This proposal is to prepare two types of fluorescent label """"""""cassettes"""""""" (two tags on a rigid support which can be linked to nucleotides) that can be conveniently used for DNA sequencing. They are designed to be extremely sensitive as a result of photonic or through-space energy transfer. Both cassette designs will be easily attached to nucleotides (more so than the energy transfer labels currently used) and compatible with both dye-primer and dye-terminator strategies. The latter aspect is important since there are no energy transfer enhanced fluorescent tags currently available for the dye-terminator approach, despite the well known advantages of labeling dideoxynucleotides rather than primers. The project will be executed in the following way. Combinatorial syntheses are proposed to generate libraries of the new cassettes. This may be the first attempt to identify new fluorescent tags from libraries, which is surprising since combinatorial approaches are ideal for self-indicating targets. Thus, model studies are proposed to develop solid phase preparations of the products, and plans are delineated to apply them to library syntheses. A strategy is then outlined for selecting the most suitable cassettes from the libraries, and carrying them through to the more experimentally demanding tests. These will include photophysical measurements, experiments to determine relative gel mobilities, kinetic studies of incorporation of cassette-ddNTPs, and, finally, applications in automated high throughput sequencing.
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