RAPID AND PRACTICAL DETECTION OF BETA-GLOBIN MUTATIONS CAUSING BETA-THALASSEMIA BY FLUORESCENCE-BASED PCR SINGLE-STRANDED CONFORMATION POLYMORPHISM ANALYSIS

We report a useful method for daily clinical examination for the diagn osis of thalassemia. We applied a fluorescence-based image analyser to a non-radioisotopic PCR-single-stranded conformation polymorphism (SS CP) analysis to detect mutations in the beta-globin gene. PCR primers were labelled with rhodamine X and the amplified fragments from the be ta-globin gene were resolved by non-denaturing polyacrylamide gel elec trophoresis. After loading, the glass plate was set in the image analy ser and scanned with a green laser. We detected four common mutations in exon I and two major mutations in intron 1 of the beta-globin gene isolated from patients with beta-thalassemia. Moreover, to discriminat e mutations and natural polymorphisms, we used primers including one b ase mismatch at the polymorphic site, which can substitute the polymor phic site by a constant base in the PCR amplified fragment. This fluor escence-based system was simple to operate and results were obtained r apidly as clear image data. Therefore, once the optimal conditions of the electrophoresis are determined, this system will be suitable for d aily clinical use, especially for screening of molecular defects and f or the prenatal diagnosis of genetic disorders.