A single molecule DNA flow stretching microscope for undergraduates
Tethering of DNA and bead (not to scale). The 5′ end of the DNA (labeled A) is labeled with a biotin moiety that binds to the streptavidin, which coats the glass coverslip surface (at the bottom of the figure). The 3′ end of the DNA (labeled B) is annealed to a primer sequence (labeled C), which is necessary to initiate DNA replication. The replication fork is formed by A, B, and C. The 3′ end labeled D is covalently linked to a digoxigenin molecule that binds to the antidigoxigenin, which coats the bead. This figure is taken from Ref. 30.
Experimental setup. Buffer is drawn from the sample through the flow cell and air spring into the syringe pump. The air spring isolates the flow cell from any mechanical noise generated by uneven pump action. The tethered DNAs are in the flow cell below the magnet. The off-axis lamp below the microscope stage creates dark field imaging. The image formed by the objective and eyepiece is viewed with the webcam. For simplicity, the microscope body is not shown.
Exploded view of flow cell. The flow channel is cut from double-sided tape (120 μm thick) and sandwiched between the quartz slide and the functionalized coverslip. Predrilled holes in the quartz slide match the tube diameter. The Y-shaped channel allows two inlet/outlet ports, which can be switched after loading beads. This figure is taken from Ref. 7.
Model of the tethered DNA and bead. The flow direction is to the right. The position of the bead center is . The magnet force and the fluid drag force are and , respectively. The functionalized coverslip is in the x - y plane.
DNA force-extension relation. The difference in extension for double stranded DNA (dsDNA) and single stranded DNA (ssDNA) is evident in this plot. This figure is adapted from Ref. 7.
DNA flow extension: comparison of experiment and theory. The horizontal bead position is plotted on the horizontal axis and the flow rate on the vertical. The curves show the calculation for four magnetic forces: 4 pN (solid line), 2 pN (dashed line), 1 pN (dotted line), and 0.5 pN (dash-dotted line). The measured values are shown with uncertainties. Uncertainties in the flow rate are assumed to be 10% (from the pump manufacturer’s specifications), and those of the horizontal extension are from standard deviations of the measured bead trajectories.
Trajectory of a bead during a DNA replication experiment. The coordinate of the bead in pixels along the flow direction versus the frame number. The direction of the flow is toward increasing pixel number. The slope of the diagonal region can be converted to the rate of motion along the DNA using the conversion factors discussed in the text. The difference in height between the plateaus before and after this region can be converted to the single molecule processivity.
Histograms of φ29 DNAP molecular motor results. Shown are the combined results of three experiments. (a) Single molecule rates. The mean speed is 59 bp/s with a standard deviation of 19 bp/s. This distribution width is a result of dispersion in the single molecule events and is not due to uncertainty in the rate determination. (b) Single molecule processivities. The data for processivities greater than 5000 bps fit to a single exponential with a decay length of 13500 bp.
The geometry of the microfluidic flow channel. Flow direction is indicated by a thick arrow. The channel height is h, the width is w, and the total length is L. Axes are oriented so that the flow is in the x direction and independent of the x coordinate.
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