BROKEN
CHAINS
SICKLED
CELLS
Causing
ANEMIA
Mystery of the Crooked Cell: Pre-lab Activities
BLOCKED
CAPILLARIES
5
Laboratory Explanation
The purpose of the laboratory component is to apply the concepts developed in the pre-lab to a clinical
test for sickle cell anemia using gel electrophoresis. The objectives of the laboratory component area as
follows:
¨
¨
¨
To perform gel electrophoresis to distinguish normal hemoglobin from sickle cell hemoglobin.
To interpret the results of gel electrophoresis.
To demonstrate the concept and process of gel electrophoresis
Before proceeding with the laboratory investigation, it is necessary to make a logical connection to the
concepts developed in the pre-lab. In doing so, the laboratory component becomes a tool in the
continuum of an ongoing problem rather than an isolated end in itself. The transitional activity that
follows links the pre-lab concepts to the ensuing laboratory investigation.
With the understanding of sickle cell anemia generated by the pre-lab, ask the students to consider ways
to test for the disease. A common response is to examine the blood and look for signs of anemia or
sickled cells. Anemia however, is not unique to sickle cell anemia nor are the blood cells necessarily
sickled unless the patient is in crisis. Furthermore, thalassemic blood samples frequently look very
similar to sickle cell blood samples (thalassemia is a hemoglobin disorder associated with the defective
synthesis of hemoglobin). Since hemoglobin is the molecule affected by the disease; the conclusion is to
observe the diseased or affected hemoglobin for characteristics that would distinguish it from normal
hemoglobin.
Developing the concept for the test
The next goal is to help the students realize the conceptual basis of the test that distinguishes normal
hemoglobin from affected hemoglobin. Raise the question by holding up a tube containing a sample of
hemoglobin and ask whether they can identify it as normal or abnormal (Use red food coloring and water
to create a light rust color which simulates the color of both normal and affected hemoglobin for this
demonstration). The students realize that they first need to see what a normal hemoglobin sample looks
like in order to identify whether the unknown is normal. Place control samples of normal hemoglobin
and abnormal hemoglobin next to the unknown sample. Again ask whether they can identify which
sample is normal and which is affected by visually comparing the three samples of hemoglobin. The
samples look exactly alike. Therefore a tool is to distinguish hemoglobin samples that look identical but
have different properties. The tool, electrophoresis, becomes the laboratory component of the
investigation.
Electrophoresis role-play
A role-play is used to demonstrate the theory behind electrophoresis. Have two groups of three students
come to the front of the room. Each group represents a hemoglobin protein and each person represents
an amino acid. Note that both molecules have the same number of amino acids and are, therefore, the
same size. Give each student a card with a number representing a charge of -1 or 0. To one group assign
two -1 charges and one 0 charge. To the other group give two people 0 charges and one person a -1
charge. Consequently one group has a net charge of -2 and the other group has a net charge of -1. Point
out that the difference in overall charge between the two molecules cannot actually be seen with the
naked eye. However the charge difference does make the hemoglobin react differently in an electric field.
Illustrate this concept by telling the class to imagine the classroom as an electrical field with the positive
pole at the back of the room and the negative pole at the front of the room. In an electrical field, the
negatively charged hemoglobin molecules migrate toward the positive pole. The group with a net
Mystery of the Crooked Cell: Laboratory Explanation
6
charge of -2 will move more quickly because it has a greater negative charge drawing it toward the
positive pole. Pretend to turn on the electricity and have the two groups of students migrate as the
molecules would. The groups can be distinguished by their different rates of migration with respect to
their net negative charge. To check student understanding, have them predict and demonstrate the
migration if the molecules both had a charge of -2.
The laboratory investigation: Protein electrophoresis
(Solution and sample preparations are given “Laboratory Preparation”)
The laboratory component incorporates the same concept as described above. Each student receives
three samples of hemoglobin:
1.
2.
3.
The patient
Normal hemoglobin
Sickle cell hemoglobin.
