Cytosine Detection by a Fluorescein‐Labeled Probe Containing Base‐Discriminating Fluorescent Nucleobase
Cytosine Detection by a Fluorescein‐Labeled Probe Containing Base‐Discriminating Fluorescent Nucleobase
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DOI：
10.1002/cbic.200400010
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发表时间：
2004-07
期刊：
影响因子：
3.2
通讯作者：
A. Okamoto;Kazuo Tanaka;Tetsuo Fukuta;I. Saito
中科院分区：
文献类型：
--
作者：
A. Okamoto;Kazuo Tanaka;Tetsuo Fukuta;I. Saito
我们报道了一种通过监测荧光素的荧光发射来检测DNA序列中特定位置的碱基的新方法。为了实现这一目标，我们开发了一种新的碱基识别荧光(BDF)核苷酸碱基--萘脱氮腺嘌呤(NDA)。当激发波长为350 nm时，以胞嘧啶碱基作为NDA的互补碱基的双链的荧光光谱在383 nm处有一个荧光峰。当NDA的互补碱基为另一种碱基时，荧光强度很低。在400-500 nm波长范围内，NDA的荧光发射光谱与荧光素的荧光激发光谱重叠。因此，我们设计了以NDA为FRET供体，以荧光素为受体的FRET-BDF探针。这两个由特定碱基对分隔的荧光团的相互作用允许有效的能量转移，从而在使用350 nm的激发波长时导致荧光素在520 nm处的主要荧光发射。只有当NDA的互补碱基为C时，才能观察到FRET-BDF探针的荧光发射，从而实现了互补DNA链上C碱基的明显区分。然而，由于NDA两侧的G/C碱基对猝灭了NDA的荧光，我们的方法的普遍实用性受到了限制。
We report on a new method for the detection of a base at a specific site in a DNA sequence by monitoring the fluorescence emission of fluorescein. To achieve this goal, we developed a new base‐discriminating fluorescent (BDF) nucleobase, naphthodeazaadenine (NDA). The fluorescence spectrum of the duplex possessing a cytosine base as a complementary base of NDA showed a fluorescence peak at 383 nm when using an excitation wavelength of 350 nm. When the complementary base of NDA was one of the other bases, the fluorescence intensity was very low. The fluorescence emission spectrum of NDA overlapped with the fluorescence excitation spectrum of fluorescein in the wavelength range of 400–500 nm. Thus, we designed FRET‐BDF probes containing NDA as the FRET donor and fluorescein as the acceptor. The interaction of these two fluorophores, which are separated by defined base pairs, allowed an efficient energy transfer that resulted in a dominant fluorescence emission of fluorescein at 520 nm when using an excitation wavelength of 350 nm. Fluorescence emission from FRET‐BDF probes was observed only when the complementary base of NDA is C, thus achieving a clear distinction of a C base on the complementary DNA strand. However, the general utility of our method is limited due to the quenching of the NDA fluorescence by a G/C base pair flanking NDA.