caa a22 4500 378898248 CHVBK 20180305123511.0 cr unu---uuuuu 161128e20041201xx s 000 0 eng 10.1515/CCLM.2004.255 doi (NATIONALLICENCE)gruyter-10.1515/CCLM.2004.255 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 [Elektronische Daten] [Magali Louis, Anne France Dekairelle, Jean-Luc Gala] 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. ©2004 by Walter de Gruyter Berlin New York Medical equipment & techniques nationallicence Medical diagnosis nationallicence Diseases & disorders nationallicence minor groove binder nationallicence mutation detection nationallicence real-time polymerase chain reaction (PCR) nationallicence single nucleotide polymorphism nationallicence thrombosis nationallicence Louis Magali Laboratory of Applied Molecular Technology, Center for Human Genetics, Université catholique de Louvain, Brussels, Belgium aut Dekairelle Anne France Laboratory of Applied Molecular Technology, Center for Human Genetics, Université catholique de Louvain, Brussels, Belgium aut Gala Jean-Luc Laboratory of Applied Molecular Technology, Center for Human Genetics, Université catholique de Louvain, Brussels, Belgium and Defense Laboratories Department, Belgian Armed Forces, Brussels, Belgium aut Clinical Chemical Laboratory Medicine Walter de Gruyter 42/12(2004-12-01), 1364-1369 1434-6621 42:12<1364 2004 42 cclm https://doi.org/10.1515/CCLM.2004.255 text/html Onlinezugriff via DOI 1 research article jats NATIONALLICENCE 856 40 https://doi.org/10.1515/CCLM.2004.255 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Louis Magali Laboratory of Applied Molecular Technology, Center for Human Genetics, Université catholique de Louvain, Brussels, Belgium aut NATIONALLICENCE 700 1- Dekairelle Anne France Laboratory of Applied Molecular Technology, Center for Human Genetics, Université catholique de Louvain, Brussels, Belgium aut NATIONALLICENCE 700 1- Gala Jean-Luc Laboratory of Applied Molecular Technology, Center for Human Genetics, Université catholique de Louvain, Brussels, Belgium and Defense Laboratories Department, Belgian Armed Forces, Brussels, Belgium aut NATIONALLICENCE 773 0- Clinical Chemical Laboratory Medicine Walter de Gruyter 42/12(2004-12-01), 1364-1369 1434-6621 42:12<1364 2004 42 cclm CC0 http://creativecommons.org/publicdomain/zero/1.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-gruyter