Journal
ANALYTICAL BIOCHEMISTRY
Volume 432, Issue 2, Pages 106-114Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.09.015
Keywords
Isothermal; Nonisothermal; Molecular beacon; Thermostable RNase H; Nucleic acids; Cyclic amplification; Forster resonance energy transfer (FRET); Real-time PCR
Funding
- National Institutes of Health [HL80186, HL80186-5S1]
- Life Science Discovery Fund [1876237]
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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 (similar to 5.7 x above background) due to an opening of the probe and a concomitant reduction in the Forster 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 x above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 degrees 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. (C) 2012 Elsevier Inc. All rights reserved.
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