We have been developing a system for real-time single molecule DNA sequencing. System components include novel """"""""charge-switched"""""""" nucleotides, an adapted DNA polymerase, a method for isolating and handling single DNA molecules, a microfluidics controller and flowcell for sorting molecules by charge, and a microscope for imaging individual dye molecules. This proposal will develop a 4-color single-molecule fluorescence microscope based on an existing 1-color platform. In Phase I, a panel of dyes will be screened for chemical, physical and fluorescence properties; optical requirements for a 4-color microscope will be developed around an optimal dye set. In Phase II, hardware options will be evaluated and a four-color instrument will be constructed. An key advantage of single-molecule sequencing is the elimination of cloning and the laboratory infrastructure associated with high-throughput operations. This, along with reduced reagent consumption, results in sequencing costs orders of magnitude lower compared to existing methods. Read lengths will be tens of kilobases to simplify shotgun sequence assembly and preserve haplotype information. Applications include whole-genome sequencing, SNPs, haplotyping, genotype-trait associations, long-read SAGE for expression profiling, analysis of alternative mRNA splicing patterns, and comparative genomics within or between species. Fields-of-use include research, diagnostics and personalized medicine. Public health applications include rapid sequencing (without cloning) of """"""""designer germs"""""""" carrying engineered genomes that could cause novel symptoms.
