Fluorescence detection of single-nucleotide polymorphisms using a thymidine-based molecular beacon

We have developed a universal molecular beacon (T7-MB-T7) for the detection of single-nucleotide polymorphisms (SNPs). The beacon, which contains a 19-mer loop and a stem comprising a pair of seven thymidine (T) bases, forms double-stranded structures with target DNA molecules, leading to increases in the fluorescence of ethidium bromide (EthBr) as a result of intercalation. The interactions of the beacon with perfectly matched (DNApm) and single-base mismatched (DNAmm) DNA strands are stronger and weaker, respectively, than those with Hg2+ ions. As a result, the fluorescence of a solution containing T7-MB-T7, DNApm, EthBr, and Hg2+ is higher than that of a corresponding solution containing T7-MB-T7, DNAmm, EthBr, and Hg2+, because the former has a greater number of intercalation sites for EthBr. Under the optimal conditions (100 nM T7-MB-T7, 20 mM NaCl, 5.0 μM Hg2+, and 300 nM EthBr in 5.0 mM Tris–HCl solution, pH 7.4), the plot of the fluorescence intensity against the concentration of DNApm was linear over the range 5.0–100 nM (R2 = 0.98). A similar probe, T7-MBt-T7, is sensitive and selective for the detection of a gene associated with hereditary tyrosinemia type I. Relative to conventional MBs, our new probe offers the advantages of higher selectivity toward DNA, less nonspecific binding toward single-stranded-DNA-binding protein, greater resistance to nuclease digestion, and low cost; therefore, we suspect that this system holds great potential for practical studies of SNPs.