Fragmentation and Desorption of Surface-Immobilized DNA on PMMA and PAA Substrates for Sequencing Applications

Producing ordered fragments of DNA would greatly simplify sequencing’s assembly problem. We studied the enzymatic cutting and desorption of surface-immobilized λ-DNA molecules on PMMA and PAA coated silicon wafers. Surfaces were dipped into 0.5-5.0 μg/mL DNA solutions and retracted at 1-10 mm/s speeds to stretch and immobilize (“molecularly comb”) DNAs on the surface. 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 PAA, which switches water-solubility by immersion in CaCl₂ or NaCl solutions, complete desorption into saline solution by PAA dissolution was observed.
Relative to previous soft lithography methods, we employed a microfluidic method to improve DNA fragmentation. Holes in 1mm thick PDMS membranes were produced by piercing the PDMS with microneedles (d =250-400 μm) or by curing PDMS around the microneedles. PDMS layers with holes were pressed onto adsorbed DNAs and a DNAse I cutting enzyme solution was deposited on top. By applying a vacuum (125 Torr) above the solution, the low-volume chambers fill with solution and efficiently cut the DNA, as confirmed by fluorescence microscopy.