Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.
Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.
复制标题
DOI：
10.1016/j.ab.2012.09.015
复制
发表时间：
2013-01-15
影响因子：
2.9
通讯作者：
Dong WJ
中科院分区：
文献类型：
--
作者：
Jacroux T;Rieck DC;Cui R;Ouyang Y;Dong WJ
描述了一种在等温或非等温条件下可操作的快速测定法，其中通过利用热稳定的RNA酶H酶促切割由DNA茎和RNA环组成的MB（R/D-MB）来提高典型分子信标（MB）系统的灵敏度。在R/D-MB与靶DNA杂交后，由于探针的打开和伴随的Förster共振能量转移效率的降低，荧光强度适度增加（高于背景约5.7倍）。添加热稳定的RNase H导致RNA环的切割，这消除了能量转移。切割步骤还释放了结合的靶DNA，使其能够与另一个R/D-MB探针结合，并使该方法成为循环扩增方案。R/D-MB的完全处理使荧光信号最大化（高于背景12.9倍），导致添加RNase H后在50 °C等温条件下观察到的信噪比增加约2-2.8倍。该探针还用于通过测量R/D-MB与扩增的pUC 19模板稀释液结合后供体荧光的增强来监测实时PCR反应。因此，R/D-MB-RNase H方案可以应用于广泛的核酸扩增方法。
A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. 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 (12.9x above background), resulting in a ~2–2.8 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 PCR reactions by measuring enhancement of donor fluorescence upon 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.
登录
查看更多内容
影响因子：
15
作者：
Du, H;Disney, MD;Krauss, TD
通讯作者：
Krauss, TD
影响因子：
46.9
作者：
Tyagi, S;Marras, SAE;Kramer, FR
通讯作者：
Kramer, FR
影响因子：
46.9
作者：
Dubertret, B;Calame, M;Libchaber, AJ
通讯作者：
Libchaber, AJ
影响因子：
2.9
作者：
Harvey, JJ;Lee, SP;Han, MK
通讯作者：
Han, MK
通过携带核糖核苷酸的分子信标和热稳定性 RNase HII 对双链 DNA 靶标进行新型单核苷酸多态性检测
影响因子：
2.9
作者：
Liu, Xi-Peng;Hou, Jing-Li;Liu, Jian-Hua
通讯作者：
Liu, Jian-Hua