The patient sample may represent sickle cell hemoglobin, normal hemoglobin, or both in the case of a
carrier. The samples of hemoglobin are put into an electrical field and the rates of migration compared.
Procedures vary depending on the electrophoresis system used. General instructions are as follows:
§ Prepare a 1.3% agarose gel
Dissolve by heating the appropriate amount of agarose in the electrophoresis buffer and cast the gel.
Introduce the function of the gel by comparing it to a track that provides a matrix for the migration
of the hemoglobin. The wells act as a starting gate while the gel itself holds the hemoglobin and
helps us visualizes it.
§ Prepare the gel electrophoresis box
Orient the gels in the electrophoresis box with the wells at the negative pole. Slowly pour the
hemoglobin electrophoresis buffer into the electrophoresis box. Fill the electrophoresis box until the
gels are covered with a 2 to 3 mm layer of buffer.
§ Load the samples
Put 15µl of the patient sample, 15µl of a normal hemoglobin, and 15µl of sickle cell hemoglobin into
separate wells. You can make the patient sample normal, sickled or carrier for each gel. Create a
mixture of patient samples for each class so students can compare the results.
§ Electrophoresis
Connect the cables and run the gels at 100 volts until the bromphenol blue dye has migrated about
30 mm from the wells.
Mystery of the Crooked Cell: Laboratory Explanation
7
Interpretation:
Results will vary. Some patient samples will display two bands representative of sickle cell trait, others
will be positive for sickle cell anemia, while some will be negative for sickle cell anemia. The bands on
the gel representing sickle cell trait usually present as one large band with the leading edge in line with
normal hemoglobin and the trailing edge in line with affected hemoglobin. Students write their analysis
in a notebook with evidence to support their results.
Schematic representation of hemoglobin gel electrophoresis results
Negative
Patient 1
positive
Patient 2
Positive
N
P
N = Normal
P = Patient
Negative
N
P
N = Normal
P = Patient
Mystery of the Crooked Cell: Interpretation
8
Negative
Patient 3
Positive
N
P
N = Normal
P = Patient
To facilitate discussion, choose a representative gel of each outcome and put the gels on the overhead
projector. Highlight the bands projected on the board with a marker. Some sample questions for
discussion include:
What can be inferred from the results of the test?
How can the presence of two bands in some patient samples be explained?
Mystery of the Crooked Cell: Interpretation
9
Laboratory Preparation
Ordering Information
Item
Company
Hemoglobin
A*
Hemoglobin
S*
Glycine
Phone No.
Catalog
No.
Quantity
Price
(As of 8/26/02)
Sigma
(800) 325-3010
H0267
100 mg
$104.05
Sigma
(800) 325-3010
H0392
100 mg
$119.70
Sigma
(800) 325-3010
5 kg
$141.80
Tris
American Bioanalytical
(800) 443-0600
G7126
AB0200001000
1 kg
$56.71
Glycerol
Sigma
(800) 325-3010
500 ml
Contact sales
representative
G7893
Bromphenol
Sigma
(800) 325-3010 B3269
5 mL
$9.25
blue
Agarose
VWR
(800) 932-5000 IB70042
100 g
$110.00
*Occasionally, Sigma is out of stock of hemoglobin and it takes several months to manufacture, so please
predict your needs and order early.
Sample Preparation
Hemoglobin Samples
1
2
Prepare 500 mL of 1.5M Tris, pH=9.2
1.1
Weigh out 90.86 g of Tris and place in a 1 L beaker
1.2
Add approximately 400 ml of dH20 to the beaker
1.3
Add magnetic stir bar and mix on stir plate until dissolved
1.4
Measure pH and adjust until pH is 9.2 (using HCl)
1.5
Pour into a 500 ml graduate cylinder and add dH20 until final volume is 500 ml
1.6
Pour into a 500 ml bottle, label, and use as needed
Note:
This solution will last several months. Therefore, it is recommended that the solution be
autoclaved or filtered in order to keep it free of contaminants (and remain sterile). By
doing this you will avoid remaking this solution every time you run out of hemoglobin.
