Many developments related to DNA diagnostics or therapeutics will require the detection of specific DNA or RNA sequences with extremely high sensitivity. The development of reporter groups and the ability to place them in multiple or specific positions in a nucleic acid sequence will not only facilitate detection by procedures which are rapid and inexpensive but will also allow the study of nucleic acids or nucleic acid protein complexes which are responsible for the nucleic acid related diseases or for their cure. This proposal will concentrate in two areas: (1) The incorporation of multiple fluorescent labels into nucleic acid sequences containing phosphorothioate diesters in a procedure we designate """"""""post-assay"""""""" labeling, that is, the label is only attached after DNA sequencing or hybridization procedures. """"""""Post-assay"""""""" labeling by the procedures described allows the detection of nucleic acid sequences in the low femtomolar (10 15 mole) range without the use of sophisticated electronic detection. With this level of sensitivity, fluorescent labeling offers an alternative to the use of radioisotopes, thereby eliminating the associated health and environmental hazards. (2) Using phosphorothioate diesters, reporter groups can be placed at specific locations for the study of the structure and dynamics of unusual nucleic acids as well as protein - nucleic acid complexes.
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