Probing single DNA mobility with fluorescence correlation microscopy

Fluorescence correlation spectroscopy combined with microscopy (FCSM) is used to study the mobility of DNA fragments in aqueous solution and tissue models on the single molecule level. The effective hydrodynamic radius was measured for various lengths of ds-DNA chains and obeyed the theoretically inveterate ${\left[\mathrm{DNA}\text{length}\right]}^{0.5}$ relationship. Hindered diffusion of ds-DNA through the gel matrix of various densities is thought of as an extension of Kramer’s problem for a flexible polymer chain. With increasing DNA length the average barrier crossing time rises as ${\left[\mathrm{DNA}\text{length}\right]}^2$ and this agrees with theory predictions for polymer molecules surmounting an entropic barrier.

Figure 1

(Color online). Optical scheme of experimental setup based on the up-right research microscope Nikon E800 with additional home-built detection scheme to carry out the spectroscopic measurements.

Figure 2

(a) The experimental autocorrelation function for $5\mathrm{kb}$ DNA and its fitting showing the validity of the model used. (b) Theoretical models (solid and dashed lines) used to describe the polymer dynamics in diluted solutions and experimental data (shown by points with error bars and fitting shown by dotted line) of diffusion of DNA fragments acquired by FCS.

Figure 3

Experimental log-log data for diffusion of DNA molecules in low concentration agarose gel (0.2 and 0.5%). The top-dashed line serves as a reference on data received for free DNA diffusion in aqueous solution. On the inset (top right corner) the ratio of diffusion coefficient in aqueous solution to diffusion coefficient in gel is shown as a function of polymer length for two subsequent gel concentrations.