Characterization and application of CataCleave probe in real-time detection assays
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.
[Show abstract] [Hide abstract] ABSTRACT: Background Accurate measurement of BCR–ABL1 fusion transcripts is critical for therapeutic stratification in patients with chronic myelogenous leukemia (CML). Previous studies have reported the variable performance of the existing quantitative reverse transcription polymerase chain reaction (RQ-PCR). Here, we developed a one-step multiplex RQ-PCR method based on the catalytically cleavable fluorescence probe technology for quantification of BCR–ABL1 transcripts. Methods Performance was evaluated with respect to the limit of detection (LoD), linearity, precision, and comparison on the VIIA7 Real-Time PCR system. Multiplex RQ-PCR was performed by the one-step and one-well reaction without the hands-on time. Results Our assay showed a LoD of 1.5 pg with linearity in the range of more than 4 logs of dilution. Intraassay, interassay, and total percent CVs at the concentration of 150 ng were 12.8%, 22.6%, and 28.0%, respectively. The assay correlated well with Asuragen's BCR/ABL1 Quant™ kit over a 6 log concentration range (r = 0.9967). Conclusion Our assay demonstrated comparable performance characteristics in comparison with previous RQ-PCR based on the TaqMan probe technology. We conclude that our method could be a reliable tool in the clinical setting.0Comments 1Citation
- "Excellent performance of our assay could be attributed to the application of CataCleave probe technology. Until now, CataCleave technology has been reported to be used to detect of target DNA, such as SNP, deletion, and partially methylated DNA as well as microorganisms [18,26]. For the first time, we showed the clinical application of CataCleave probe technology for quantification of human RNA such as BCR–ABL1 fusion transcripts in a multiplex assay format. "
[Show abstract] [Hide abstract] 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.0Comments 7Citations
- "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). "
- [Show abstract] [Hide abstract] ABSTRACT: Not available Cellular and Molecular Biology, Institute for0Comments 0Citations