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.
Sasvari-Szekely
M
M
Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University Medical School, Budapest, Hungary.
Gerstner
A
A
Ronai
Z
Z
Staub
M
M
Guttman
A
A
D001331:Automation; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005165:Factor V / Q000032:analysis / Q000235:genetics*; D005838:Genotype; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012307:Risk Factors; D013927:Thrombosis / Q000235:genetics
D017931:DNA Primers; C095381:factor V Leiden; D005165:Factor V