DETECTION OF POLYPHOSPHATE USING FLUORESCENTLY LABELED POLYPHOSPHATE ACCEPTOR SUBSTRATES

摘要

Provided herein are methods and compositions for fluorogenic detection of a polyphosphate released from a nucleoside polyphosphate by the enzymatic action of a nucleic acid polymerase. The methods and compositions are based on the transfer of a free polyphosphate (or polyphosphate labeled with a fluorescence dye) to a polyphosphate acceptor molecule that is modified with a moiety that facilitates fluorogenic detection of a product that indicates the release of polyphosphate by the enzymatic action of a DNA or RNA polymerase. In one aspect, fluorogenic detection is facilitated by forming a fluorescent donor/acceptor pair on the polyphosphate acceptor substrate. The combination of the donor and acceptor pair provides a differentially detectable fluorescent product. In another aspect, fluorogenic detection is facilitated by releasing a fluorescent dye from the polyphosphate acceptor substrate when the free polyphosphate is transferred to the acceptor substrate. When the fluorescent dye is released form the acceptor substrate, it is dequenched, thereby providing for fluorescent detection.