Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated PCR and multiple- injection capillary electrophoresis

Abstract

The C677T mutation of the methylenetetrahydrofolate reductase gene and the G1691A (Leiden) mutation of the factor V gene are established risk factors for thromboembolic disease. We here present an assay for the simultaneous genotyping of these common genetic variants. The assay involves a strategy based on multiplex mutagenically separated PCR performed in a single tube containing six primers. Separation of the resulting four PCR products (197, 207, 233, and 246 bp) was performed by capillary electrophoresis coupled with laser-induced fluorescence detection. The time for the automated electrophoresis was reduced to 2.5 min per sample by performing the capillary electrophoresis analysis in a multiple-injection mode.