Analysis of Double-Stranded DNA by Capillary Electrophoresis with Laser-Induced Fluorescence Detection Using the Monomeric Dye SYBR Green I

Abstract

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/μl to 30 ng/μl). 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 a fluorescent dye well suited for efficient separation and quantitative, sensitive, and precise determination of dsDNA by CE–LIF.