A Novel Method for UV Thin-Film Backside Development for Ordered DNA Fragmentation

Conventional DNA sequencing is limited by loss of sequence order during fragmentation, which makes ordered DNA fragmentation methodology attractive. We investigate the use of E. coli transposase Tn5 and DNA bridging [1] (for Tn5 steric clearance [2]) in ordered fragmentation of mouse DNA strands. The adhesion surface for DNA molecular combing was poly-methyl methacrylate(PMMA) spuncast on silicon wafers, then UV-patterned at 254 nm. Fluorescent microscopy reveals that 7:3 isopropyl alcohol : water developer disturbs DNA deposition, since DNA precipitation likely weakens surface interaction. Through our novel "backside" development, we reduced prolonged contact between developer and DNA. In this process, silicon wafers were spuncast with an initial 4% polyvinyl alcohol solution, then a second PMMA layer. This PMMA layer is floated off and deposited on another wafer with a hole, which allows development of exposed PMMA from "below". We found an optimal film thickness of 3400Å. 2,200—2,700Å tore during development, while < 2200Å often tore during UV patterning. Future work includes investigating different well patterns or photoresist materials, like SU-8, and optimizing experimental parameters.