Entropic unfolding of DNA molecules in nanofluidic channels.

Single DNA molecules confined to nanoscale fluidic channels extend along the channel axis in order to minimize their conformational free energy. When such molecules are forced into a nanoscale fluidic channel under the application of an external electric field, monomers near the middle of the DNA molecule may enter first, resulting in a folded configuration with less entropy than an unfolded molecule. The increased free energy of a folded molecule results in two effects: an increase in extension factor per unit length for each segment of the molecule, and a spatially localized force that causes the molecule to spontaneously unfold. The ratio of this unfolding force to hydrodynamic friction per DNA contour length is measured in nanochannels with two different diameters.

Conformation, length, and speed measurements of electrodynamically stretched DNA in nanochannels.

A method is presented to rapidly and precisely measure the conformation, length, speed, and fluorescence intensity of single DNA molecules constrained by a nanochannel. DNA molecules were driven electrophoretically from a nanoslit into a nanochannel to confine and dynamically elongate them beyond their equilibrium length for repeated detection via laser-induced fluorescence spectroscopy. A single-molecule analysis algorithm was developed to analytically model bursts of fluorescence and determine the folding conformation of each stretched molecule. This technique achieved a molecular length resolution of 114 nm and an analysis time of around 20 ms per molecule, which enabled the sensitive investigation of several aspects of the physical behavior of DNA in a nanochannel. lambda-bacteriophage DNA was used to study the dependence of stretching on the applied device bias, the effect of conformation on speed, and the amount of DNA fragmentation in the device. A mixture of lambda-bacteriophage with the fragments of its own HindIII digest, a standard DNA ladder, was sized by length as well as by fluorescence intensity, which also allowed the characterization of DNA speed in a nanochannel as a function of length over two and a half orders of magnitude.