High Surface Area Substrates for DNA Arrays
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The
approach integrates two key technologies: light-directed combinatorial chemistry, which enables
the synthesis of hundreds of thousands of discrete compounds at a high resolution in precise locations on a substrate, and laser confocal fluorescence scanning, which permits measurement of molecular interactions on the array. A schematic which shows how a target molecule is recognized by an array is shown in Figure 1.
Figure 1: Schematic of fluorescence analysis on a DNA array. A fluorescently labeled target binds to complementary sequences on the chip. Darker regions represent features where the probes are complementary to the target. 371
Mat. Res. Soc. Symp. Proc. Vol. 576 © 1999 Materials Research Society
Fluorescence microscopy is used to detect the binding of the target oligonucleotide to complementary probes on the array. The ability to detect the hybridization of target to complementary probes on the array is dependent on the quantity and density of immobilized probe molecules on the surface, as well as the thermodynamics of hybridization. The ability to detect target molecules may be limited under certain conditions, such as when target concentrations are very low, and hybridization kinetics becomes limiting, or in the case of weakbinding target sequences, such as those containing a high percentage of A, T bases, which form less stable duplexes than sequences that are rich in G, C bases. Porous surface layers are a potential route to increasing the signal from DNA arrays, as they increase the total surface area on which probes can be attached, and hence the capacity for bound target molecules. Thin films of organic polymer gels have previously been used as a support for immobilizing arrays of pre-synthesized oligonucleotide probes [2]. Flat glass substrates have typically been used for fabricating high-density probe arrays by light-directed synthesis, in which single bases are added in a combinatorial fashion to construct arrays in situ. In this work we use porous inorganic thin films as supports for photolithographic patterning techniques pioneered by Affymetrix to synthesize the DNA probes. Photolithographic patterning allows the construction of probe arrays with extremely high density and information content [1 ]. Inorganic surfaces have the advantage that they are similar to the original glass substrate, so that array fabrication protocols can be used. Additionally, dip and spin coating techniques for depositing thin inorganic layers have already been established [3]. One route for fabricating a porous layer is the "subtractive" method in which a porous layer is etched into the surface of a phase-separating glass. The miscibility gap in the Si0 2B2 0 3-Na2O system is well documented. Annealing causes the glass to separate into acid soluble (B20 3,Na 2O rich) and acid insoluble (Si0 2 rich) phases. The soluble phase can be leached with an acid such as hydrochloric or hydrofluoric. This process has been used for many years as a route to high purity silica glass in the Vycor process [4,5]. A second rou
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