Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation.
This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.
Yan
Xiaomei
X
Bioscience Division, Los Alamos National Laboratory, NM 87545, USA.
Hang
Wei
W
Majidi
Vahid
V
Marrone
Babetta L
BL
Yoshida
Thomas M
TM
D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D009696:Nucleic Acids; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D004396:Coloring Agents; D009696:Nucleic Acids; D004247:DNA