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Sickle Cell Anemia: Diagnosis Using Simulated
Restriction Analysis of DNA.
PreLab Reading and Questions

Background, Sickle Cell Anemia

Read the background information provided in the handout, Sickle Cell Anemia and Genetics: Background Information.

Background, DNA Restriction Analysis as a Diagnostic Tool:

DNA obviously differs from one individual to another. However, some areas of DNA contain quite a bit of sequence variation due to point mutations, deletions, insertions, and repetitions. These areas are often referred to as polymorphic regions (or "many-form places"). Alleles of a gene are a familiar example of a polymorphism, for example, the A and S alleles of the b globin gene. However, polymorphic regions usually do not code for peptide products.

When human DNA is digested with a particular restriction enzyme, a polymorphic region yields fragments of different sizes, called RFLPs (pronounced "riflips", meaning "restriction fragment length polymorphisms" -whew!). The fragments are separated by gel electrophoresis. In a technique called Southern Analysis, special probes are then used to bind to these fragments. (A probe is a molecule that can help you find the molecule you are interested in by binding to it. Often, probes are ëtaggedí with a label such as radioactivity.) The patterns resulting on the gel can be used to identify criminals or settle paternity cases. They can also be used in captive breeding programs of endangered species (cheetahs and California condors, for example) to identify genetically dissimilar parents and avoid inbreeding.

Another use of RFLP analysis is diagnosis of genetic diseases and identification of disease carriers. If a polymorphic region is close to the area responsible for a disease, they are said to be linked. Sometimes the polymorphic region that is capable of being cut with a restriction enzyme is known WITHIN the gene responsible for a disease. This is the case with sickle cell anemia. In 1978, Yuet Wai Kan and Andrees Dozy of the University of California-San Francisco showed that the restriction enzyme Mst II, which cuts normal b globin DNA at a particular site, will not recognize (and therefore will not cut) DNA that contains the sickle cell mutation.

Mst II recognized the sequence CCTNAGG (where N = any nucleotide). Sickle cell disease is due to a single point mutation in the b globin gene on chromosme 11 that changes CCTGAGG to CCTGTGG. Therefore, the A to T mutation that causes sickle cell anemia also causes the loss of the recognition site for the restriction enzyme Mst II!

Thus, the DNA from normal homozygous individuals (AA), heterozygous carriers of the trait (AS), and homozygous sickle cell patients (SS) produces different sizes of restriction fragments when cut with Mst II. In Southern blot analysis, these RFLPs are detected as characteristic banding patterns, using a radioactive b globin gene probe.

PreLab Questions: Answer the following questions in your lab notebook in complete thoughts:

1. What are RFLPs and how are they used?

2. How can a restriction enzyme help identify carriers of sickle cell anemia?

3. If a person has sickle cell anemia and his or her beta globin DNA is cut with Mst II, will the fragments be longer or shorter than those from an individual without the disease? Explain!

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