Real-Time Monitoring of RNA and DNA Reactions by Fluorescence Detection

Conventional analysis of nucleic acid reactions (cleavages, ligations) is performed with a (radioactive) labeled substrate, and reaction products (one aliqout for each time-point) are analyzed by gel electrophoresis followed by detection (X-ray film + densitometer or phosphoimager). This is a cumbersome approach, and involves frequent preparation of fresh labeled substrate, and rather limited time resolution. Real-time monitoring with non-decaying fluorescent labels overcomes these problems. Two analysis methods are presented here. Fluorescence polarization with a very broad range of applications, can be used for any type of RNA or DNA conversion with significant change of mol wt (including “gel shift”). FRET (fluorescence resonance energy transfer) depends on intramolecular interaction (or in a multimolecular complex) of a fluorescent dye (“reporter”) with a quencher moiety (a guanine base or a second dye). Applications include detailed kinetic studies and optimizing reactions with a wide range of conditions and highly specific nucleic acid sequence detections, challenging other hybridization-based methods. Special applications are the introduction of catalytic nucleic acids for real-time monitoring of nucleic acid amplification reactions such as NASBA and PCR.