Sickle Cell Anemia  -- A classroom simulation of DNA diagnosis
Donna L. Daniels
WisTEB, Univ. of Wisconsin
1994, 1997, 1999
 
Sickle cell Anemia is a very painful, often fatal condition.  It is one of the best understood genetic diseases.  It is caused by an autosomal recessive gene.  In the following activity, students are given a pedigree, showing the sickle cell anemia phenotype of mom, dad, and two children, one of whom suffers from sickle cell anemia.  They are asked to use what they know about genetics together with DNA analysis to determine the genotypes of the four individuals and to assess the sickle cell status (genotype and future phenotype) of a third child, a currently an in utero fetus.  There are two versions.  In the lab version, students are given the scenario and the pedigree and 6 DNA samples (each individual on the pedigree and a size standard).  They load and run an electrophoresis gel, stain and photograph the results, and use their own gel results to determine the genotype and phenotype of the members of this family.  In the class activity version, the DNA banding pattern is given to the students for analysis.  Both activities can segue into discussions of social, ethical, and legal issues related to genetics and genetic testing.  All directions given here assume that you (the teacher) know how to run DNA on an agarose electrophoretic gel and that the students have already been given instructions on how to do DNA electrophoresis.
DNA Sample Prep directions
Classroom Activity
Lab Activity
Notes for teachers
Results from some high schoolers
 
 
 

DNA Sample Prep directions
 
 Making DNA samples for simulation of Sickle Cell diagnostics for lab activity
  "Using DNA for Diagnosis of Sickle Cell Anemia"
Scheme   (Use this to design you own simulation)

Digest any plasmid with an enzyme that cuts once. This will make one band and represent the Sickle Cell version of the B-globin gene. In a separate tube, digest the plasmid with an enzyme that cuts twice (preferably into a large and a small piece). This will make two bands and represent the wild type version of the B-globin gene. Stop the reactions with EDTA. Mix some of the two different digests in equal portions into a third tube to represent the homozygous individual's DNA (having both versions of the gene).

Overview   (Use this to figure out how to make the student DNA kits from the ingredients that you have or will purchase)

1) Make two digests of pBR322 DNA, one with Hind III and one with Hinc II.
2) Run digests on a gel to assess

3) If incomplete, add more enzyme and reincubate. Repeat test gel.
4) If complete, add EDTA as a preservative and to inactivate the enzymes.
5) Make 3 tubes at "appropriate" concentrations by mixing digests, TE, and loading dye. 6) load mixtures onto gel to test and verify
7) Aliquot and label tubes. Sort into "kits". Mom: heterozygous
Dad: heterozygous
Child 1: heterozygous
Child 2: homozygous sickle cell
Child 3: homozygous normal
 
Details and step by step directions (Exact details will depend on concentration of DNA and enzymes available.  All volumes are given in microliters.  DNA is given in micrograms. DNA concentration is given in micrograms per microliter.  Enzyme concentration is in Units per microliter.)     If you have: Follow the following detailed directions.

1) Make two digests of 10 ug of pBR322 DNA, one with Hind III and one with Hinc II, each in a total volume of 100 ul (0.1 ug/ul)   by following the pipetting grid below.
  pBR322 digested w Hind III pBR322 digested w Hinc II
DNA 10 ul (10 ug) 10 ul (10 ug)
Enzyme 3 ul (30U Hind III) 3 ul (30U Hinc II)
10X buffer 10 ul 10 ul
water 77 ul 77 ul
     
Total volume 100 ul 100 ul
Incubate for 60 min at 37 degrees C.

 

2) Run small amounts of the digests on a gel to assess a) is the digest complete? and b) how much is appropriate to load for staining conditions chosen. To do this, pour a gel, similar to the gel to be used in the classroom (approx. same size wells); mix the appropriate amount of DNA digest (from the chart below) with loading dye; load and run.
Lane 1 size standard
Lane 2 0.5 ul Hind III digest
Lane 3 1 ul
Lane 4 2 ul
Lane 5 0.5 ul of Hinc II digest
Lane 6 1 ul
Lane 7 2 ul
 

After electrophoresis, stain with Ethidium Bromide and photograph.     (or Stain with Methylene Blue. )

Decide on appropriate amount to load

Decide if digest is complete.

 
4) If complete, add EDTA as a preservative and to inactivate the enzyme. (i.e., Add 10 ul of 0.5 M EDTA to each tube of digest -final is 50 mM EDTA).

5) Make 3 tubes at "appropriate" concentrations by mixing digests, TE, and loading dye.

Details will depend on results. For example, if you decided that appropriate load was 1.5 ul (0.15 ug) and your wells hold 12 ul and you want to give students tubes of 10 ul and say, "load it all" , then mix up as follows:
  Heterozygous Homozygous. normal Homozygous Sickle cell
Hinc II digest 67 23  
Hind III digest 67   23
TE 166 77 77
loading dye 150 50 50
       
Total (15 kits) 450 (45 loads) 150 (15 loads) 150 (15 loads)
 

6) Load a gel to test the mixtures.  (i.e., load 10 ul of each mixture onto a gel, electrophoresis, stain, photograph).  If everything looks good, proceed to the next step.   look at gel

7) Label tubes and aliquot 10 ul into tubes. Divide into kits.
 
