A method for homogeneous color-compensated genotyping of factor V (G1691A) and methylenetetrahydrofolate reductase (C677T) mutations using real-time multiplex fluorescence PCR

Objectives: To set up an optimized multiplex polymerase chain reaction for real-time genotyping of the prothrombotic risk factors methylenetetrahydrofolate reductase C677T and factor V Leiden on the LightCycler. Design and methods: Novel primer and probe sets were designed on the basis of thermodynamic double-strand DNA stability calculations. Detection probes were labeled with LC-Red640 or Cy5.5 dye. Results: The polymerase chain reaction efficiency was reduced in multiplex polymerase chain reaction but this could be overcome by the design of novel amplification primers. The selection of detection probes with a lower melting temperature (Tm) and high ΔTm improved the discrimination of heterozygous samples. Color compensation was not compromised by either the use of the Cy5.5 dye or different fluorescein linker chemistries. Conclusions: Probes with a ΔTm of 5 °C or more between the matched and mismatched state are desirably for genotyping. Such probes can be selected by using a priori calculations based on the thermodynamic nearest neighbor model.