A Microfluidic System for Large DNA Molecule Arrays

Abstract

Single molecule approaches offer the promise of large, exquisitely miniature ensembles for the generation of equally large data sets. Although microfluidic devices have previously been designed to manipulate single DNA molecules, many of the functionalities they embody are not applicable to very large DNA molecules, normally extracted from cells. Importantly, such microfluidic devices must work within an integrated system to enable high-throughput biological or biochemical analysis-a key measure of any device aimed at the chemical/biological interface and required if large data sets are to be created for subsequent analysis. The challenge here was to design an integrated microfluidic device to control the deposition or elongation of large DNA molecules (up to millimeters in length), which would serve as a general platform for biological/biochemical analysis to function within an integrated system that included massively parallel data collection and analysis. The approach we took was to use replica molding to construct silastic devices to consistently deposit oriented, elongated DNA molecules onto charged surfaces, creating massive single molecule arrays, which we analyzed for both physical and biochemical insights within an integrated environment that created large data sets. The overall efficacy of this approach was demonstrated by the restriction enzyme mapping and identification of single human genomic DNA molecules.

High-resolution human genome structure by single-molecule analysis

“…1) to generate shotgun single-molecule restriction maps from the genomes of a complete hydatidiform mole (37) (CHM1h-TERT) and three lymphoblast-derived cell lines (GM15510, GM10860, GM18994). High molecular-weight genomic DNA was extracted from the cells with a gentle liquid lysis, then deposited on charged glass surfaces by an array of microfluidic capillary channels (26). The immobilized DNA molecules were digested in situ with the methylation-insensitive restriction endonuclease SwaI, chosen because its moderate average restriction fragment size balances good restriction map resolution with accurate fragment sizing.…”

“…A device comprising an array of microfluidic channels was fabricated using soft lithography and adhered to an Optical Mapping surface. A dilute DNA solution was pumped through the microchannels under parabolic flow conditions (26), causing the DNA to adhere to and stretch along the Optical Mapping surface via electrostatic interactions and flow-mediated forces. Thus presented and immobilized, the DNA was digested with the restriction endonuclease SwaI (New England Biolabs), then stained with YOYO-1 fluorescent dye (Invitrogen) and imaged on a Zeiss 135M inverted microscope (Carl Zeiss MicroImaging) at 63× magnification.…”

“…Thus presented and immobilized, the DNA was digested with the restriction endonuclease SwaI (New England Biolabs), then stained with YOYO-1 fluorescent dye (Invitrogen) and imaged on a Zeiss 135M inverted microscope (Carl Zeiss MicroImaging) at 63× magnification. The micrographs were analyzed by our automated machine-vision processing pipeline whose ultimate output was a set of ordered restriction maps of single DNA molecules (26,31).…”

Resolution limit for DNA barcodes in the Odijk regime

“…To determine the genomic distance between barcode markers, the DNA needs to be stretched from its equilibrium, coiled conformation. Several competing technologies have arisen to accomplish this task: using a receding contact line and subsequent binding to the surface (molecular combing), [8][9][10] extensional flow (direct linear analysis), [11][12][13] and nanochannel confinement. [5][6][7]14,15 The genomic distance between neighboring barcodes is then determined by first locating the centers of the sequence-specific labels and then integrating the total backbone fluorescence intensity between these two locations.…”

Anisotropic particles align perpendicular to the flow direction in narrow microchannels

“…There are many different types of anisotropic particles that are of high relevance for such studies, including semiflexible polymer chains, carbon nanotubes, fibrous proteins, rod-like nanoparticles, and DNA (10)(11)(12)(13)(14). For our first experiments, we chose cylindrical polymer micelles, a particularly well-suited model system for investigating the shear orientation of anisotropic particles (15).…”