Resuspend Hemoglobin
2.1
Dissolve 100 mg of HgA and/or 100 mg HgS in 5 ml of 1.5M Tris, pH = 9.2
Note:
Hemoglobin is purchased in 100mg aliquots. Add 5 ml 1.5M Tris, pH = 9.2 directly to the
bottle the hemoglobin was shipped in and gently swirl to mix. Make five-1 ml aliquots
and freeze at -20oC. This will reduce the number of times the hemoglobin has to be
handled.
Mystery of the Crooked Cell: Laboratory Preparation
10
3
4
Prepare 2X Sample Dye by mixing the following:
3.1
3.2
3.3
3.4
Glycerol
1.5M Tris, pH = 9.2
Deionized H2O
Bromphenol Blue
Note:
Store at 4oC
8ml
4ml
28ml
0.01g
Prepare Hemoglobin Sample Mixes:
HgA HgA/S HgS
1.5 M Tris, pH 9.2 270µl 230µl
270µl
2X Sample dye
500µl 500µl
500µl
HgA
230µl ----Hgs
--230µl
230µl
Note: This will make approximately 50 aliquots of each type.
4.1
Aliquot Hemoglobin Samples
4.1.1
Label microcentrifuge tubes “N” and “P”
4.1.2
Into tubes labeled “N” add 18µl of HgA mix
4.1.3
Into tubes labeled “P” add 18µl of either HgA, HgA/S, HgS mix
Note:
Each student will load 15µl of Sample “N” and 15µl of Sample “P”. Sample “N”
is the normal control and Sample “P” is the patient they are testing. Patient
samples should be distributed amongst the students so that all results (normalHgA, carrier-HgA/S, and sickle-HgS) will be represented.
Electrophoresis Buffer
1
Prepare 10X Buffer Stock
Tris
Glycine
1.1
1.2
1.3
1.4
1.5
1 liter
160 g
72 g
2 liters
320 g
144 g
Weigh out Tris and Glycine for either 1 liter or 2 liters according to chart below and place
into a flask
Add approximately 700 ml (or 1400 ml if preparing 2 liters) of dH2O
Add magnetic stir bar and mix on stir plate until dissolved
Pour into a 1 liter (or 2 liters) graduated cylinder and add dH20 until final volume is 1
liter (or 2 liters)
Pour into a storage jug labeled accordingly
Note: This buffer can be stored indefinitely at room temperature
2
Dilute 1X Working Stock
2.1
2.2
2.3
2.4
2.5
Add 2 liters of 10X stock to an empty 20 liter carboy
Add dH20 to 20 liter “fill line” for a final volume of 20 liters
Shake very well
Label student bottles “Hemoglobin Electrophoresis Buffer”
Fill Student bottles with 500 ml of 1X Working Solution
Mystery of the Crooked Cell: Laboratory Preparation
11
Note:
Do not prepare 1X-working solution in 20-liter carboy until carboy is completely empty.
Other volumes may be prepared as needed. This buffer can be stored indefinitely at
room temperature.
Laboratory Station Set – up
¨
¨
¨
¨
¨
¨
¨
¨
¨
¨
¨
¨
¨
¨
¨
Set p20s at 15µl – please leave in racks
Pipette aid
10 ml pipette
2 test tubes in glass beaker
spatula
gel mold
2 combs
2 glass plates
gel box
buffer
yellow tips
practice gel
practice loading dye
manual
2 data/observation sheets
Preparation of the gel
If available, use E-gels to run the samples. E-gels are convenient pre-prepared agarose gels that come
with blue stain or ethidium bromide. Follow the manufacturer’s recommendations for loading and
running the gels.
If Eels are not available, prepare a one percent agarose gel by combining 1 g of agarose for every 100
mL of 1X TAE. Heat in a microwave until the agarose has completely dissolved. Add ethidium
bromide if that is the stain the class will use (10 mg/mL) to a final concentration of 0.2 mg/mL and
mix. If methylene blue will be used to stain the gel (see step 5), do not add ethidium bromide to the
agarose.
