Deposition of DNA onto Polymethacrylate (PMMA) Thin Film Gratings in Preparation for the Tagmentation of DNA with Tn5 Transposase

In recent years, next-generation sequencing (NGS) has significantly advanced high-throughput sequencing. However, the NGS library preparation process can result in the loss of information regarding spatial organization, making reassembly difficult [1]. This study examines the application of depositing DNA onto flat polydimethylsiloxane (PDMS) samples that are then contact-printed onto PMMA-coated thin film gratings to suspend the DNA (bridging) and provide steric clearance for the Tn 5 transposase to simultaneously cut and label the DNA strands, a process termed tagmentation.<br/><br/>Samples of PDMS were dipped into solutions of λ DNA (NEB) in either DNase I reaction buffer (NEB) or Tris-EDTA (TE) buffer with sodium chloride to promote DNA adsorption to PDMS. Glycerol was also added to some solutions at a 1% (v/v) concentration to encourage the DNA to transfer from the PDMS to the Si grating. The samples were then imaged with a LEICA fluorescence microscope at 10x and 63x magnification. Samples with linearly stretched DNA were stamped onto a PMMA-coated Si wafer for various lengths of time up to 180 seconds using various weights up to 70 g. Results indicated that PDMS cured in a 60°C oven for 48 hours increased the adhesion of DNA onto the PDMS. However, this made it too difficult for the DNA to be stamped and transferred onto the Si wafer. PDMS that was cured for less time (4 hours) resulted in improved separation of adsorbed strands. Additionally, the NaCl made it difficult to transfer the DNA, causing the DNA to tear and not bridge. To address this problem, glycerol was added instead of salt to aid transfer. Furthermore, it was found that finer gratings were preferable because the DNA did not need to stretch as far to bridge. A solution with a concentration of 2.5 ng λ DNA per µL of DNase I reaction buffer with 1% (v/v) added glycerol deposited on PDMS and stamped across an 8.6 µm linear grating for 15 seconds and no weight yielded the best results thus far. Further development of this method will allow NGS to be analyzed with minimal risk of losing important genome information.<br/><br/>We acknowledge the Morin Charitable Trust for funding.<br/><br/>[1] Cho, NaHyun & Goodwin, Sara & Budassi, Julia & Zhu, Ke & Mccombie, W. & Sokolov, Jonathan. (2017). Fragmentation of Surface Adsorbed and Aligned DNA Molecules using Soft Lithography for Next-Generation Sequencing. Journal of Biosensors & Bioelectronics. 08. 10.4172/2155-6210.1000247.