Binary probe system for sensitive detection of target analytes

We suggest a novel PCR-free assay that allows fluorescent detection of specific nucleic acid sequences with high selectivity and sensitivity. The assay can discriminate signal nucleotide substitution in an analyzed nucleic acid with high accuracy even at ambient temperatures. The high sensitivity of the assay is predetermined by the possibility of fluorescent signal amplification due to the catalytic action of an enzyme, which recognizes only the complex of the probe with the analyte and not the probe itself. The assay is cost-efficient, which is especially beneficial for the analysis of multiple nucleic acid analytes: two short unmodified oligonucleotides need to be synthesized for each new analyte sequence, while all other reagents are analyte-independent and, therefore, can be obtained in bulk amount and utilized efficiently. The assay is adaptable for the microarray format and do not require any alterations of the existing techniques for the microarray production