Allow the agarose to cool to approximately 55 °C. Prepare and seal the ends of the gel mold
according to the manufacturer’s instructions. Also position the desired comb to cast the wells. Pour
the cooled, liquefied agarose into the gel mold and allow it to solidify. Gels should be 5 –0 8 mm
thick.
After the gel has solidified place it in the electrophoresis chamber and carefully remove the comb.
Add a sufficient volume of the 1X TAE buffer used to make the gel to the electrophoresis chamber to
cover the gel by 1 – 2 mm.
Mystery of the Crooked Cell: Laboratory Preparation
12
Extension Activities
Discussion Topics
The investigation can serve as the centerpiece for a variety of topics to be explored further:
Central dogma
Using the amino acid sequence of the affected and normal hemoglobin, the students can identify the
mutation from glutamate to valine that results in the change in net negative charge of the affected
hemoglobin. Working back through the central dogma, they can identify the point mutation in the DNA
resulting in the amino acid alteration.
Inheritance
Genotypes can be derived from the phenotypic results expressed on the gel and the probability of
inheriting sickle cell anemia can be predicted given the genotypes of the parents.
Natural Selection
The sickle cell allele is more prevalent in races whose gene pools originate in tropical areas. People of
African, Asian, and Hispanic-Caribbean descent have a higher incidence of sickle cell anemia. Selective
pressure for the allele results from its ability to decrease the mortality rate of people infected with
malaria. Malaria is caused by a protoctist in the genus Plasmodium, which is transmitted to human hosts
by mosquitoes. Plasmodia infiltrate red blood cells where they multiply and eventually rupture the cell.
Cells with sickle cell hemoglobin are less susceptible to infection by Plasmodia. Therefore carriers
(heterozygotes) benefit from the presence of sickle cell hemoglobin while remaining largely asymptotic
(some heterozygous individuals may show mild symptoms) with respect to sickle cell anemia.
Random mutation/Selective Pressure
Interestingly, plasmodium did not infect humans 10,000 years ago. It was an avian pathogen. However,
a mutation in plasmodium enabled it to jump species and infect humans. Ask the students whether they
think anyone could have had sickle cell anemia prior to 10,000 years ago. It often leads to a discussion
concerning random mutations and the distinction between a Lamarkian and Darwinian perspective on
evolution.
Treatments
Ask the students how they would treat sickle cell anemia based on their knowledge of the disease. Bone
marrow transplants, blood transfusions, and gene therapy are often mentioned. Recently attention has
been given to turning on the fetal hemoglobin gene, which is turned off shortly after birth. Hydroxyurea,
which has been used to treat cancer and blood disorders, has been found to stimulate the production of
fetal hemoglobin.
Suggested Extension Activities
§
Have the students write a letter to the fictional patient explaining the results of the test and the
precautions the patient may want to consider taking.
§
Use the documents at the end of the student section titled A Closer Look at the Cause of Sickle Cell
Anemia. This activity shows students how the sickle cell trait is determined at the genetic level.
§
Sickle cell anemia is an example of one genetic condition for which there is a test but no cure.
Have groups of students research other inherited conditions for which there is a test but no cure,
for example; cystic fibrosis, Huntington’s disease, muscular dystrophy, and fragile x syndrome.
Each group can make an informative display about the disease. The display could be in the form
of a poster, mobile, booklet, radio broadcast, interview, role-play etc. After each group has made
Mystery of Crooked Cell: Extension Activities
13
a presentation about the disease, create a role-play in which a genetic counselor presents a
scenario, for example:
Both parents are carriers for sickle cell anemia, cystic fibrosis, or any recessive disorder. The
couple decides whether or not to try to have children.
One spouse’s parent has been diagnosed with Huntington’s disease. The couple has two
young children. The counselor asks whether they want to be tested.
The role-plays can lead to interesting discussions among the students. The teacher can
facilitate by writing down ideas and issues on the board as they come up.
