title: Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.
abstract: A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods.
authors.affiliation: Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jacroux
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Rieck
authors.forename: Rong
authors.initials: R
authors.lastname: Cui
authors.forename: Yexin
authors.initials: Y
authors.lastname: Ouyang
authors.forename: Wen-Ji
authors.initials: WJ
authors.lastname: Dong
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D016914:Ribonuclease H / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D013696:Temperature
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D012313:RNA; D004247:DNA; D016914:Ribonuclease H