β-Globin mutation detection by tagged single-base extension and hybridization to universal glass and flow-through microarrays

To test the feasibility of developing a diagnostic microarray for a specific disease, we selected all pathogenic changes of the β-globin gene occurring at a frequency ⩾1% in the multi-ethnic Dutch population for analysis. A tagged single-base extension (SBE) approach was used to detect 19 different mutations causing β-thalassemia or abnormal hemoglobins. In the SBE reaction, the primers were elongated at the 3′site with a fluorescently labeled dideoxyribonucleotide triphosphate (ddNTP) complementary to the mutation, following tag hybridization to a glass or flow-through microarray. We compared the performance of a generic glass array and a porous system, by testing each mutation separately using heterozygous carriers and by screening a cohort of 40 unknown β-thalassemia carriers and patients. The results were verified by direct sequencing. The microarray system was able to detect 17 β-globin mutations simultaneously with >95% accuracy in a single SBE reaction. The flow-through array performed slightly better (96%), but the main advantages of the system included real-time data recording and a considerable time saving achieved through a reduced hybridization time.