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 (T-m) and high DeltaT(m) 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 DeltaT(m) of 5 degreesC 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. Copyright (C) 2000 The Canadian Society of Clinical Chemists.