Thrust 3: Presentation of Large DNA Molecules for Analysis as Nanoconfined Dumbbells
Kristy L. Kounovsky-Shafer, Juan P. Hernández-Ortiz, Kyubong Jo, Theo Odijk, Juan J. de Pablo, and David C. Schwartz
The analysis of very large DNA molecules intrinsically supports long-range, phase sequence information, but requires new approaches for their effective presentation as part of any genome analysis platforom. Using a multipronged approach that marshaled molecular confinement, ionic environment, and DNA elastic properties-buttressed by molecular sinulations-we have developed an efficient 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 molecule 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 hydrodynamiceffects 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 strat 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.
icrochannel-nanoslit device supporting the formation of molecular dumbbells (see Supplemental Movie S1). (A) PDMS device adhered to cleaned glass coverslip, immersed in buffer (not shown), for electrokinetic loading of DNA molecules. (B) Dumbbells form when loaded T4 DNA molecules (166 kb; 74.5 μm, dye adjusted contour length) exceed the nanoslit length (28 μm); molecule ends flanking nanoslits become relaxed coils within the microchannels (lobes), thereby enhancing the stretch of intervening segments within the nanoslits to (0.85 ± 0.16, I = 0.51 mM); yellow traces show fluorescence intensity variations along molecular backbones. (C) λ−DNA molecules (48.5 kb, 21.8 μm) are too short to form dumbbells and are thus completely confined within the nanoslits; a lower stretch (S/L = 0.62 ± 0.08, I = 0.48 mM) is further evidenced by uneven fluorescence intensity profiles.