A single-molecule barcoding system using nanoslits for DNA analysis

Abstract

Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro-and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.

Wall depletion length of a channel-confined polymer

“…6 is met if the experimental data are to be compared to theory. For example, working at very low ionic strengths [4,13,19] is a facile strategy to increase DNA stretching through a combination of electrostatic repulsion from the walls (which reduces the effective channel size) and the increased persistence length of the DNA backbone [44][45][46]. In such experiments, it may prove more challenging to match the experimental data to theories for neutral polymers because the DNA-wall electrostatic interactions are sensible throughout most of the channel cross-section.…”

“…Fortunately, for most experimental systems, the Debye length is small compared to the channel cross section and the surface potential of the walls is not too high, so this condition is met. However, there is a small body of experimental data [4,13,19] that uses low ionic strength buffers where the Debye lengths are a substantial fraction of the channel.…”

“…DNA has played a key role in the experimental tests of theories of a polymer confined to a slit [1][2][3][4][5][6][7][8][9][10][11][12][13][14] or a channel [15][16][17][18][19][20][21][22][23][24][25][26][27], in particular the models by de Gennes and coworkers for weak confinement [28,29] and Odijk for strong confinement [30,31]. The key results of this body of work have been summarized in several recent reviews [32][33][34].…”

Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics

“…Recently, several groups have used nanofluidic channels to investigate the physical properties of nanoconfined DNA-protein complexes [12][13][14][15][16] and for optical mapping of single DNA molecules [17][18][19][20][21] .…”

An angle on sticky films

“…Other examples include the phenomenon of wrinkling 3,4 , which also has potential for material characterization. The use of the elasticity of confined DNA 5 as a means to stretch DNA in microchannels 6 -aimed at developing cheap sequencing devices -is also related. This list is obviously not exhaustive but is certainly indicative of the success of the impressionist style of theoretical physics and its sometimes unexpected applications.…”