Characterization and applications of CataCleave probe in real-time detection assays
Characterization and applications of CataCleave probe in real-time detection assays
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DOI：
10.1016/j.ab.2004.05.037
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发表时间：
2004-10-15
影响因子：
2.9
通讯作者：
Han, MK
中科院分区：
文献类型：
--
作者：
Harvey, JJ;Lee, SP;Han, MK
循环探针技术（CPT）是一种利用嵌合DNA-RNA-DNA探针和RNase H的快速等温探针扩增系统，用于检测靶DNA。在探针与其靶DNA杂交后，RNA酶H切割DNA/RNA杂交体的RNA部分。利用CPT，我们设计了一种含有两个内部荧光团的催化可切割荧光探针（CataCleave探针）。由于福斯特共振能量转移，探针本身的荧光强度较弱。在其靶DNA的存在下，通过RNase H切割探针引起供体荧光增强，但在非特异性靶DNA中未观察到这一点。此外，RNA酶H与CataCleave探针的反应表现出对靶DNA的催化剂量依赖性响应。这证实了通过信号放大过程直接检测特异性靶DNA的能力。此外，CataCleave探针也是检测DNA扩增过程的理想选择，如聚合酶链反应（PCR）和等温滚环扩增（RCA）。事实上，我们观察到信号增强与形成的RCA产物的量成比例。我们还能够通过测量供体荧光的增强来监测实时PCR。因此，CataCleave探针可用于实时监测等温和温度循环核酸扩增方法。(C)2004年爱思唯尔公司All rights reserved.
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 Forster 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. (C) 2004 Elsevier Inc. All rights reserved.