Detection of the Sickle Mutation

Sickle cell disease results from a specific point mutation ( A > T) causing the substitution of glutamic acid by valine at amino acid six in the beta-globin gene. This disease is inherited as an autosomal recessive disorder and has a high carrier frequency of about 8% among Americans of African origin. Carriers are said to have sickle cell trait and are usually asymptomatic while individuals affected with sickle cell disease usually have serious medical complications. As shown in the figure below, the mutation can be detected by a DNA-based test with 100% accuracy due to the loss of a specific recognition site for the restriction endonuclease Dde I (or Mst II).

In the prenatal testing example shown below, lane 1 shows DNA size markers, lane 2 a no DNA control, and lane 3 a full length PCR product of the beta-globin gene. Lanes 4 - 8 show beta- globin PCR products digested with the restriction endonuclease Dde I. Lane 4 shows a no DNA control, lane 5 shows a normal individual (HbB/HbB), lane 6 a carrier mother (HbB/HbS), lane 7 her carrier fetus (HbB/HbS), and lane 8 the carrier father (HbB/HbS). HbS carriers are detected by the presence of the 381 bp DNA fragment. An affected individual (HbS/HbS) would not have any 201 bp and 180 bp DNA fragments.