Reverse dot‐blot detection of Thai β‐thalassaemia mutations

Abstract

Pending curative therapy, newborn screening and prenatal diagnosis are essential to the management of beta thalassaemia. Diagnosis using electrophoretic methods is difficult in the presence of composite phenotypes and high Hb F levels. Direct DNA detection of mutant alleles circumvents both problems, but the enormous diversity of beta-thalassaemia mutations poses challenges for this approach. Among PCR-based tests, the reverse dot-blot method enables screening several mutations with a single hybridization reaction. Unfortunately it has often been targeted to only the common mutations of a particular ethnic population, necessitating the use of more arduous detection methods for the less common mutations. We developed a reverse dot-blot strip for the 10 beta-thalassaemia mutations, including the beta-thalassaemic haemoglobinopathies Hb E and Hb Malay, that account for 96% of beta thalassaemia in Thailand, and another strip for six less common Thai mutations. The second strip precludes the need for more technically challenging methods. To avoid problems associated with secondary structure of amplified full-length target DNA, we amplified and labelled beta-globin DNA as two shorter fragments that encompassed all known Thai mutations. Reverse dot-blotting is a rapid, accurate method for detecting beta-thalassaemia mutations.