The application of fluorescence correlation spectroscopy in detecting DNA condensation

Abstract

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHβAPr-1-neo (10 kbp, contour length 3.4 μm) and pBluescript SKt (2.96 kbp, contour length 1.02 μm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10 −5 M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0×10 −6 M and 2.0×10 −6 M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D 10 kbp=9.6×10 −13 m 2 s −1 and D 2.96 kbp=2.5×10 −12 m 2 s −1, respectively. When the condensing agent in a concentration higher than 1.10×10 −5 M is added to pHβAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D 10 kbp=8.0×10 −12 m 2 s −1 (spermine) and D 10 kbp=5.5×10 −12 m 2 s −1 (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D 2.96 kbp=9.2×10 −12 m 2 s −1 (spermine) and D 2.96 kbp=8.2×10 −12 m 2 s −1 (HTAB).