The phase I work proposed is the development of devices and methods for sequencing of DNA by hybridization. Sequencing by hybridization (SBH) is a method in which a target DNA is sequenced by separate hybridization to a large number of short oligos [1]. In the work proposed, we will develop a novel simultaneous multiple oligonucleotide synthesizer (SMOS) capable of synthesizing tens of thousands of oligos, such as all possible 8-mer oligos (65,536) needed to perform the SBH on unknown target DNAs. The proposed manually operated multiple oligonucleotide synthesizer will be capable of synthesizing all 8-mer oligos in as little as 3 days on a 0.05 micromole scale. We will develop methods to simultaneous blot arrays of the 8-mer oligos onto hybridization membranes measuring less than six square inches. These membranes will be used to develop SBH methods that can be performed in an ordinary laboratory without expensive equipment. The SBH method will reduce the cost of sequencing DNA substantially and lend itself to simplified automation. Actual sequencing work will be carried out in phase II during which we will optimize sequencing protocols and devise data interpretation algorithms. We will also develop a simple software program for a desk top computer that recognizes data from digitally scanned autoradiographs, translates this data into oligo sequences, and reconstructs target sequences from hybridization data.
