TOWARDS AUTOMATIC DETECTION OF POINT MUTATIONS - USE OF SCINTILLATINGMICROPLATES IN SOLID-PHASE MINISEQUENCING

Simplification of molecular genetic techniques is one of the main feat ures of large-scale clinical applications of mutation analysis. The so lid-phase minisequencing method, which is based on single-nucleotide p rimer extension by a DNA polymerase on a solid support, is an easy way of detecting point mutations of previously known locations. Here the procedure was further- simplified by the use of microplates made of sc intillating plastics, a microplate format scintillation counter and an automatic micrroplate washer DNA samples from patients with either a hereditary aspartylglucosaminidase (AGA) gene point mutation or an acq uired N-ras gene mutation were analyzed by three different minisequenc ing detection procedures utilizing tritiated nucleotides. The new coun ting method with scintillating plates was compared to traditional liqu id scintillation. counting in scintillation vials or to another microp late format procedure, which requires addition of scintillation liquid . In all three methods, normal individuals, heterozygous carriers of t he AGA mutation and homozygous patients could be unequivocally discrim inated. The N-ras mutation in leukemic blasts could also be detected w ith high resolution. The coefficients of variation and reproducibility of the scintillating microplate method were almost identical to those of the traditional liquid scintillation assay, which was used as a re ference method. The technical innovations adopted here for performing minisequencing assays reduce significantly the labor required without affecting the quality of the results.