Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

Thomas Jacroux; Daniel C Rieck; Rong Cui; Yexin Ouyang; Wen-Ji Dong

doi:10.1016/j.ab.2012.09.015

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Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

Journal article

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Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

Thomas Jacroux, Daniel C Rieck, Rong Cui, Yexin Ouyang and Wen-Ji Dong

Analytical biochemistry, Vol.432(2), pp.106-114

01/15/2013

DOI: https://doi.org/10.1016/j.ab.2012.09.015

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https://hdl.handle.net/2376/107656

PMCID: PMC3522425

PMID: 23000602

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Abstract

Isothermal

Real-time PCR

Nonisothermal

Molecular beacon

Cyclic amplification

Thermostable RNase H

Nucleic acids

Förster resonance energy transfer (FRET)

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.

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Title: Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection
Creators: Thomas Jacroux - Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA
Daniel C Rieck - Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA
Rong Cui - Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164, USA
Yexin Ouyang - Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164, USA
Wen-Ji Dong - Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA
Publication Details: Analytical biochemistry, Vol.432(2), pp.106-114
Academic Unit: Chemical Engineering and Bioengineering, School of
Publisher: Elsevier Inc
Identifiers: 99900546746001842
Language: English
Resource Type: Journal article