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Single molecule -DNA stretching studied by microfluidics and single particle tracking
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View: Figures


Image of FIG. 1.
FIG. 1.

(Color online) Schematic of the experimental setup and the bead-DNA-substrate construction. (a) The experimental setup (not drawn to scale). The microfluidic device was mounted on an inverted fluorescent microscope with a objective. The hydrodynamic force was used to pull on the bead. (b) Schematic representation of the bead-DNA-substrate construct. The DNA was annealed and ligated to the cohesive ends with oligo 1 (one end tagged with biotin), oligo 2, and oligo 3 (one end tagged with digoxigenin). The DNA molecule was bound to glass surface at one end by biotin∕streptavidin link and the other end to a fluorescent bead ( diameter) by digoxigenin∕antidigoxigenin link as illustrated.

Image of FIG. 2.
FIG. 2.

(Color online) Steady state velocity profile viewed in the cross section of the microfluidic channel. The profile was simulated using FLUENT 6. The velocity was normalized by setting 0 at the boundaries and 100 at the center. The region in which tethered beads were monitored is labeled in the figure.

Image of FIG. 3.
FIG. 3.

(Color online) Point-spread function (PSF) of an individual fluorescent microbead tethered to a single DNA at flow rate . A 2D Gaussian (solid lines) fits the PSF surface very well and this enables the center to be determined with uncertainty within .

Image of FIG. 4.
FIG. 4.

Flow rate∕force vs extension for dsDNA and ssDNA in a microfluidic channel. The flow rate was increased (stretching) or decreased (relaxing) every . The dsDNA stretching data is compared to a WLC model (dash lines) with persistence length of and contour length of .

Image of FIG. 5.
FIG. 5.

vs extension for a single dsDNA molecule. The linear relation between the two in the region of supports strong stretching WLC behavior. Only the data between 14 and was used to generate the trend line.

Image of FIG. 6.
FIG. 6.

(Color online) Flow rate∕force vs extension for a number of single dsDNA molecules in a microfluidic channel. The flow rate was increased every . (a) The force vs extension curves of several single dsDNA molecules. (b) These curves compare very well to WLC model (dash lines) when the hydrodynamic force is lower than .


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Single molecule λ-DNA stretching studied by microfluidics and single particle tracking