The ability to produce ordered fragments of DNA would greatly simplify the assembly problem of sequencing. We have studied the enzymatic cutting and desorption of surface-immobilized λ-DNA molecules deposited on PMMA and PAA coated silicon wafers. Surfaces were dipped into DNA solutions of 0.5-5.0 μg/ml and retracted at speeds of 1-10 mm/s to stretch and immobilize DNAs on the surface (a process termed “molecular combing”). Desorption experiments were performed on both surfaces. For PMMA, desorption was done in heated buffer solutions (DNase buffer, NEBuffer 3.1, pH-altered NEBuffer 3.1 (pH ≈ 10) at 50-70°C for varying times). For PAA, a polymer that exhibits a solubility switch from water-insoluble to water-soluble by immersion in CaCl<sub>2</sub> or NaCl solutions respectively, complete desorption into saline or buffer solution by PAA dissolution was observed.<br/>A microfluidic method was used to improve the application of a cutting enzyme to the DNA molecules on the surface (relative to a previously used soft lithographic stamping method). A pattern of holes in 1mm thick PDMS membranes was produced either by piercing the PDMS with microneedles (d = 400 μm) or forming and curing PDMS around the microneedles (d = 250 μm). The latter method avoided the irregular holes and cracks observed using the piercing method. PDMS layers with holes were pressed into contact with substrates having adsorbed DNAs and a solution containing DNase I cutting enzyme was deposited on top of the PDMS. By applying a vacuum (125 Torr) above the solution, air is made to escape the holes and the low-volume holes fill with the DNase I solution. The enzyme efficiently cuts the DNA, as confirmed by fluorescence microscopy.

Fragmentation and desorption of surface-immobilized DNA on PMMA and PAA substrates for sequencing applications

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.