High-Sensitivity Capillary Electrophoresis of Double-Stranded DNA Fragments Using Monomeric and Dimeric Fluorescent Intercalating Dyes

• Published in: Analytical chemistry (Washington) 1994-07, Vol.66 (13), p.1941-1948
• Main Authors: Zhu, Huiping, Clark, Steven M., Benson, Scott C., Rye, Hays S., Glazer, Alexander N., Mathies, Richard A.
• Format: Article
• Language: English
• Subjects: 400105 - Separation Procedures; 550400 - Genetics; Analytical, structural and metabolic biochemistry; BASIC BIOLOGICAL SCIENCES; Biochemistry; Biological and medical sciences; CARBOHYDRATES; CELLULOSE; CLATHRATES; COMPLEXES; Deoxyribonucleic acid; DNA; DNA - isolation & purification; Dna, deoxyribonucleoproteins; DYES; ELECTROPHORESIS; FLUORESCENCE; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; Intercalating Agents; LASERS; LUMINESCENCE; NUCLEIC ACIDS; ORGANIC COMPOUNDS; POLYSACCHARIDES; SACCHARIDES
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac00085a004

Fluorescence-detected capillary electrophoresis separations of phi X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from phi X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. The optimum concentrations of 9AA for the separation of complexes preformed with the dimeric dyes TOTO, EthD, TOTAB, and YOYO were determined to be 100, 1, 1, and 0.5 microM, respectively.