Fluorescent DNA Probes for Detection of Mycobacteria

This developmental project describes the utilization of novel enzymatic methods for synthesizingpermanently fluorescent nucleic acid probes and descibes their use in developing automatablesolution-phase hybridization assays for the detection of mycobacteria, specifically the Mycobacteriumtuberculosis complex from human clinical specimens. Two proprietary technologies have been developedwhich incorporate fluorescent nucleotide analogs into probes during enzymatic synthesis. Thetechnology relies upon the complete replacement at each occurrence of a specific nucleotide with itscorresponding fluorescent structural analog. This substituted oligonucleotides hybridize to theirrespective target sequences with the same specificity and reaction kinetics as the normal non-substitu-ted oligonucleotides. The fluorescent probes can be subsequently incorporated into diagnostic assaysfor infectious disease pathogens. Fluorescent nucleotide analogs have been chosen as the detectablelabel because their emission is stable over long periods, and the multiple substitutions provide a systemof label amplification, thereby enhancing sensitivity by several orders of magnitude over existingdiagnostic assays.