Nucleotide analogs labeled with detectable moieties, such as fluorescent dyes, are widely used in research and diagnostic assays. Chain terminating nucleotide analogs (dideoxynucleotides) are especially useful in DNA sequencing applications and primer extension assays, such as minisequencing or genetic bit analysis (GBA). They propose to develop a unique set of pro-fluorescent dideoxynucleotide analogs that will enable more rapid and sensitive mutation detection as well a provide unique substrates for polymerase activity assays, such as for cell viability. The proposed analogs are non-fluorescent as free nucleotide, however, upon incorporation into DNA by a DNA polymerase, a fluorophore is released into solution. The amount of fluorescence released into solution is directly correlated with polymerase activity, thus providing a real-time assay for incorporation of a specific base analog. They propose to synthesize and test the concept of pro-fluorescent dideoxynucleotide analogs during Phase I of this grant and if successful, develop all four base analogs and assay conditions during Phase II. Successful completion of this project will result in a series of pro-fluorescent nucleotide analogs with broad ranging utility in genetic and biochemical research and diagnostics.
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