Real-time quantification of RNA polymerase activity using a "broken beacon".
A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.
Blair
Rebecca H
RH
InDevR LLC, Boulder, CO 80301, USA.
Rosenblum
Eric S
ES
Dawson
Erica D
ED
Kuchta
Robert D
RD
Kuck
Laura R
LR
Rowlen
Kathy L
KL
D002232:Carbocyanines / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D007700:Kinetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D015203:Reproducibility of Results
D002232:Carbocyanines; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; C089187:cyanine dye 3; D012313:RNA; D012321:DNA-Directed RNA Polymerases