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 polymorphism (SNP) analysis using bidirectional allele‐specific amplification with polymerase chain reaction (PCR) in a single‐step/single‐tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2—5% of the human population. The rationale of our method is the production of allele‐specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply 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. Preferential production of the allele‐specific products was achieved by a hot‐start, time release PCR system. Specificity was increased by introducing a mismatch in the 3′‐antepenultimate position of the allele‐specific primers. This method made possible the large‐scale screening for the factor V Leiden mutation using single‐tube PCR followed by automated ultrathin‐layer agarose gel electrophoresis, with real‐time detection of the “in migratio” ethidium‐bromide‐labeled fragments.

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