探索SYTOX橙色染料与双链DNA结合的动力学，对DNA分析具有重要意义。,Analytical Chemistry

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探索SYTOX橙色染料与双链DNA结合的动力学，对DNA分析具有重要意义。
Analytical Chemistry ( IF 6.7 ) Pub Date : 2005 Jun 1 , DOI: 10.1021/ac050306u
Xiaomei Yan ₁ , Robert C. Habbersett ₁ , Thomas M. Yoshida ₁ , John P. Nolan ₁ , James H. Jett ₁ , Babetta L. Marrone ₁

Affiliation

在DNA片段大小流式细胞仪上揭示了SYTOX Orange染色剂与双链DNA（dsDNA）的快速结合动力学。我们首次证明了染料分子可以被吸附到毛细管表面上，并且天然DNA片段在通过毛细管时可以被动态染色。在染色，稀释或混合后立即分析的DNA样品获得了高质量的突发大小分布直方图。这些观察结果表明，SYTOX Orange染料分子与dsDNA之间存在快速相互作用。设置了停止流动的荧光设备，以捕获与dsDNA结合的嵌入染料的快速缔合痕迹。推导了动力学方程，以拟合关联曲线以确定缔合速率，并对动态染色，稀释，插入染料染色的DNA样品的混合过程。插入dsDNA的SYTOX Orange和PicoGreen染色的缔合率约为10（8）M-1 s-1，表明存在扩散控制的过程。模拟表明，染色，稀释或混合后几秒钟即可达到重新平衡。深入了解DNA结合染料的动力学将有助于在DNA分析中实施有效的样品处理实践，包括DNA片段大小流式细胞仪。

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Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis.

Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.

更新日期：2017-01-31

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