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.
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.
Louis
Magali
M
Laboratory of Applied Molecular Technology, Center for Human Genetics, Université catholique de Louvain, Brussels, Belgium.
Dekairelle
Anne France
AF
Gala
Jean-Luc
JL
D004252:DNA Mutational Analysis / Q000379:methods; D005165:Factor V / Q000235:genetics*; D005838:Genotype; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics*; D009154:Mutation; D015345:Oligonucleotide Probes / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011516:Prothrombin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin; D042965:Methylenetetrahydrofolate Reductase (NADPH2)