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.
Liu
Jianwei
J
DuPont Pharmaceuticals, Wilmington, Deleware 19880, USA. jianwei.liu@astrazeneca.com
Feldman
Patricia
P
Chung
Thomas D Y
TD
D012321:DNA-Directed RNA Polymerases / Q000032:analysis*; D023381:Endpoint Determination / Q000379:methods; D005453:Fluorescence; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry; D015345:Oligonucleotide Probes / Q000737:chemistry*; D012313:RNA / Q000032:analysis / Q000096:biosynthesis; D015203:Reproducibility of Results; D014158:Transcription, Genetic
D020319:Oligodeoxyribonucleotides, Antisense; D015345:Oligonucleotide Probes; D012313:RNA; D012321:DNA-Directed RNA Polymerases