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Recent
developments in top-down lithography have made
possible the use of nanoconfined environments
for the separation and analysis of DNA molecules.
Separation systems are based an energy barrier
caused primarily by the lower entropy confined
state. The propensity of DNA to pass through
this entropic barrier varies for different molecule
sizes. In contrast, genomic analytical systems,
which present individual DNA molecules rely
on the low entropy environment to effectively
decrease the number of possible conformations
of DNA and ease its observation. Until recently,
most fabricated devices were aimed at separations;
however, engineering concerns for directing
the movement of DNA within a device designed
for DNA presentation requires vastly different
considerations. As such, it is desirable to
finely control the motion of DNA within these
devices for optimizing measurements reporting
biochemical states reflecting sequence composition,
protein or drug binding events within the context
of developing systems for genome analysis.
Towards
this goal, I am studying the diffusion, relaxation,
and local melting behavior of DNA within nanochannels
whose dimensions are on the order of that of
DNA's persistence length. The external and internal
diffusion and conformation will be measured
as functions of channel dimensions and solution
conditions. Two fluorophores will be attached
to the DNA molecule; one will be intercalated
into the DNA backbone while the other will be
attached to specific loci of the DNA. The two
dyes will interact through a process known as
FRET. Using fluorescence microscopy, the intercalated
dye will allow for the visualization of the
molecule while the second dye will allow for
the measurement of the position of specific
locations of the DNA chain in space. Under the
influence of an electric field, DNA will diffuse
into the nanochannels. After the electric field
has been switched off, the motion of the center
of mass as well as the motion of the marked
locations along the DNA backbone will be measured
through time-lapse imaging. In addition, local
melting behavior will be studied using a double
stranded molecule of DNA whose composition consists
of a poly-A region surrounded by two poly-G
regions. The poly-A region is expected to melt
under milder conditions than the poly-G region
forming a bubble within the molecule. By correlating
the internal and external motion of DNA in a
nanochannel with channel dimensions and different
solution conditions, a better understanding
of the effects of its confinement will be gained
and this new knowledge applied for design of
next generation platforms that will leverage
nanoconfinement effects.
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