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 (DNA_pm) and single-base mismatched (DNA_mm) DNA strands are stronger and weaker, respectively, than those with Hg²⁺ ions. As a result, the fluorescence of a solution containing T₇-MB-T₇, DNA_pm, EthBr, and Hg²⁺ is higher than that of a corresponding solution containing T₇-MB-T₇, DNA_mm, 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 DNA_pm was linear over the range 5.0-100 nM (R² = 0.98). A similar probe, T₇-MB_t-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.