Electrophoresis has been a longstanding, useful
technique for separating DNA. It is the basis for many important techniques in
molecular biology, including DNA restriction fragment mapping, DNA sequencing,
Southern blotting, DNA fingerprinting and Dnase footprinting. It has typically
been performed in slab gels, but the advent of capillary electrophoresis (CE)
has expanded the use of DNA separations by reducing the time of separation.
Typically, gel media have been used to effect a length-dependent separation in
CE. In early work, we demonstrated that slow reptation-based DNA separations in
gels can be replaced by the use of dilute polymer solutions as the separating
agent in CE. We have shown that the mechanism of DNA separation is based a
transient entanglement coupling of DNA with polymer, where the release time of
the DNA is a function of DNA length, thereby providing length-dependent
resolution.
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We have confirmed this new
separation mechanism using epifluorescence video microscopy. By observing
single-molecule DNA-polymer entanglements directly as DNA electrophoreses
through a capillary, we see that DNA/polymer collisions depend on the size and
concentration of each species, and that multiple entanglements are possible. We
have obtained data on the entanglement time as a function of the Porod-Kratky
persistence length of the polymer (a measure of its stiffness), the collision
frequency and probability distribution of multiple entanglements. Using these
data, a mechanistic model for DNA separations has been developed, and is able to
predict DNA separations as a function of DNA size and polymer properties. |
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