DNA POLYMERASES FOR SINGLE MOLECULE DNA SEQUENCING
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AbstractAPPLICANT'S DESCRIPTION: DNA polymerases with altered substrate specificity are needed to support a new approach to single molecule DNA sequencing. Single molecule methods can be applied to DNA isolated directly from a subject organism, eliminating the need to clone, map and sort DNA fragments prior to sequencing. The need for electrophoretic separation is eliminated and reagent consumption is minimal. Single molecule detection has been developed and practiced by physicists and chemists for over 10 years. The crucial optical and detection systems needed for single molecule sequencing now exist. To implement this technology, novel nucleotides labeled at two different atomic positions have been synthesized and tested as substrates for DNA polymerases. Whereas some enzymes utilize nucletides labeled at the first position and other enzymes utilize nucleotides labeled at the second position, we could not identify any natural polymerase able to efficiently incorporate nucleotides labeled at both positions (double-labeled nucleotides). In Phase I, we will develop methods for the directed evolution of DNA polymerases adapted to the utilization of double-labeled nucleotides. Directed evolution will be used in Phase II to select for highly processive DNA polymerases having the desired substrate specificities. PROPOSED COMMERCIAL APPLICATION: A genomic DNA sequencing technology that reduces cost and eliminates the need for cloning, physical mapping and electrophoresis would have great appeal to both the academic and industrial genomic research communities.
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