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


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Abstract

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.

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