Mixed confinement regimes during equilibrium confinement spectroscopy of DNA

Abstract

We have used a combination of fluorescence microscopy experiments and Pruned Enriched Rosenbluth Method simulations of a discrete wormlike chain model to measure the mean extension and the variance in the mean extension of λ-DNA in 100 nm deep nanochannels with widths ranging from 100 nm to 1000 nm in discrete 100 nm steps. The mean extension is only weakly affected by the channel aspect ratio. In contrast, the fluctuations of the chain extension qualitatively differ between rectangular channels and square channels with the same cross-sectional area, owing to the "mixing" of different confinement regimes in the rectangular channels. The agreement between experiment and simulation is very good, using the extension due to intercalation as the only adjustable parameter.

Distribution of distances between DNA barcode labels in nanochannels close to the persistence length

“…(10) appears to be a good fit to the experimental data. However, corrections to L for intercalation, 30,42 even at rather low dye:bp ratios, would tend to reduce the fractional extension by increasing L, so we would expect that the classic Odijk regime is in fact an overestimate of the experimental data. This result is consistent with expectations at ξ ≈ 1, since the classic Odijk regime is only valid for ξ ≫ 1.…”

“…[27][28][29] While the imaging time is similar to the decorrelation time for λ-DNA in 100 nm channels, 30 the successful application of localization methods for measuring positions with sub-diffraction limit spatial resolution [27][28][29] indicates that motion blur is not a substantial problem. Nevertheless, we expect that there should be some error in the localization due to motion of the fluorophores during imaging.…”

Wall depletion length of a channel-confined polymer

“…In most cases, experiments compare the mean span and/or the variance obtained in experiments to theory or simulation, for example our recent work using confinement spectroscopy [22,23]. In these cases, the question of the exact value of the depletion length remains an open question for quantitative comparisons [22,23]. However, we cannot ascribe any discrepancy between the experiments and theory (or simulations) to a possible problem with the concept of a depletion length -even if there is double-layer overlap in the experimental system.…”

“…DNA has played a key role in the experimental tests of theories of a polymer confined to a slit [1][2][3][4][5][6][7][8][9][10][11][12][13][14] or a channel [15][16][17][18][19][20][21][22][23][24][25][26][27], in particular the models by de Gennes and coworkers for weak confinement [28,29] and Odijk for strong confinement [30,31]. The key results of this body of work have been summarized in several recent reviews [32][33][34].…”

“…Intercalation also increases the DNA contour length relative to the rise of naked DNA [55]. However, uncertainties in contour length can be minimized by (i) ensuring uniform staining of the DNA [56] and (ii) using funnel-shaped channels that allow one to interrogate the properties the same DNA molecule in a range of confinement [18,22,23,25,57]. For long chains [31], the thermodynamic variables become extensive quantities, so funnel-shaped channels permit a single-molecule test of various scaling laws independent of the contour length.…”