7 research outputs found
Presentation of Large DNA Molecules for Analysis as Nanoconfined Dumbbells
The analysis of very large DNA molecules
intrinsically supports
long-range, phased sequence information, but requires new approaches
for their effective presentation as part of any genome analysis platform.
Using a multipronged approach that marshaled molecular confinement,
ionic environment, and DNA elastic propertiesbuttressed by
molecular simulationswe have developed an efficient and scalable
approach for presentation of large DNA molecules within nanoscale
slits. Our approach relies on the formation of DNA dumbbells, where
large segments of the molecules remain outside the nanoslits used
to confine them. The low ionic environment, synergizing other features
of our approach, enables DNA molecules to adopt a fully stretched
conformation, comparable to the contour length, thereby facilitating
analysis by optical microscopy. Accordingly, a molecular model is
proposed to describe the conformation and dynamics of the DNA molecules
within the nanoslits; a Langevin description of the polymer dynamics
is adopted in which hydrodynamic effects are included through a Green’s
function formalism. Our simulations reveal that a delicate balance
between electrostatic and hydrodynamic interactions is responsible
for the observed molecular conformations. We demonstrate and further
confirm that the “Odijk regime” does indeed start when
the confinement dimensions are of the same order of magnitude as the
persistence length of the molecule. We also summarize current theories
concerning dumbbell dynamics
Presentation of Large DNA Molecules for Analysis as Nanoconfined Dumbbells
The analysis of very large DNA molecules
intrinsically supports
long-range, phased sequence information, but requires new approaches
for their effective presentation as part of any genome analysis platform.
Using a multipronged approach that marshaled molecular confinement,
ionic environment, and DNA elastic propertiesbuttressed by
molecular simulationswe have developed an efficient and scalable
approach for presentation of large DNA molecules within nanoscale
slits. Our approach relies on the formation of DNA dumbbells, where
large segments of the molecules remain outside the nanoslits used
to confine them. The low ionic environment, synergizing other features
of our approach, enables DNA molecules to adopt a fully stretched
conformation, comparable to the contour length, thereby facilitating
analysis by optical microscopy. Accordingly, a molecular model is
proposed to describe the conformation and dynamics of the DNA molecules
within the nanoslits; a Langevin description of the polymer dynamics
is adopted in which hydrodynamic effects are included through a Green’s
function formalism. Our simulations reveal that a delicate balance
between electrostatic and hydrodynamic interactions is responsible
for the observed molecular conformations. We demonstrate and further
confirm that the “Odijk regime” does indeed start when
the confinement dimensions are of the same order of magnitude as the
persistence length of the molecule. We also summarize current theories
concerning dumbbell dynamics
Presentation of Large DNA Molecules for Analysis as Nanoconfined Dumbbells
The analysis of very large DNA molecules
intrinsically supports
long-range, phased sequence information, but requires new approaches
for their effective presentation as part of any genome analysis platform.
Using a multipronged approach that marshaled molecular confinement,
ionic environment, and DNA elastic propertiesbuttressed by
molecular simulationswe have developed an efficient and scalable
approach for presentation of large DNA molecules within nanoscale
slits. Our approach relies on the formation of DNA dumbbells, where
large segments of the molecules remain outside the nanoslits used
to confine them. The low ionic environment, synergizing other features
of our approach, enables DNA molecules to adopt a fully stretched
conformation, comparable to the contour length, thereby facilitating
analysis by optical microscopy. Accordingly, a molecular model is
proposed to describe the conformation and dynamics of the DNA molecules
within the nanoslits; a Langevin description of the polymer dynamics
is adopted in which hydrodynamic effects are included through a Green’s
function formalism. Our simulations reveal that a delicate balance
between electrostatic and hydrodynamic interactions is responsible
for the observed molecular conformations. We demonstrate and further
confirm that the “Odijk regime” does indeed start when
the confinement dimensions are of the same order of magnitude as the
persistence length of the molecule. We also summarize current theories
concerning dumbbell dynamics
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Electrostatic confinement and manipulation of DNA molecules for genome analysis
Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte “issues” as exploitable advantages by our invention and characterization of the “molecular gate,” which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an “electrostatic bottle.” This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasma florum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis