| abstract |
An assay for polynucleotides employing total internal reflection of excitation radiation at a coating bonded to the surface of an optically conductive glass cell. The coating initially includes single-stranded polynucleotides coupled to individual attachment sites on the surface of the cell, such polynucleotides being complementary, at least in part to the single-stranded form of the polynucleotide that is being assayed. Each molecule of the coupled polynucleotide is connected to the cell surface through a spacer connected to an irreversibly conjugated polyadenine/polythymidine sequence at one end of the coupled polynucleotide. n When the surface of the coated cell is contacted with a sample that contains single-stranded polynucleotide complementary to the bound polynucleotide, renaturation will occur, forming a double-stranded form of the polynucleotide of interest. A fluorochrome dye specific to that double-stranded form is coupled to the latter, the dye including a chromophore that the excitation radiation will excite into fluorescence. The induced fluorescence is then gathered and measured. |