Characterization and application of CataCleave probe in real-time detection assays
Seoul National University, Sŏul, Seoul, South Korea Analytical Biochemistry
(Impact Factor: 2.22).
11/2004; 333(2):246-55. DOI: 10.1016/j.ab.2004.05.037
Cycling probe technology (CPT), which utilizes a chimeric DNA-RNA-DNA probe and RNase H, is a rapid, isothermal probe amplification system for the detection of target DNA. Upon hybridization of the probe to its target DNA, RNase H cleaves the RNA portion of the DNA/RNA hybrid. Utilizing CPT, we designed a catalytically cleavable fluorescence probe (CataCleave probe) containing two internal fluorophores. Fluorescence intensity of the probe itself was weak due to Förster resonance energy transfer. Cleavage of the probe by RNase H in the presence of its target DNA caused enhancement of donor fluorescence, but this was not observed with nonspecific target DNA. Further, RNase H reactions with CataCleave probe exhibit a catalytic dose-dependent response to target DNA. This confirms the capability for the direct detection of specific target DNA through a signal amplification process. Moreover, CataCleave probe is also ideal for detecting DNA amplification processes, such as polymerase chain reaction (PCR) and isothermal rolling circle amplification (RCA). In fact, we observed signal enhancement proportional to the amount of RCA product formed. We were also able to monitor real-time PCR by measuring enhancement of donor fluorescence. Hence, CataCleave probe is useful for real-time monitoring of both isothermal and temperature-cycling nucleic acid amplification methods.
Available from: Daniel C Rieck
- "As demonstrated in Fig. 4, the R/D-MB–RNase H system can be applied as a conventional MB without the addition of RNase H if desired, with good performance still being obtained. It may also be seen that RNase-H-mediated enhancement of the S:N ratios seen in our study is greater than that obtained by Harvey and coworkers with the CataCleave approach  . Even with 200 nM CataCleave probe at the highest [target ssDNA] tested, Harvey and coworkers obtained an S:N ratio of approximately 6, whereas S:N ratios as high as approximately 30 were seen in our study (Fig. 4C). "
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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.
Available from: utexas.edu
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ABSTRACT: Biochemical and biophysical analyses of reversible associations may be reduced to a few fundamental pieces of information. One is the degree to which the complexes are formed (vs. disassembled) in any particular setting (concentrations, temperature, salts, etc.) Another is the docking geometry and conformation of such complexes. The latter information can be gleaned from structural techniques such as X-ray crystallography solution, NMR spectroscopy, SANS, or SAXS (or in some cases, cryo-EM studies), but many of those detailed methods are performed in conditions that change the equilibria we mean to study in the first place; i.e., either concentrations or temperatures are outside of the physiologically interesting regime. The fact that interesting and relevant structures still emerge is a tribute to artistry in those methods and an indication that many complexes are hardy and long-lived. It would be advantageous, however, to also work with techniques that are capable of working on low concentration samples (needed to assess the stoichiometry of tight interactions) and that are sensitive to short-lived, flexible, fragile, or sparsely populated complexes.
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