Stable triple-stranded DNA formation and its application to the SNP detection.

We have found that a short stretch (30mer or larger) of triple-stranded DNA structure formed at the terminus (or very near) of linear DNA molecules is unusually stable, withstanding heat treatment at as high as 95 degrees C. The stable triple-stranded structure is formed only when deoxyoligonucleotides are complementary to the strand terminating with 5'-phosphate and not to the strand terminating with 3'-OH. Presence of a single mismatched base in the complementary deoxyoligonucleotides drastically reduces the stability. We show that these unique properties of the terminal triple-stranded structure can be applied to the detection of single nucleotide polymorphisms in genomes without DNA dissociation and/or hybridization.