 


Classroom Activity
 
  Using DNA for Diagnosis of Sickle Cell Anemia
 
Scenario

A couple has two children. The first child is healthy but the second child suffers from sickle cell anemia. The woman is pregnant with their third child and wants to know the sickle cell status of the fetus. The situation is diagrammed below, along with the results of DNA testing. Remember, sickle cell anemia occurs in individuals homozygous for the HbS form of the gene for beta-globin. That is, sickle cell anemia is caused by an autosomal recessive gene. For each individual in the pedigree state their genotype at the beta-globin locus and the sickle cell anemia phenotype. (homozygous dominant -- healthy and has only wild type hemoglobin in their blood stream, heterozygous- healthy but has both versions of hemoglobin in their blood stream and is a genetic carrier, or homozygous recessive -- sickle cell anemia and has only the HbS version of hemoglobin in their blood stream). What DNA pattern indicates each of these situations? For each individual in the pedigree state their genotype at the beta-globin locus and the sickle cell anemia phenotype.  Speculate about what the map of the DNA at the beta-globin gene might look like. (Hint:  Remember each person has two maps, one for each chromosome.  The DNA pattern is a mixture of the DNA from the two chromosomes.  Homozygous individual have the same map on each chromosome.  Heterozygous individuals have two different maps.)  Draw the proposed map in this region for each of the two chromosomes in each individual.

Pedigree and DNA Pattern
 


 
 

  


Lab Activity
 
  Using DNA for Diagnosis of Sickle Cell Anemia

Scenario

A couple has two children. The first child is healthy but the second child suffers from sickle cell anemia. The woman is pregnant with their third child and wants to know the sickle cell status of the fetus. The situation is diagrammed below.

Pedigree

 

 
Lab instructions

Obtain 6 tubes of DNA. Five of these DNA samples simulate the RFLP pattern of each of the five people in the pedigree. The sixth is a size standard.  Load the samples on an agarose electrophoresis gel and electrophorese the DNA until the Bromphenol Blue Dye marker is near the bottom of the gel.  Stain the gel and photograph it. Label the photograph and tape it below.

Gel results
see more gels
 Your gel should look something like this                                              Paste your photograph here

 Analysis

Diagram the results. Explain the pattern you see and make conclusions about the Sickle Cell status of child 3. Remember, sickle cell anemia occurs in individuals homozygous for the HbS form of the gene for beta-globin. That is, sickle cell anemia is caused by an autosomal recessive gene. For each individual in the pedigree state their genotype at the beta-globin locus and the sickle cell anemia phenotype. (homozygous dominant -- healthy and has only wild type hemoglobin in their blood stream, heterozygous- healthy but has both versions of hemoglobin in their blood stream and is a genetic carrier, or homozygous recessive -- sickle cell anemia and has only the HbS version of hemoglobin in their blood stream). What DNA pattern indicates each of these situations? Speculate about what the map of the DNA at the beta-globin gene might look like. Draw the proposed map in this region for each of the two chromosomes in each individual.

  



 
Notes for teachers
  Using DNA for Diagnosis of Sickle Cell Anemia

I have used this lab activity with HS students including  9th or 10th grade Biology I, 11 grade Biology II, AP Biology, and biotechnology.  I have used the non-lab version of  it with 6th, 7th, and 8th grade science classes, and with 4th-5th TAG classes.  How successfully it engages students seems to depend primarily on their background understanding of fundamental principles of genetics including that humans are diploid, one gene copy coming from each parent. Useful vocabulary for the students to already know includes genotype, phenotype, homozygous, heterozygous, dominant, recessive.

Students usually come up with an approach to the analysis by themselves.  If they don't, I review fundamental principles of genetics (so they know what it means to say that sickle cell anemia is a genetic disease caused by an autosomal recessive gene.)  I then help them through the analysis as needed.  Look at the pedigree. Child 2 has sickle cell anemia, so what is her genotype? (homozygous recessive).  Mom and Dad have an affected child, but they are both heathy, so what are their genotypes?  (both heterozygous, ie carriers).  Now look at the DNA banding pattern.  What pattern shows heterozygous? Are Mom and Dad's patterns the same as each other?  Anybody else have that pattern? (child 1 has that pattern, so child one must be heterozygous, ie a carrier.  Child 1 is healthy, so it all fits)   What is the DNA pattern for homozygous recessive? Anybody else have that pattern?  (no, not in this pedigree)   What is the third possible genotype? (homozygous dominant).  What is child three's genotype and phenotype.  (Banding pattern is not that of heterozygous nor of homozygous recessive, so it must be homozygous normal, ie healthy and not a carrier.)

A "map" of the DNA is the globin gene region could look like this.
 


 
 

I have also used this activity with numerous high school teachers in WisTEB summer courses.  One teacher, once, complained that use of sickle cell anemia as the example disease in so many scenarios and activities was racially insensitive, if not actually racist.  Another complained that having an in utero fetus in the scenario, even though the results show the "happy outcome", was a potential problem because of the pro-life/pro-choice issue.
 
 

Comments?  Questions?  Suggestions? Data?  email  Donna Daniels
 
 
 

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