CPIMA Seed - Flow-Enhanced Single Molecule DNA Hybridization Studies
Susan J. Muller, Chemical Engineering, University of California, Berkeley
This project leverages ongoing research on the dynamics of DNA within CPIMA. We have developed microfluidic devices and sequence-specific probes for studies of DNA hybridization kinetics that may also allow DNA genotyping with unprecedented read lengths. In addition, we have developed a novel microfluidic four-roll mill that allows all flow types (from extension to shear to rotation) to be accessed and used it to examine DNA tumbling in mixed flows. Ultimately, our work will provide benchmark data for extending simulations to include reaction kinetics and provide a microfluidic tool of bringing together vesicles or other supramolecular assemblies in a controlled way.
The dynamics of DNA in mixed flows that vary from pure shear to pure rotation have been examined through a combination of Brownian dynamics simulations and experiments. Brownian dynamics simulations, performed in collaboration with Shaqfeh, reveal a new vane-like tumbling pathway and a universal scaling law for the tumbling period in rotation-influenced flows. A unique microfluidic four-roll mill has allowed us to access rotation-dominated flows for the first time; experiments in the four-roll mill confirm the pathway and the scaling law (Figure 3).
Figure 3. DNA tumbling in mixed flows that vary from shear to rotation. DNA rotation. (a) The four-roll mill design. (b) Images of l-DNA in partially (Wi=4.65, a=-0.37) and purely (Wi=2.95, a=-1.0) rotational flow during a tumbling period. (c) Master curves of dimensionless tumbling period (T/t0) in rotational flow regime using Wieff. Shown are BD simulation (open symbols) and experimental observations (closed circles) for 1.1<Wi<18.7 and -1 <a< -0.3.
An enzyme with specific recognition sites on the DNA strand is covalently attached to a fluorescent nanoparticle. On incubation with DNA in the absence of Mg2+, the enzyme binds to but does not cleave the DNA. The DNA-probe complex is then introduced into a microfluidic cross-slot where it is stretched and imaged; the probe positions along the DNA backbone then serve to indicate the presence and location of the sequence of interest (Figure 4).
Figure 4. YOYO-1 stained, double-stranded l-DNA stretched in the stagnation point of a cross slot and visualized by epifluorescence microscopy. Left: Two fluorescent beads observed on hydrodynamically extended DNA molecules stretched at De = 0.8. Right: One bead observed; DNA extended at De = 1.2.
