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We have developed an assay based on solid-phase minisequencing to screen for the following seven point mutations in the gene CYP21 encoding 21-hydroxylase: Pro30Leu, I2-splice, Ile172Asn, Cluster-E6, Val281Leu, Gln318Stop, and Arg356Trp. 5'-Biotinylated PCR products of CYP21 are bound to streptavidin-coated microtiter wells, where the minisequencing reaction takes place after denaturation of DNA. Depending on the sequence investigated, one specific 3H-labelled deoxyribonucleotide is incorporated to extend a detection primer. By using an appropriate set of detection primers, it is possible to screen the gene for several mutations within the same PCR amplificate. This fast and reliable method very clearly distinguishes between DNA from homozygous mutant, heterozygous, and normal individuals and is well suited for routine diagnosis of patients with 21-hydroxylase deficiency and for carrier detection.