Diagnosis of Sickle Cell Anemia and β-Thalassemia with Enzymatically Amplified DNA and Nonradioactive Allele-Specific Oligonucleotide Probes

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and β-thalassemia. The procedure involves the selective amplification of a segment of the human β-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for β-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets. (N Engl J Med 1988; 319:537–41.) SICKLE cell anemia and β-thalassemia are inherited hématologie disorders that are widespread in regions of the world where malaria was once endemic. 1 Both these autosomal recessive diseases are caused by mutations in the β-globin gene, a gene encoding a major protein component of hemoglobin A. These mutations generally involve the replacement, insertion, or deletion of one to four nucleotide bases from the DNA sequence of the normal (ßA) gene. 2 , 3 Sickle cell anemia, which is found primarily in African populations, is caused by homozygosity for a unique DNA base-pair substitution (βS) in the sixth codon of the gene. 4 β-Thalassemia . . .