Real-time monitoring in vitro transcription using molecular beacons.

A homogeneous fluorescence-based molecular beacon (MB) method has been developed for real-time monitoring of in vitro transcription reactions. MB probes are structured as target-specific antisense oligodeoxynucleotides containing a proximate fluorophore-quencher pair. Upon binding to its target sequence, the probe undergoes a structural rearrangement that separates the proximate pair, thus dequenching fluorescence. We demonstrate that this simple, inexpensive, rapid, and homogeneous fluorescence-based assay permits real-time monitoring of in vitro transcription and end-point measurement of RNA. The results from the RNA MB assay were comparable to those from other methods.