Whitcombe D, Theaker J, Guy S P, Brown T, Little S
AstraZeneca Diagnostics, Gadbrook Park, Rudheath, Northwich, Cheshire CW9 7RA, UK.
Nat Biotechnol. 1999 Aug;17(8):804-7. doi: 10.1038/11751.
Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.
分子诊断正在从低通量、异质性、大多为手工操作的技术向高通量、闭管和自动化方法发展。荧光是此类检测中最受欢迎的信号技术，许多技术依赖于荧光团与近端猝灭分子之间的能量转移。在这些方法中，双标记探针与扩增子杂交，并检测荧光团猝灭的变化。我们描述了一种新技术，它使用简单，能提供高度特异性的信息，并且避免了替代方法的主要困难。它使用带有完整尾巴的引物来探测引物的延伸产物。对靶序列的探测因此转化为单分子事件，这在动力学、热力学、检测设计和探针可靠性方面具有显著优势。