Suggested readings:
§
Pelehach, Laura. (1995).Understanding Sickle Cell Disease. Laboratory Medicine (26), 720-725
§
Ogamdi, S. and White, G. (1993).Sickle Cell Disease. Clinician Reviews (February), 65-82.
§
The Sickle Cell Information Center: www.emory.edu/PED/SICKLE/news.htm
Mystery of Crooked Cell: Extension Activities
14
STUDENT ACTIVITY HANDOUTS
&
LABORATORY PROTOCOL
Patient Description
In 1904, a student from the West Indies came to a Chicago Physician, Dr. James Herrick, with a puzzling
condition. Below is a summary of some of the observations Dr. Herrick made. Your job is to learn more
about this condition and to find out how the disease affects the body. Read the description below and
underline the information that you think may provide important clues that will help you understand the
disease.
The patient reports feeling well most of the time. But he also reports
odd reoccurring events. For instance, one day after a short swim he
became so tired that he could hardly move. He became short of breath
and complained of pain in his joints and muscles, especially the arms
and legs. He felt unusually weak and required bed rest lasting a few
weeks. These symptoms occurred repeatedly during his youth. He
also had frequent fevers and infections.
The patient complained of fatigue and soreness in the joints. Upon
inspection, the whites of his eyes had a yellowish tint. He complained
of pain in the left abdominal area, which was tender to the touch.
A family history reveals that he has two brothers and three sisters.
None of them have this condition. His uncle and his grandmother
often had similar symptoms. His grandmother died a young woman.
His parents do not have this condition.
James Herrick
Patient Description
Recorder_________________________
Reader___________________________
Station A
At this station you will use a microscope to observe blood samples magnified 1000X. The slide marked P
represents the patient’s blood sample. The slide marked N represents a normal blood sample.
Describe (in writing or pictures) the differences you see between the two blood samples.
Station A Guide Sheet
Recorder__________________________
Reader____________________________
Station B
The tubing at this station represents the pathways of blood in the body. Models representing the
patient’s red blood cells are also given. Red blood cells must flow freely through the body in order for
the blood to do its job of delivering oxygen and picking up wastes. Use these models to show the effect
the patient’s red blood cells will have on the flow of blood.
Station B Guide Sheet
Recorder________________________________
Station C
Reader__________________________________
At this station you will be given two sets of models. Each model represents a blood cell. One model
represents a patient’s blood cell and is labeled “P”. The other model represents a normal blood cell and is
labeled “N”. The pieces inside the blood cells represent blood proteins called hemoglobin. Hemoglobin
is the oxygen-carrying component of blood. This model uses only a few pieces to represent the millions
of hemoglobin units found in real blood cells. Scientists have discovered that abnormal hemoglobin units
connect with one another when oxygen levels in the blood are low.
Use these models to investigate what happens to the shape of red blood cells with abnormal hemoglobin
when the oxygen levels in the blood are low. Record your results below.
Station C Guide Sheet
Recorder____________________
Reader______________________
Station D
Based on the family history given below, how do you think the patient got the disease? Record your
answer on the back or this page.
I
II
III
IV
Patient
Key to symbols
Male
Female
Deceased
Male affected
Female affected
Parents
Offspring
Station D Guide Sheet
A Closer Look at the Cause of Sickle Cell Anemia
Laboratory Protocol
1
2
Loading Samples
1.1
Use a micropipette (p20) to put 15µl of hemoglobin from sample N into a well. Keep a
record of the well you put sample N into on your data sheet.
1.2
Use a micropipette (p20) to put 15µl of hemoglobin from sample P into a well. Keep a
record of the well you put sample P into on your data sheet.
1.3
Follow the instructor to begin electrophoresis of your samples.
Removal of the gel from the electrophoresis box
Wait for an instructor to demonstrate removal of the gel and clean up.
3
2.1
Turn the power off.
2.2
Disconnect the cables and return them to the bag.
2.3
Open the lid of the electrophoresis box.
2.4
Gently lift the casting tray and the gel out of the box.
