Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis

We report a novel, high-throughput genotyping method by single nucleotide p olymorphism (SNP) analysis using bidirectional allele-specific amplificatio n with polymerase chain reaction (PCR) in a single-step/single-tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation wa s chosen as a model system for SNP detection, as this is one of the most co mmon inherited risk factors of thrombosis, effecting 2-5% of the human popu lation. The rationale of our method is the production of allele-specific PC R fragments, different in size, which was achieved by bidirectional amplifi cation, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simpl y determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Prefere ntial production of the allele-specific products was achieved by a hot-star t, time release PCR system. Specificity was Increased by introducing a mism atch in the 3'-antepenultimate position of the allele-specific primers. Thi s method made possible the large-scale screening for the factor V Leiden mu tation using single-tube PGR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the "in migratio" ethidium -bromide-labeled fragments.