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Molecular Basis of Heredity: Part 4. Gene Identification and Tests

Author(s): Raye L. Alford, PhD

Restriction Enzymes II

Because of the specificity of their DNA sequence recognition, restriction enzymes can also be used to detect mutations associated with genetic diseases. In the image shown in this slide, the restriction enzyme DdeI is used to identify the mutation in the beta-globin gene that is associated with sickle cell disease in a fragment of DNA amplified from patients by polymerase chain reaction (PCR). In this case, the mutation associated with sickle cell disease changes the DNA sequence of the beta-globin gene in such a way that it is no longer recognized by DdeI (Panel A). As a result, normal, non-sickle, beta-globin genes are digested by DdeI while sickle cell-associated alleles are not (Panel B).

When DNA from patients is amplified by PCR and the PCR products are digested with DdeI and run on agarose gel electrophoresis, a person with two normal beta-globin genes will demonstrate a two band pattern, indicating that the beta-globin DNA was cut by DdeI (Panel C, Row 1). A person with two copies of the sickle cell-associated gene will show only an uncut band, indicating that neither of the beta-globin genes was cut by DdeI (Panel C, Row 2). A carrier for sickle cell disease will demonstrate both cut and uncut bands (Panel C, Row 3), indicating that he or she has one normal gene and one sickle cell-associated gene.

Understanding the molecular genetic basis of a disorder and developing a DNA-based methodology for mutation detection enable precise DNA-based diagnosis of the disorder, permitting identification of affected and unaffected individuals, and carriers of recessive diseases. In this example, the individual in row 2 of Panel C is affected by sickle cell disease, while the individual in row 3 is a carrier for sickle cell disease.