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Sickle Cell Disease Diagnosis Lab
Student Instructions and Questions

Objective: To simulate the diagnosis of sickle cell anemia with DNA restriction analysis.

Background: The "DNA" you will receive has, in this simulation, already been "cut" by the Mst II restriction enzyme. You will separate the resulting fragments of DNA by gel electrophoresis in order to diagnose the genotypes of all members of a family (mother, father, teenager, and fetus). Known samples will also be run for comparison.

DNA and dyes are ëchargedí molecules that can be separated by gel electrophoresis. The dyes we will use are charged in solution, just as DNA is. They will therefore move from the BLACK cathode (- end) to the RED anode (+ end). (Remember, negatively charged molecules such as DNA "run towards the red.")

Procedure:

1. Receive the seven "DNA" samples from your teacher. Record whether you have Family #1 or Family #2 in your lab notebook. The tubes are coded in the following manner:

Mother	M
Father	F
Teenager	T
Fetus	O
Known Normal	N
Known Carrier	C
Known Sickle Cell Patient	S

2. Slide the gel into the box, wells facing up and closest to the black electrode.

3. Using a P-20 micropipet set to 15 ml, load each well in the gel with the samples. Take turns loading with others in your group, making sure to use a new tip each time.

4. Draw a gel as shown below and indicate in your lab notebook which sample you put in which lane. Label this RESULTS. Draw the + and - ends of your gel so you remember the orientation.
Note: if figure below does not display properly on your browser, please view in the downloadable pdf version of this document.

Figure 1. Sample Results diagram for lab notebook.

5. If necessary, add more 1X TAE Buffer to the gel box so that the gel is adequately covered. (The buffer should cover the gel by about 1-2 mm.) Connect the electrodes to the gel box and to the power supply (red to red, black to black).

6. Turn on the power supply and set it at about 100V. Run the gel for at least 10 minutes. While your gel is running, make a second drawing of a gel showing what results you expect for the three known samples. Label this PREDICTIONS.

7. Turn off the power supply, unplug the electrodes, and open the gel box. Lift the gel and deck and slide the gel back into the dish, pouring off extra buffer. For better viewing, place the dish on white paper. Color the pattern observed into your RESULTS drawing.

8. Throw away the gel and pour back buffer!! Put all equipment back into the supply box.

Analysis:

1. Imagine that you are a genetic counselor. Based on your results, explain to your family (in the form of a letter addressed to the parents) what their options are both now and in the future. Use an understanding tone, and be aware of sensitive issues.

a. Intrepret the results of the tests:

ï Which family members have the sickle cell genotype (SS)?

ï Which family members have the carrier genotype (AS)?

ï Which family members have the normal genotype (AA)?

b. Make a Punnett Square and explain the probabilities of various genotypes and phenotypes for offspring given the parentsí genotypes. (Be sure that your square includes a key; A=normal allele, S=sickle allele).

2. Choose ONE of the following to answer.

ï Should genetic defects that cause hereditary problems be diagnosed before birth?

ï Should people with genetic disease be allowed to pass on their disease-causing genes to their offspring? Who should make such a decision?

(For your information, scientists estimate that each of us has at least six lethal recessive genes!)

3. What are some possible sources of error in this lab?

Contributed by Jeanne Ting Chowning, BioLab, Seattle, WA
Provided jointly by the GENETICS Project and the Genetics Education Partnership http://genetics-education-partnership.mbt.washington.edu