Single DNA molecule stretching in sudden mixed shear and elongational microflows

Abstract

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.

Collision of a DNA Polymer with a Small Obstacle

“…[23][24][25] In a broader context, similar disentanglement processes occur in polymers flowing over a polymer brush, 45 DNA separations using dilute neutral polymers as a sieving medium, 46 and flows of polymer solutions. 47 Finally, in direct linear analysis, 48,49 DNA collisions with posts may be a useful way of preconditioning DNA configurations before attempting to completely stretch DNA in an elongational flow [48][49][50] or electric field. 28,51 …”

Nanofluidic structures for single biomolecule fluorescent detection

“…The second half of this review discusses ways in which nanofluidic structures can be used to measure the mechanical properties of single DNA molecules [15][16][17][18] and perform sequence analysis. [19][20][21][22] As with the structures that are used to confine light, the devices that perform these tasks can be placed into one of two categories. They are either ''nanoslits,'' which allow a DNA strand to exist in a quasi two-dimensional (2D) plane, or they are called ''nanochannels'' and confine the polymer to a line.…”

Simulation of electrophoretic stretching of DNA in a microcontraction using an obstacle array for conformational preconditioning

“…It consists of two straight channels of different widths connected by a hyperbolic contraction. The wide inlet channel has a width of w 1 = 200 m, and the outlet channel has a width of w 2 = 3.8 m. The shape of the hyperbolic contraction was chosen to create a uniform strain rate within the contraction 17,19 and has a length of ᐉ c =80 m. In front of the contraction are three rows of posts with each post having a 1 m radius. The posts are spaced 4 m center-to-center within each row, and the distance between rows is also 4 m center-to-center.…”

“…15,16 In DLA devices, the molecules are typically stretched by field gradients in microcontractions. 2,3,[17][18][19][20] However, in these strain-limited devices, molecular individualism 21 leads to a large population of molecules that do not reach full extension. Recent studies have shown that the effects of molecular individualism can be mitigated by "preconfiguring" the initial conformation of a molecule before it is stretched.…”