Fluorescence Energy Transfer between Fluorescein Label and DNA Intercalators to Detect Nucleic Acids Hybridization in Homogeneous Media
Fluorescence Energy Transfer between Fluorescein Label and DNA Intercalators to Detect Nucleic Acids Hybridization in Homogeneous Media
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DOI：
10.1366/000370203321535132
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发表时间：
2003-02
影响因子：
3.5
通讯作者：
E. Talavera;R. Bermejo;L. Crovetto;Á. Orte;J. M. Alvarez-Pez
中科院分区：
文献类型：
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作者：
E. Talavera;R. Bermejo;L. Crovetto;Á. Orte;J. M. Alvarez-Pez
提出了一种通过稳态荧光测量来检测均质介质中核酸序列的通用方法。该方法结合使用荧光标记的单链 DNA 模型探针、互补的单链 DNA 靶标和 DNA 嵌入剂。该探针用荧光素标记在聚核糖胞苷酸 (5')、poly(C) 中胞嘧啶氨基的 N4 位置处的间隔臂上，充当模型 DNA 探针。互补链是聚核糖肌苷酸（5'），聚（I），作为靶标的模型，能量转移受体是嵌入剂，溴化乙锭或乙锭同二聚体。在之前的论文中，我们已经证明，当标记的 Poly(C) 与 Poly(I) 杂交时，荧光素标记的荧光强度会降低，这种荧光猝灭可用于检测均质介质中的 DNA 杂交或复性。在本文中，我们证明了当单链 Poly(C) 与 Poly(I) 杂交时，标记到 Poly(C) 的荧光素与嵌入剂之间会发生荧光共振能量转移 (FRET)，并且我们展示了荧光能量转移如何进一步降低标记的稳态荧光强度，从而提高该方法的检测限。这项工作的主要目的是开发一种真正均质的检测系统，用于使用稳态荧光和 FRET 在溶液中进行特定核酸杂交，但其优点是只需用能量供体标记探针，因为能量受体是自发插入的。而且，位点标记并不重要，可以在DNA链中随机标记。因此，该方法比之前发表的基于 FRET 的方法更简单。实验以直接形式和竞争形式进行。
A general approach to detecting nucleic acid sequences in homogeneous media by means of steady-state fluorescence measurements is proposed. The methodology combines the use of a fluorescence-labeled single-strand DNA model probe, the complementary single-strand DNA target, and a DNA intercalator. The probe was fluorescein labeled to a spacer arm at the N4 position of the cytosine amino groups in polyribocytidylic acid (5′), poly(C), which acts as a model DNA probe. The complementary strand was polyriboinosinic acid (5′), poly(I), as a model of the target, and the energy transfer acceptor was an intercalator, either ethidium bromide or ethidium homodimer. In previous papers we have shown that the fluorescence intensity of the fluorescein label decreases when labeled poly(C) hybridizes with poly(I), and this fluorescence quenching can be used to detect DNA hybridization or renaturation in homogeneous media. In this paper we demonstrate that fluorescence resonance energy transfer (FRET) between fluorescein labeled to poly(C) and an intercalator agent takes place when single-stranded poly(C) hybridizes with poly(I), and we show how the fluorescence energy transfer further decreases the steady-state fluorescence intensity of the label, thus increasing the detection limit of the method. The main aim of this work was to develop a truly homogeneous detection system for specific nucleic acid hybridization in solution using steady-state fluorescence and FRET, but with the advantage of only having to label the probe with the energy donor since the energy acceptor is intercalated spontaneously. Moreover, the site label is not critical and can be labeled randomly in the DNA strand. Thus, the method is simpler than those published previously based on FRET. The experiments were carried out in both direct and competitive formats.