Detection of point mutations using pyrene-labeled DNA probes

Pyrene-labeled oligodeoxyribonucleotide probes were shown to be suitable for the detection of point mutations. Reagents based on homochiral 2,4-dihydroxybutyramides were used to introduce pyrene residues at the 3"- and 5"- ends of oligonucleotide pairs. The oligonucleotide pair forms a tandem complex with a complementary target, giving rise to an excimer signal (λmax 470—490 nm) in the fluorescence spectra when the pyrene residues come into close proximity. The maximum ratio of the intensity of the excimer signal to the monomer signal (λmax 380—400 nm) is attained when (S)-N-(1-pyrenylmethyl)-3,3-dimethyl-2,4-dihydroxybutyramide is used to introduce the pyrene residue. The excimer fluorescence completely disappears with an increase in the distance between the pyrene residues (upon the introduction of an additional nucleotide in the target) or in the presence of a mismatch near the contact site of the probes.