AUTOMATED DNA SEQUENCING INSTRUMENTATION

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We request funding for instrumentation to be used in automated DNA sequencing. We have determined that a centralized sequencing facility will save considerable time and consumable costs for the 19 major users and others in the chemistry and biology divisions at Caltech, while at the same time contributing data crucial to the success of the projects. Large amounts of sequencing are carried out in the day to day projects, and it all must be done by manual methods based on radioactivity by individual students, post-doctoral fellows and technicians, many of whom have no previous experience in DNA sequencing. This leads to lack of efficiency and loss of competitiveness. We are requesting three items of related equipment: an automated DNA sequencer that runs and reads electrophoresis gels, a robotic station to carry out the polymerization reactions on which DNA sequencing technology is based, and computer hardware and software to update an existing Sequencing Analysis Facility to make it able to cope with the increased load that will arise with the advent of the automated sequencing facility here. Automated sequencing based on fluorescent labels has at least four advantages over manual methods based on radioactivity: saving in consumable costs, labor saving, safety, and, most important, quality of results. Perhaps the most useful aspect of a central sequencing facility based on automated methods is the extent to which the automated sequence can be integrated with computer systems. The facility will, thus, enhance the productivity and quality of the studies of the major users. There are 19 major users documented here and additional users are mentioned. The laboratories houses over 300 investigators, students, post-doctoral fellows and technicians. The major applications of the instruments will be projects that involve extensive cloning and sequencing of novel genes and/or construction of genetically engineered constructs that require sequence analysis. The projects cover, among others, sequencing of genes involved in RNA splicing, in molecular control of neural crest development, in the survival and differentiated function of neurons in Drosophila, in membrane excitability, in controlling body segment development in Drosophila, in cell cycle regulation, in DNA replication in yeast, in cell fate and morphogenesis in several organisms. One project involves sequencing large numbers of randomly-chosen cDNA clones involved in floral development in Arabidipsis.