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Sickle Cell Anemia Lab:
Diagnosis Using Simulated Restriction Analysis of DNA
Teacher Information
Materials, per team:
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•gel electrophoresis box with casting tray and comb •power supply •P-20 micropipet and tips •rack for tubes •250 ml beaker for used tips •permanent marker •agarose (1%) |
•"DNA" samples (dye mixtures of xylene cyanole and napthol blue)* •1X TAE buffer, 150 ml (more if making a gel also) •acetate and blotter paper •plastic wrap •gloves and goggles |
*To make dye mixtures, dissolve 0.025 grams xylene cyanole or napthol blue in 10 ml water and 1 ml glycerol. For the heterozygote mixture, mix together equal amounts of each dye solution.
In most cases, you should pour and set up gels before class starts. However, depending on the length of your class period, you may be able to make and run the gels on the same day. For this lab, it is assumed that students have prior knowledge of the principles behind electrophoresis and restriction enzymes.
Napthol blue and xylene cyanole are dyes that are similar in color yet run differently enough through the gel such that two distinct bands can be seen. They are both negatively charged and thus ‘run towards the red’ as DNA does. The dyes will diffuse quickly so make sure students record their results right away!
Xylene cyanole (XC) mimics the larger-fragment band (1350 bp, representing
the S allele).
Napthol blue (NB) mimics the smaller-fragment band (1150 bp, representing the A
allele).
A 1:1 mixture (XC + NB) of the two dyes mimics the carrier heterozygous
genotype.
|
Individual |
Label |
Family 1 Key |
Family 2 Key |
|
Mother |
M |
Carrier (XC+NB) |
Carrier (XC+NB) |
|
Father |
F |
Carrier (XC+NB) |
Normal (NB) |
|
Teenager |
T |
Carrier (XC+NB) |
Normal (NB) |
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Fetus |
O |
Sickle (XC) |
Carrier (XC+NB) |
|
Known Normal |
N |
Normal (NB) |
Normal (NB) |
|
Known Carrier |
C |
Carrier (XC+NB) |
Carrier (XC+NB) |
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Known Sickle Cell Patient |
S |
Sickle (XC) |
Sickle (XC) |
Extension Ideas:
For ease of lab preparation, only two families have been included; however, more variations could be created.
Have students try to determine the mode of inheritance of a disease based on restriction analysis of members of a large family.
Use a hypothetical/imaginary disease and actual DNA to model disease diagnosis. Have students create diagnostic tests using available enymes and the sequence of the disease gene.
Sources:
Keeton, W., and J. Gould, Biological Science, W.W. Norton and Co., NY, NY, 1986.
Leary, W., Sickle Cell Trial Called Success, Halted Early, New York Times, January 31, 1995. pp. B5, B8.
Lewis, R., Case Workbook in Human Genetics, Wm C. Brown Publishers
, Dubuque, IA, 1994.Micklos, D., and G. Freyer, DNA Science, Cold Spring Harbor Laboratory Press, Burlington, NC, 1990.
Modern Biology Inc. Catalog, Dayton Indiana, 1995.
Pines, M., Blood: The Bearer of Life and Death, Howard Hughes Medical Institute, Chevy Chase, MD, 1993.
Tortora, G., B. Funke, and C. Case, Microbiology, Benjamin Cummings Publishing Co., Inc., Redwood City, CA, 1992.
Contributed by Jeanne Ting Chowning, Juanita High School, Lake Washington School District, Kirkland, WA
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Last updated 7/18/00