Biology Reference
In-Depth Information
approach is very simple and requires no further flow during data acquisi-
tion. Other methods of confining DNA close to the surface have been
achieved by tagging one end of the DNA with biotin and conjugating to
surface-immobilized streptavidin.
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Alternatively, lipid-immobilized DNA
molecules can be spatially aligned on the coverslip such that under flow
they elongate into ''curtains''.
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The disadvantages of these methods are,
first, that they all require flow (with the exception of combing), which will
tend to bias the dataset toward sliders as proteins that dissociate from the
DNA are likely to be carried away by the flow. In addition, the effects of the
surface are unknown: Are moving complexes stalling due to the binding of
the target site or because of interaction with the surface? Finally, as these
structures are often located within the same focal plane as the surface, it
becomes difficult to distinguish between surface-immobilized beads and
those that are immobile but attached to the DNA. Stopping and starting
flow using DNA curtains helps to circumvent this latter problem.
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4. Elevated DNA platforms or ''tightropes''
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offer perhaps the best com-
promise between many of the methods described here as they are microns
away from the surface and they elongate the DNA, and anything that
appears stationary for long enough to see within the visual field must be
bound to DNA (acquisitions are performed using high numerical aperture
objectives, which shorten the depth of field) (see
Fig. 3
). These structures
can be simply constructed by sequentially flowing the components into a
surface-passivated observation flowcell.
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The simple flow sequence for
setting up tightropes is as follows: (i) poly-l-lysine-coated 5
m
m sized
F
IG
. 3. Oblique angle fluorescence permits a high signal to noise view of DNA tightropes. To
generate OAF (top right), a standard TIRF (bottom right) optical path is steered to a subcritical
angle, resulting in a far-field illumination beam emerging at a steep angle. Although this was
achieved using a custom-built system (left), this is possible using off-the-shelf systems with little
difficulty.
