A method for homogeneous color-compensated genotyping of factor V (G1691A)and methylenetetrahydrofolate reductase (C677T) mutations using real-time multiplex fluorescence PCR
N. Von Ahsen et al., A method for homogeneous color-compensated genotyping of factor V (G1691A)and methylenetetrahydrofolate reductase (C677T) mutations using real-time multiplex fluorescence PCR, CLIN BIOCH, 33(7), 2000, pp. 535-539
Objectives: To set up an optimized multiplex polymerase chain reaction for
real-time genotyping of the prothrombotic risk factors methylenetetrahydrof
olate 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 melt
ing temperature (T-m) and high DeltaT(m) improved the discrimination of het
erozygous 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 matc
hed and mismatched state are desirably for genotyping. Such probes can be s
elected by using a priori calculations based on the thermodynamic nearest n
eighbor model. Copyright (C) 2000 The Canadian Society of Clinical Chemists
.