Rapid combined genotyping of factor V, prothrombin and methylenetetrahydrofolate reductase single nucleotide polymorphisms using minor groove binding DNA oligonucleotides (MGB probes) and real-time polymerase chain reaction

    loading  Checking for direct PDF access through Ovid


Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors (factor V Leiden [Arg506Gly], G20210A in the 3'-untranslated region of factor II) and variant C677T of the methylenetetrahydrofolate reductase (MTHFR) are often involved and co-inherited. These three factors were genotyped simultaneously in the same 96–well plate, using a real-time polymerase chain reaction (PCR) Taqman® assay and minor groove binding DNA oligonucleotides (MGB probes). While primers and MGB probes matched their corresponding single nucleotide polymorphism (SNP), the real-time MGB program was identical for each target gene. Homozygous wild-type (WT; −/−), heterozygous (+/−) or homozygous (+/+) variants (n = 362) were selected for factor V (n = 115, with −/−, 40; +/−, 40; +/+, 35), factor II (n = 122, with −/−, 60; +/−, 60; +/+, 2), and MTHFR (n = 120, with −/−, 40; +/−, 40; +/+, 40), according to the results of conventional PCR-restriction fragment length polymorphism (PCR-RFLP), but the allelic discrimination was performed blind. Results of the real-time MGB and PCR-RFLP assays were identical. This new assay was easy and fast with high throughput, without risk of molecular carryover, and cost-effective for laboratories utilizing the Taqman or related fluorescence reading methods. These advantages make it particularly suitable for large-scale combined genotyping of several polymorphisms in the routine setting.

    loading  Loading Related Articles