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Synthetic modification of silica beads that allows for sequential attachment of two different oligonucleotides.

We developed a simple and elegant synthesis strategy that enables us to attach controlled (equimolar) amounts of two different oligonucleotides onto one silica bead. The method involves addition of orthogonally protected lysine followed by activation and derivatization of each amino group with a different moiety. This sequential oligonucleotide attachment enables the use of a combinatorial scheme to generate millions of bead types, each characterized by its two oligo tags. (In our randomly assembled arrays each bead type can then be identified by a series of hybridizations of fluorescently labeled decoder oligos to the address tags.) To demonstrate feasibility of such a scheme we created over 1000 bead types, which were characterized by their two oligo tags. The method enables genotyping or gene expression assays at multiplex levels of hundreds of thousands to millions.