ANALYTICAL BIOCHEMISTRY 231, 359–365 (1995) Analysis of Double-Stranded DNA by Capillary Electrophoresis with Laser-Induced Fluorescence Detection Using the Monomeric Dye SYBR Green I Jarle Skeidsvoll 1 and Per Magne Ueland Department of Clinical Biology, University of Bergen, Armauer Hansens Hus, N-5021 Bergen, Norway Received April 10, 1995 acids in terms of speed, resolution, quantitation, repro- The monomeric fluorescent dye, SYBR Green I, was ducibility, and automation compared with conventional investigated and compared with the dyes YO-PRO-1 gel electrophoresis systems (1,2). Highly efficient sepa- and thiazole orange (TO) for their application in capil- rations of single- and double-stranded DNA ranging in lary electrophoresis (CE) with laser-induced fluores- size from a few nucleotides to millions of base pairs cence (LIF) detection of double-stranded DNA have been obtained by CE (3,4). (dsDNA). DNA fragments were injected by hydrody- Due to the minute sample volumes injected in typical namic pressure and separated in a replaceable matrix CE analyses (nanoliter range), the practical application of hydroxypropyl methylcellulose. For all 3 dyes, opti- of CE is highly dependent on sensitive detection sys- mal concentrations were established and efficient sep- tems. Detection by uv absorbance is commonly used in arations of DNA fragments ranging in size from 75 to CE analyses of DNA, but confers limited sensitivity. 12216 bp were obtained. The most promising results Extremely high sensitivity can be obtained by laser- in terms of linear detection range were achieved with induced fluorescence (LIF) detection (5). This approach SYBR Green I. At the optimal dye concentration, fluo- requires fluorescent DNA derivatives obtained by ei- rescence intensity versus DNA concentration was lin- ther covalent binding of fluorophores or physical ab- ear over more than three orders of magnitude (4 pg/ml sorption of fluorogenic dyes. to 30 ng/ml). Limit of detection (LOD) with SYBR Green I was approximately 80 fg of dsDNA (240 zmol of a 200- Characteristics of an ideal fluorescent dye for CE – bp fragment). Similar LOD was obtained with YO-PRO- LIF analysis of DNA have been summarized by 1, whereas TO resulted in lower sensitivity. Precision Schwartz and Ulfelder (6) as (1) excitation maximum in both fluorescence intensity and migration time was of the DNA–dye complex close to a available laser high (relative standard deviation, RSD õ 3.6%; n Å 10) wavelength, (2) low intrinsic fluorescence of dye not for dsDNA fragments complexed with SYBR Green I. complexed with DNA, (3) large fluorescence enhance- In conclusion, SYBR Green I is a fluorescent dye well ment upon binding of dye to DNA, and (4) high fluores- suited for efficient separation and quantitative, sensi- cence quantum yield of the DNA–dye complex. Addi- tive, and precise determination of dsDNA by CE – LIF. tional important characteristics for quantitation of  1995 Academic Press, Inc. DNA in unknown samples are (5) uniform and not se- quence specific binding, (6) constant fluorescence inten- sity and electropherographic profile over a wide range of DNA–dye ratios, and (7) large linear detection Capillary electrophoresis (CE) 2 represents a signifi- range. cant improvement in electrophoretic analysis of nucleic A series of new fluorescent dyes with improved physi- cal and spectroscopic properties have recently been de- 1 To whom correspondence should be addressed. Fax: /47-55- 974605. veloped (7–10). The dyes can be classified as mono- 2 Abbreviations used: CE, capillary electrophoresis; LIF, laser-in- meric and dimeric. The dimeric dyes have several duced fluorescence; YO-PRO-1, 1-(4-[3-methyl-2,3-dihydro-(benzo- favorable features, including high affinity for and large 1,3-oxazole) - 2 - methylidene] - quinolinium) - 3 - trimethylammonium fluorescence enhancement upon binding dsDNA (10). propane diodide; TO, thiazole orange; LOD, limit of detection; DMSO, dimethyl sulfoxide; HPMC, hydroxypropyl methylcellulose. However, efficient separations are only obtained over 359 0003-2697/95 $12.00 Copyright  1995 by Academic Press, Inc. All rights of reproduction in any form reserved.