Tn5-Mediated Ordered DNA Fragmentation on UV-Patterned SU-8 Substrates

Current DNA sequencing methods require extensive fragmentation and may form erroneous sequences. Ordered DNA fragmentation mitigates these concerns by preserving sequence. 

For immobilizing DNA, we examined the viability of patterned negative photoresist thin film of SU-8. KemLab’s SQ2 (thickness 2µm) was spuncast onto 5.5mmx20mm Si wafer slivers using the recommended program[1]. SQ2-coated silicon wafers were positioned over a striped soda-lime glass photomask and exposed to 365 nm LED (10-15mW) from a 3-in distance for 3 minutes. 

E. coli transposase Tn5 was used for DNA cleavage[2]. Patterned SQ2 wafers were dipped in mouse DNA solution and then developed in PGMEA for 1 minute. DNA solution of mouse DNA solution (1 mg/mL), 5 µL10x-diluted SYBR Gold dye[3], and 1390 µL DNase reaction buffer. Droplets of 3 μL of 5.26% loaded Diagenode Tn5 transposase solution (4 µL 2 mg/mL Tn5, 36 µL 2x tagmentation buffer, and 36 µL water) were deposited onto samples. Heating samples at 55°C for 7 minutes in a low-volume space, initiated DNA cleaving. Tn5 was finally digested for 20 min in 15 µL Proteinase K in 985 µL DNase buffer. Fluorescence microscopy gauged cleavage and digestion efficacy.

Overall, tests demonstrated successful DNA bridging, particularly over gaps <20 µm, with moderately efficient DNA fragmentation. 5.26% Tn5 concentration is effective for DNA cutting.Further directions of work include using PMMA, optimizing Tn5 scission, proteinase or SDS digestion of Tn5, and selective DNA removal.