Wavelength-shifting molecular beacons
Wavelength-shifting molecular beacons
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DOI：
10.1038/81192
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发表时间：
2000-11-01
影响因子：
46.9
通讯作者：
Kramer, FR
中科院分区：
文献类型：
--
作者：
Tyagi, S;Marras, SAE;Kramer, FR
我们描述了波长移位的分子信标，这是一种核酸杂交探针，它以各种不同的颜色发光，但受到普通单色光源的激发。从激发光吸收能量和以荧光的形式发射能量的双重功能被分配给同一探头中的两个独立的荧光团。这些探针包含在单色光源的波长范围内强吸收的收集器荧光团、所需发射颜色的发射体荧光团和非荧光猝灭剂。在没有互补核酸目标的情况下，探针是暗的，而在目标存在的情况下，它们发出荧光--不是在吸收光线的收集器荧光团的发射范围内，而是在发射器荧光团的发射范围内。发射光谱的这种变化是由于通过荧光共振能量转移将吸收的能量从收集器荧光团转移到发射器荧光团，而且这种变化只发生在与目标结合的探测器中。波长移动的分子信标比传统的分子信标要亮得多，因为传统的分子信标包含的荧光团不能有效地从可用的单色光源中吸收能量。我们描述了波长移位分子信标的光谱特性，并演示了它们的使用如何改进和简化多重遗传分析。
We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.