A double-stranded molecular probe for homogeneous nucleic acid analysis
A double-stranded molecular probe for homogeneous nucleic acid analysis
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DOI：
10.1039/b804853c
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发表时间：
2008-01-01
期刊：
影响因子：
4.2
通讯作者：
Wong, Pak Kin
中科院分区：
文献类型：
--
作者：
Meserve, Daniel;Wang, Zhaohui;Wong, Pak Kin
本文报道了一种双链分子探针的设计和优化，用于特异性核苷酸序列的均匀检测。用荧光团或猝灭剂标记探针，使得探针杂交使两个标记接近，这在没有目标时减弱了荧光信号。在目标存在的情况下，荧光团探针被热力学驱动从其杂化形式解压缩并与目标结合。平衡分析成功地描述了该测定的所有主要特征，没有任何拟合参数，进行了推广探针的设计。研究了影响分析性能的几个关键参数。研究表明，探针浓度、猝灭与荧光团比和探针链序列可以优化检测的动态范围和信噪比。通过适当的序列设计，探针可以在一个步骤中识别单个核苷酸错配，而无需任何分离步骤或熔化pro的测量。
This paper reports the design and optimization of a double-stranded molecular probe for homogeneous detection of specific nucleotide sequences. The probes are labeled with either a fluorophore or a quencher such that the probe hybridization brings the two labels into close proximity, and this diminishes the fluorescence signal in the absence of a target. In the presence of a target, the fluorophore probe is thermodynamically driven to unzip from its hybridized form and bind with the target. An equilibrium analysis, which successfully describes all the major features of the assay without any fitting parameter, is performed to generalize the design of the probe. Several key parameters affecting the performance of the assay are examined. We show that the dynamic range and the signal-to-noise ratio of the assay can be optimized by the probe concentration, the quencher-to-fluorophore ratio, and the probe strand sequence. By proper design of the sequence, the probe discriminates single nucleotide mismatches in a single step without any separation step or measurement of melting pro.