Fabrication of Inverted High-Density DNA Microarrays in a Hydrogel

Abstract

Current techniques for making high-resolution, photolithographic DNA microarrays suffer from the limitation that the 3′ end of each sequence is anchored to a hard substrate and hence is unavailable for many potential enzymatic reactions. Here, we demonstrate a technique that inverts the entire microarray into a hydrogel. This method preserves the spatial fidelity of the original pattern while simultaneously removing incorrectly synthesized oligomers that are inherent to all other microarray fabrication strategies. First, a standard 5′-up microarray on a donor wafer is synthesized, in which each oligo is anchored with a cleavable linker at the 3′ end and an Acrydite phosphoramidite at the 5′ end. Following the synthesis of the array, an acrylamide monomer solution is applied to the donor wafer, and an acrylamide-silanized acceptor wafer is placed on top. As the polyacrylamide hydrogel forms between the two wafers, it covalently incorporates the acrydite-terminated sequences into the matrix. Finally, the oligos are released from the donor wafer upon immersing in an ammonia solution that cleaves the 3′-linkers, thus freeing the oligos at the 3′ end. The array is now presented 3′-up on the surface of the gel-coated acceptor wafer. Various types of on-gel enzymatic reactions demonstrate a versatile and robust platform that can easily be constructed with far more molecular complexity than traditional photolithographic arrays by endowing the system with multiple enzymatic substrates. We produce a new generation of microarrays where highly ordered, purified oligos are inverted 3′-up, in a biocompatible soft hydrogel, and functional with respect to a wide variety of programable enzymatic reactions.

Spatial Transcriptomics Sequencing of Mouse Liver at 2µm Resolution Using a Novel Spatial DNA Chip

“…Additionally, utilizing markers from specific subcellular compartments, such as pre-mRNA, and merging high-resolution microscopy might further refine cellular boundary determinations in tissues with intricate architectures 30 . manufactured using light-directed synthesis on silicon wafers then transferred to hydrogels as previously described 20 . Capture regions are confluent 2 x 2 µm squares.…”

“…High-resolution spatial DNA chips were manufactured as previously described 20 . Briefly, semiconductor photolithographic manufacturing techniques were used in conjunction with highly-efficient light-directed DNA synthesis to prepare high-density DNA arrays on silicon wafer substrates.…”

“…Briefly, semiconductor photolithographic manufacturing techniques were used in conjunction with highly-efficient light-directed DNA synthesis to prepare high-density DNA arrays on silicon wafer substrates. Positional information is encoded in the DNA sequences on the silicon chips photolithographically 20 . High-resolution chips are fabricated by sequential exposures of lithography masks, each one directing a single base addition.…”

“…Chips are singulated and subsequently inverted and transferred to a thin polyacrylamide gel (10-20 µ m) cast on a silanized glass slide. This process has been previously described 20 . Each of the 10mm x 10mm chips contains 5,000 x 5,000 features that are 2 µ m x 2 µ m each and juxtaposed adjacently without interstitial spaces.…”

Recent Advances in Biomaterial‐Based High‐Throughput Platforms

“…After cleavage of the linker, the hydrogel is freed from the donor‐wafer with a free 3’‐end (3’‐up) providing scope for many enzymatic reactions (Figure 1B). [ 21 ] This technique demonstrated a hydrogel‐based 3’‐up DNA microarray with high positional fidelity and high potential in applications employing enzymes, for example, sequencing. In comparison to conventional techniques, this technique opens up possibilities for reactions other than hybridization.…”