Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves.

A microvolume fluorimeter integrated with a rapid thermal cycler allows both amplification and point mutation detection from genomic DNA in approximately 30 min. This homogeneous method combines rapid cycle DNA amplification with allele-specific fluorescent probe melting profiles for product genotyping. The amplification reaction includes a primer internally labeled with Cy5 and a 3'-fluorescein-labeled probe that spans the region of interest. During asymmetric amplification, the probe hybridizes to excess Cy5-labeled strand and is observed as fluorescence resonance energy transfer. Resonance energy transfer increases each cycle as product accumulates during amplification. When fluorescence is monitored as the temperature increases through the Tm of the probe/product duplex, a characteristic melting profile for each genotype is obtained. Fluorescence genotyping of the common C677T base substitution in the methylenetetrahydrofolate reductase gene in 110 DNA samples correlated perfectly with genotyping by restriction enzyme digestion and gel electrophoresis. The relatively stable G:T mismatch of this example gave a 3 degrees C difference in Tm from complete Watson-Crick pairing, suggesting that this homogeneous fluorescence method can be used for all single-base mismatches.