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 58-bound x-rhodamine and 38-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 (Tm), 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 Tms 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 Tm 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.
Kay M. Parkhurst,
Lawrence J. Parkhurst,
"Detection of point mutations in DNA by fluorescence energy transfer," Journal of Biomedical Optics 1(4), (1 October 1996). https://doi.org/10.1117/12.250674