Reverse dot-blot detection of the African-American β-thalassemia mutations

DNA-based diagnosis of the β thalassemias provides accuracy to newborn screening, genetic counseling, and prenatal diagnosis. However, the use of polymerase chain reaction (PCR)-based methods is challenged by the great number of different β-thalassemia mutations that exist even within defined ethnic groups. In this regard, the reverse dot-blot method offers a means of screening for several mutations with a single hybridization reaction. We have applied the reverse dot-blot method to the detection of the β-thalassemia mutations of African-Americans. We used two biotin-labeled primer pairs in a duplex reaction to amplify and label two β-globin target DNA fragments that encompass all known African-American β-thalassemia mutations. The PCR products were denatured and hybridized to polyT-tailed, membrane-fixed, allele-specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 β- thalassemia mutations and their corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic strip, and seven less common β-thalassemia mutations were included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these alleles. Reverse dot-blot diagnosis of the less common β-thalassemia mutations precludes the need for alternative, more technically challenging methods. This method provides a rapid, accurate method for diagnosis of β thalassemia among African-Americans and other ethnic groups in which β thalassemia occurs.