We have developed a microfluidic device that works in concert with positively charged surfaces to deposit stripes consisting of elongated DNA molecules. Normally, large DNA molecules exist in solution as random coils with conformations akin to a floppy "ball of yarn,'' so that stable elongation of molecules becomes necessary for the high-resolution imaging of molecular barcodes. The current device boasts 48 parallel lanes for high throughput, and loads DNA solutions via capillary action onto positively charged glass surfaces. Normally relaxed DNA molecules are deposited on surfaces in an elongated state (stretched), because of the combination of capillary driven flow and their electrostatic interactions with the charged surface. Since electrostatic interactions bind molecules to the surface, enzymatic action is unhindered by common covalent bonding approaches requiring chemical linkers for the amelioration of deleterious surface interactions. Although each electrostatic bond is weak in comparison to covalent ones, the net effect of electrostatic bonding is additive, and on long molecules their sum greatly exceeds covalent bond
Fig. 1. The optical mapping system. (A) Genomic DNA is extracted from lysed cells. (B) DNA molecules are elongated and immobilized onto a positively charged glass surface using a microfluidic device. (C) A restriction enzyme cleaves cognate sites of the surface-bound DNA molecules. (D) The digested single DNA molecules are stained with a fluorescent dye and images are collected using a fully automated fluorescent microscope scanning system and a high resolution digital camera. (E) Single images are processed and merged together. (F) The flattened and overlapped single DNA molecule images are analyzed by machine vision software to generate single DNA molecule optical maps. (G) The single DNA molecule optical maps are formed into contigs based on overlapping restriction sites. Whole genome optical maps are constructed using ensembles of single DNA molecule maps. The dark gray barcode represents the consensus map, which includes data measurements from every single molecule map (light gray) in the underlying contig.
strengths. Accordingly, local electrostatic interactions are weak (spanning kb, compared to the thermal environment) fostering a dynamic equilibrium of "absorbed" DNA segments within a single molecule to shuttle between bound and free solution environments - only a portion of a molecule is bound at any given instant. This equilibrium supports vigorous enzymatic operations on "surface-bound" molecules enabling use of most DNA modification enzymes including restriction endonucleases. Restriction enzymes produce double-stranded "cuts" at specific cognate sequences, and these cleavage events are visualized by fluorescence microscopy as gaps (~1 mm) due to coil relaxation occurring at nascent ends; consecutive restriction fragments remain in register producing an ordered restriction map (barcode) after sizing operations.
Was this article helpful?