RFLP Analysis and Genetic Screening
Restriction fragment length polymorphisms (RFLPs) can be used to identify genetic disorders. A single nucleotide change in a sequence of DNA can destroy a recognition site for a restriction enzyme and thereby result in fragments of different lengths compared to a piece of DNA with the original sequence. When searching for a gene that indicates a genetic disease, researchers examine fragment patterns of families with the genetic disorder. Eventually, researchers find a special fragment pattern that is usually inherited with the disease. The special fragment pattern is called a “marker.” Once a particular pattern is known to be inherited with a particular disease, RFLP analyis can be used to detect the presence of the disease-causing allele in the DNA of an individual.
The technique uses a restriction enzyme to cut DNA near the gene and produce a series of fragments which can be sorted using gel electrophoresis. By analyzing the bands on the gel we can determine the genotype of the individual. The difference in the DNA sequence is called a polymorphism and the resulting differences in fragment lengths is called a restriction fragment length polymorphism.
Strand A and B give part of the DNA sequence for the normal and sickle cell hemoglobin genes.
Strand A (normal)
1 10 20 30
A A G G T C T C C T C T A A T T G G T C T C C T T A G G T C T C C T T
Strand B (sickle cell)
1 10 20 30
A A G G T C T C C T C T A A T T G G T C A C C T T A G G T C T C C T T
The restriction site for enzyme Mst II is GGTCTCC and it cuts the DNA between the first T and first C of that sequence.
1. Determine the sizes of the fragments from an Mst II digest of these two samples.
2. Draw a gel with three lanes to show the position of fragments for individual A who is normal, individual B who has sickle cell trait, and individual C who is a carrier.
3. Why is Mst II used to distinguish sickle cell from normal hemoglobin?
4. Mrs. Smythe is pregnant with her first child. It is known that Mr. Smythe is a carrier for sickle cell
anemia so Mrs. Smythe decides to be tested for the condition. She learns she is also a carrier. The Smythe's then decide to have a prenatal test performed on the fetus. How could we use RFLP analysis to determine the genotype of the fetus?
5. Should this type of screening be used routinely on embryos or unborn fetuses?
6. Should insurance companies have access to the results of such screening?