Optimized in situ DNA synthesis on patterned glass
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Optimized in situ DNA synthesis on patterned glass Ishtiaq Saaem, Kuo-Sheng Ma, and Jingdong Tian Department of Biomedical Engineering and Institute for Genome Sciences and Policy. Duke University, Durham, NC 27708 ABSTRACT This paper describes studies of patterned arrays on glass surfaces and their use as spatially separated reactors for in situ synthesis of DNA using an inkjet synthesizer. Photolithographic methods were employed to fabricate arrays composed of homogenous circular features containing a hydroxyl-terminated silane coupled to the surface of the glass via a siloxane bond. Features are embedded within a background matrix composed of a fluorosilane attached to the glass. Due to the differential wettability of the two silanes, whereby the hydroxyl-terminated silane and fluorosilane are hydrophilic and hydrophobic respectively because of their head groups, the patterned circular features are able to constrain liquid within a defined site. The silanization result was analyzed using X-ray photoelectron spectroscopy (XPS) to optimize silanization time and solvent. Synthesis was then performed using a custom-built inkjet system using phosphoramidite chemistry. Base-by-base analysis using fluorescent labeling showed consistent coupling efficiency on synthesis of a 50-mer homopolymer. INTRODUCTION The synthesis of a large library of molecules is a crucial task for modern chemistry and biology. Combinatorial synthesis and usage of oligonucleotides and pharmaceutical compounds are common place today [1, 2]. A spatially constrained array on a planar surface is the most exploited platform for most uses. Glass microscope slides are commonly used and their surfaces contain Si-OH groups for functionalization. However, the chemistry achievable on functionalized glass is not easily optimized as on silicon due to a lack of ordered structure. This lack of order gives rise to issues regarding microscale reactions such as reactivity of surface functional groups and whether the groups are in an accessible conformation at a sufficiently high surface density. The work presented here dwells on the effect of time and solvent on silanization of patterned microscope slides. Using a simple photolithographic approach, we present the fabrication of the patterned glass slides. We find that a tenable surface for in situ synthesis is determined by using an appropriate silanization time and solvent. We then prepare oligonucleotide/DNA arrays on these slides using in situ synthesis that uses standard nucleoside phosphoramidite chemistry and dimethoxytrityl-based protection. Determination of coupling efficiencies are then made using a fluorescence assay employing a labeled terminating nucleotide for synthesizing homopolymers of various sizes [3]. MATERIALS AND METHODS Positive photoresist Shipley 1813 and developer were purchased from MicroChem Corp. (Newton, MA, USA). Nucleoside phosphoramidites (Pac-dA-CE, Ac-dC-CE, iPr-Pac-dG-CE
and dT-CE), 5-ethylthio-1H-tetrazole, Cap Mix A (5% Phenoxyacetic anhydride in THF) and oxidiz
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