Probing the Kinetics of SYTOX Orange Stain Binding to Double-Stranded DNA with Implications for DNA Analysis
- 15 April 2005
- journal article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 77 (11), 3554-3562
- https://doi.org/10.1021/ac050306u
Abstract
Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 108 M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.Keywords
This publication has 17 references indexed in Scilit:
- Sizing of single fluorescently stained DNA fragments by scanning microscopyNucleic Acids Research, 2003
- DNA Fragment Sizing by Single Molecule Detection in Submicrometer-Sized Closed Fluidic ChannelsAnalytical Chemistry, 2002
- 1H NMR Studies of the Bis-Intercalation of a Homodimeric Oxazole Yellow Dye in DNA OligonucleotidesJournal of Biomolecular Structure and Dynamics, 1998
- New technique for high resolution DNA sizing in epi-illuminationCytometry, 1998
- Characterization of DNA Size Determination of Small Fragments by Flow CytometryAnalytical Chemistry, 1995
- Optical and Photophysical Properties of the Oxazole Yellow DNA Probes YO and YOYOThe Journal of Physical Chemistry, 1994
- Characterization of the Binding of the Fluorescent Dyes YO and YOYO to DNA by Polarized Light SpectroscopyJournal of the American Chemical Society, 1994
- InstrumentationAnalytical Chemistry, 1993
- Rapid sizing of individual fluorescently stained DNA fragments by flow cytometryNucleic Acids Research, 1993
- Stable dye–DNA intercalation complexes as reagents for high-sensitivity fluorescence detectionNature, 1992