The objective of the proposed research is to design and synthesize a large library of combinatorial fluorescent energy transfer (CFET) tags with unique fluorescence emission signatures that can be analyzed using a simple optical system. A fundamental limitation in fluorescent genetic analysis is the availability of fluorescent tags with distinctive fluorescence emissions. The PIs will develop a novel fluorescent-labeling scheme that uses a limited number of fluorescent molecules to create a large number of CFET tags with unique fluorescence signatures. The approach is based on the fluorescence energy transfer principle, the combinatorial concept, and that the energy transfer efficiency is dependent on the separation distance between the donor and acceptor. Synthetic procedures will be developed to construct a library of at least 20 of these CFET tags, and the spectroscopic properties of the CTET tags will be evaluated. The CFET tags will be generated using combinations of a number of different fluorescent molecules that will be linked through a rigid polymeric scaffold. The scaffold will be assembled by solid phase synthesis and solution coupling chemistry. The fluorescent signatures and chemical properties of the CFET tags will be carefully characterized to identify the most useful members of the collection. The tags will have particular utility in the massive high-throughput approaches of the genomics revolution, including DNA sequencing, identification of individual variations in the genome that are responsible for human disease, detection of microbial pathogens, and even drug screening, essentially any approach that can benefit by multiplexing operations. The utility of this library of CFET tags with unique fluorescence emission signatures will be validated by labeling a multiplex set of oligonucleotides for genetic mutation detection in a model system, as well as for genome-wide chromosome analyses.