2.5
Hold the clear plastic bag provided at your lab bench and label the top of the bag with
your initials (your partner should do the same with the other bag).
2.6
Slide the gel off the casting tray into the plastic bag.
Analysis of the gel
3.1
Observe the banding patterns on your gel and record the results on your data sheet.
3.2
What is your conclusion? Write your answer on your data sheet.
Laboratory Protocol
Name_________________________
Test Tube Number_______
Mystery of the Crooked Cell: Data/Observation sheet
Preparation of the Agarose Gel
1.
What is the function of the agarose gel?
2.
Predict what would happen if you used 0.02 g of agarose instead of 0.2 g. What effect would
that have on your experiment?
3.
What is the function of the comb?
Preparation of the Gel Electrophoresis Box
1.
Predict what would happen if you put the agarose gel at the opposite pole?
2.
Why is the gel in the electrophoresis buffer?
3.
Describe what is occurring in the gel when the electric current is applied.
4.
What must you be careful of when loading the samples into the wells?
5.
Diagram the final results as they would appear on the gel if the patient (sample P) has sickle cell
anemia.
Data/Observation Sheet
6.
Diagram the expected results if the patient (sample P) does not have sickle cell anemia.
Use the diagram below to:
§
Record where you loaded each sample.
§
Record the results of the electrophoresis (sketch the results)
Conclusion
1.
What is the purpose of sample N (normal hemoglobin)?
2.
What do your results tell you about the hemoglobin in sample P?
3.
If sample P came from a person who you were testing for sickle cell anemia, what would your
diagnosis be?
4.
How would you explain the test results to the patient? Assume the patient does not know how
the test works.
Data/Observation Sheet
A Closer Look at the Cause of Sickle Cell Anemia
Your research has determined that sickle cell hemoglobin differs from normal hemoglobin in the net
negative charge on the proteins. This discovery is an important one; it identifies a characteristic that can
be used to diagnose sickle cell anemia. However, it does not tell us what causes sickle cell anemia or why
the proteins are different. Advances in molecular biology and our understanding of DNA in the past two
decades have provided us with more insights into the cause of sickle cell anemia. See if you can use the
following data obtained from research in molecular biology to uncover more information about sickle cell
anemia.
Document 1: The DNA base sequences of the first seven amino acids for both normal and sickle cell
hemoglobin.
Document 2: A chart of mRNA codons and their corresponding amino acids.
Document 3: The structural formulas for the amino acids and their corresponding charges.
A Closer Look at the Cause of Sickle Cell Anemia
Document 1
The DNA base sequences of the first seven amino acids for normal and sickle cell hemoglobin.
The DNA sequence of bases for the first 7 amino acids in normal hemoglobin is:
CACGTGGACTGAGGACTCCTC
The DNA sequence of bases for the first 7 amino acids in sickle cell hemoglobin is:
CACGTGGACTGaGGACACCTC
A Closer Look at the Cause of Sickle Cell Anemia
Document 2
A chart of mRNA codons and their corresponding amino acids
5’ End
T
C
A
G
3’ End
T
Phe
Ser
Tyr
Cys
T
Phe
Ser
Tyr
Cys
C
Leu
Ser
Stop
Stop
A
Leu
Ser
Stop
Trp
G
Leu
Pro
His
Arg
T
Leu
Pro
His
Arg
C
Leu
Pro
Gln
Arg
A
Leu
Pro
Gln
Arg
G
Ile
Thr
Asn
Ser
T
Ile
Thr
Asn
Ser
C
Ile
Thr
Lys
Arg
A
Met(Start)
Thr
Lys
Arg
G
Val
Ala
Asp
Gly
T
Val
Ala
Asp
Gly
C
Val
Ala
Glu
Gly
A
Val
Ala
Glu
Gly
G
C
A
T
A Closer Look at the Cause of Sickle Cell Anemia
THIRD BASE OF CODON
FIRST BASE OF CODON
SECOND BASE OF CODON
Document 3
The structural formulas for the amino acids.
A Closer Look at the Cause of Sickle Cell Anemia