Skeidsvoll J, Ueland P M
Department of Clinical Biology, University of Bergen, Norway.
Anal Biochem. 1995 Nov 1;231(2):359-65. doi: 10.1006/abio.1995.9986.
The monomeric fluorescent dye, SYBR Green I, was investigated and compared with the dyes YO-PRO-1 and thiazole orange (TO) for their application in capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection of double-stranded DNA (dsDNA). DNA fragments were injected by hydrodynamic pressure and separated in a replaceable matrix of hydroxypropyl methylcellulose. For all 3 dyes, optimal concentrations were established and efficient separations of DNA fragments ranging in size from 75 to 12216 bp were obtained. The most promising results in terms of linear detection range were achieved with SYBR Green I. At the optimal dye concentration, fluorescence intensity versus DNA concentration was linear over more than three orders of magnitude (4 pg/microliters to 30 ng/microliters). Limit of detection (LOD) with SYBR Green I was approximately 80 fg of dsDNA (240 zmol of a 200-bp fragment). Similar LOD was obtained with YO-PRO-1, whereas TO resulted in lower sensitivity. Precision in both fluorescence intensity and migration time was high (relative standard deviation, RSD < 3.6%; n = 10) for dsDNA fragments complexed with SYBR Green I. In conclusion, SYBR Green I is fluorescent dye well suited for efficient separation and quantitative, sensitive, and precise determination of dsDNA by CE-LIF.
研究了单体荧光染料SYBR Green I，并将其与YO-PRO-1染料和噻唑橙（TO）进行比较，以评估它们在毛细管电泳（CE）中用于激光诱导荧光（LIF）检测双链DNA（dsDNA）的性能。通过流体动力学压力注入DNA片段，并在羟丙基甲基纤维素的可替换基质中进行分离。对于所有这3种染料，确定了最佳浓度，并实现了对大小在75至12216 bp范围内的DNA片段的有效分离。就线性检测范围而言，SYBR Green I取得了最有前景的结果。在最佳染料浓度下，荧光强度与DNA浓度在超过三个数量级（4 pg/微升至30 ng/微升）的范围内呈线性关系。SYBR Green I的检测限（LOD）约为80 fg的dsDNA（200 bp片段的240 zmol）。YO-PRO-1获得了类似的LOD，而TO的灵敏度较低。与SYBR Green I复合的dsDNA片段在荧光强度和迁移时间方面的精密度都很高（相对标准偏差，RSD < 3.6%；n = 10）。总之，SYBR Green I是一种非常适合通过CE-LIF对dsDNA进行高效分离以及定量、灵敏和精确测定的荧光染料。
