A straightforward and effective PCR-based assay system is devised that allows one to reveal and identify homozygous and heterozygous point mutations. The system uses two sets of allele-specific primers. In one set, the 3′-nucleotide matches the allele under study so that the primer functions effectively only if the DNA contains the corresponding allele. To increase primer specificity, template-noncomplementary nucleotides are introduced near its 3′-end. The primers from another set invariably bear a 3′-terminal mismatch, and, in addition, the mutant nucleotides of the alleles under study form mismatches with the internal nucleotides of the primers. In such combinations primer activity is suppressed if the DNA contains a homozygous mutation. The assay system devised was utilized to reveal the Leiden mutation in the gene for factor V of the human blood clotting system in patients with thrombophilia. © 1998 MAHK Hayka/Interperiodica Publishing.