A new concept for nucleic acid probe design is reported. The extremely high selectivity of the probe is predetermined by cooperative hybridization of the two relatively short ( 10 nucleotide) DNA hairpin fragments to the analyte. A binary DNA probe fluorescently reports the presence of 0.5% of the analyte in excess amount of a single base substituted oligodeoxyribonucleotide and distinguishes single nucleotide substitutions at any position of a 20-mer oligonucleotide at room temperature.