| Rapid detection of single nucleotide polymorphisms (SNPs) has potential applications in both genetic screening and pharmacogenomics. Planar waveguide fluorescent biosensor technology was employed to detect SNPs using three analytical techniques: traditional DNA/DNA hybridization, peptide nucleic acid/DNA hybridization, and single base extension. Waveguide technology allows real-time measurements of DNA hybridization/reaction kinetics. The traditional hybridization assay only produced absolute discrimination between a perfectly matched duplex and one containing a single mismatch at temperatures that destabilized or melted the heteroduplex while maintaining the stability of the homoduplex. Such assays could be performed in 2 minutes. A lower degree of discrimination (2-fold difference in hybridization rate between the hetero- and homoduplex) was obtained at lower, less rigorous temperatures (32°C), with hybridization times of 7.5 minutes, or longer. Peptide nucleic acid/DNA hybridization assays yielded similar results to the traditional hybridization assay, but with improved attenuation (10-fold) of the mismatched sequence in a 5-minute assay at 30°C at low ionic strength. Single base extension yielded attenuation of the mismatched sequence to near background levels. Single base extension requires magnesium for enzyme activity, which also activates DNA digesting pyrogens. Single base extension would be the technique of choice for most assay requirements, because of the near complete discrimination in a 5-minute assay at 40°C. |
