Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China.
Analyst. 2012 Aug 21;137(16):3787-93. doi: 10.1039/c2an35262a. Epub 2012 Jul 4.
We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.
我们利用分子信标（MBs）和核酸染料 SYBR Green I 通过同步扫描荧光光谱法开发了一种用于特定单链 DNA 的双色荧光定量检测方法。以人类免疫缺陷病毒（HIV）的 RNA 片段的逆转录寡核苷酸序列（靶 DNA，33 个碱基）作为模型系统进行了证明。在不存在靶 DNA 的情况下，MBs 处于茎环闭合状态，5-羧基-X-罗丹明（ROX）的荧光被黑洞猝灭剂-2（BHQ-2）猝灭，并且 SYBR Green I 与 MBs 之间的相互作用非常弱。此时 ROX 和 SYBR Green I 的荧光信号都非常弱。在存在靶 DNA 的情况下，MBs 与靶 DNA 杂交形成双链结构，荧光团 ROX 与猝灭剂 BHQ-2 分离，ROX 的荧光恢复。同时，SYBR Green I 结合杂交的 dsDNA，其荧光强度显著增强。因此，可以通过同步扫描荧光光谱法实现对靶 DNA 的双色荧光定量检测。在该策略中，SYBR Green I 的荧光信号远大于 ROX 的荧光信号，因此通过 SYBR Green I 的荧光强度对靶 DNA 进行定量分析可以显著提高检测灵敏度。此外，MBs 的假阳性信号不会影响核酸染料 SYBR Green I 的荧光信号。因此，在分析复杂样品时，通过 SYBR Green I 对靶 DNA 进行定量分析可以避免 MBs 的假阳性信号，提高检测准确性。
