Detection of point mutations in DNA by fluorescence energy transfer

A method has been developed for the rapid and direct identification of a single point mutation in a DNA sequence using fluorescence resonance energy transfer (FRET). The probe was a 16-base oligomer with 5′-bound x-rhodamine and 3′-bound fluorescein (R*16*F); the two dyes acted as a donor/acceptor pair for FRET, resulting in a dramatic difference in the fluorescence emission of the R*16*F in a duplex structure (hybridized to a complementary strand) and as a single strand (melted). This difference was used to obtain the melting temperature (T_m), by spectroscopically following the transition from double to single strand, for the probe hybridized to three different strands: the 16-base complement, the 16-base complement containing a single base mismatch, and the 16-base complementary sequence in the phage DNA M13mp18(+). The T_ms thus determined for the perfectly base-paired duplexes, with R*16*F hybridized to the 16-mer complement and to M13, differed by 2°C, whereas the T_m obtained for R*16*F hybridized to the mismatched 16-mer complement was 10°C lower than that for the perfect duplex. The sharpness of the transition and the ease of detection allow single base mismatches to be reliably detected in nano- and subnanomolar concentrations in less than 1 h following hybridization.