Nucleic acid assay method

Claim 1

A method of assaying a liquid sample for a polynucleotide of interest in single-stranded form, said method consisting of the steps of: bonding a first single-stranded polynucleotide to a surface of an optically-transmissive, attenuated total internal reflection cell, said first single-stranded polynucleotide being complementary, at least in part, to said polynucleotide of interest; adding to said cell a fluorochrome dye that yields a fluorescence signal which is strongly affected by the degree of hybridization of the polynucleotide, said dye binding preferentially to a double stranded polynucleotide as compared with a single stranded polynucleotide, said binding causing a greater efficiency of fluorescence as compared with the unbound state; contacting said surface having said first polynucleotide bound thereto with said sample containing a single stranded polynucleotide of interest under conditions sufficient for any single-stranded polynucleotide which is complementary to said bound first polynucleotide to hybridize with said bound first polynucleotide; coupling said fluorochrome dye to said hybridized polynucleotide on said cell surface; internally illuminating said cell with excitation radiation so as to generate, adjacent to said surface, an evanescent wave which interacts with said fluorochrome dye to induce fluorescence from said fluorochrome dye; collecting fluorescent radiation arising from excitation of said fluorochrome dye and exiting from an end of said cell; and measuring the collected fluorescent radiation wherein said fluorescence is indicative of hybridization of the single stranded complementary polynucleotide and the single stranded polynucleotide of interest, thereby indicating the presence of the single stranded polynucleotide of interest.

Claim 2

A method of assaying a liquid sample for a polynucleotide of interest in single-stranded form, said method consisting of the steps of: bonding a first single-stranded polynucleotide to a surface of an optically-transmissive, attenuated total internal reflection cell, said first single-stranded polynucleotide being complementary, at least in part, to said polynucleotide of interest; adding to said cell a fluorochrome dye that yields a fluorescence signal which is strongly affected by the degree of hybridization of the polynucleotide, said dye binding preferentially to a double stranded polynucleotide as compared with a single stranded polynucleotide, said binding causing a greater efficiency of fluorescence as compared with the unbound state; contacting said surface having said first polynucleotide bound thereto with said sample containing a single stranded polynucleotide of interest under conditions sufficient for any single-stranded polynucleotide which is complementary to said bound first polynucleotide to hybridize with said bound first polynucleotide; coupling said fluorochrome dye to said hybridized polynucleotide on said cell surface; internally illuminating said cell with excitation radiation so as to generate, adjacent to said surface, an evanescent wave which interacts with said fluorochrome dye to induce fluorescence from said fluorochrome dye; collecting fluorescent radiation arising from excitation of said fluorochrome dye and exiting from an end of said cell; and measuring the collected fluorescent radiation wherein said fluorescence is indicative of hybridization of the single stranded complementary polynucleotide and the single stranded polynucleotide of interest, thereby indicating the presence of the single stranded polynucleotide of interest.

Claim 18

A method of assaying a liquid sample for a polynucleotide of interest in single-stranded form, said method consisting of the steps of: placing said sample in an optically-transmissive, attenuated totally internally reflecting cell having immobilized on a surface thereof a coating including a first single-stranded polynucleotide complementary, at least in part, to said polynucleotide of interest; adding a fluorochrome dye that binds preferentially to the double-stranded form of said polynucleotide of interest as compared to said single-stranded form, said binding causing a greater efficiency of fluorescence as compared with the unbound state forming a complex of said double-stranded polynucleotide and said fluorochrome dye; propagating an evanescent wave within said cell radiation which excites fluorescent radiation upon interaction with said fluorochrome dye, and detecting said fluorescence from said fluorochrome dye wherein said fluorescence is indicative of hybridization of the single stranded complementary polynucleotide and the single stranded polynucleotide of interest, thereby indicating the presence of the single stranded polynucleotide of interest.

Claim 19

A method of assaying a liquid sample for a polynucleotide of interest in single-stranded form, said method consisting of the steps of: placing said sample in an optically-transmissive, attenuated totally internally reflecting cell having immobilized on a surface thereof a coating including a first single-stranded polynucleotide complementary, at least in part, to said polynucleotide of interest; adding a fluorochrome dye that binds preferentially to the double-stranded form of said polynucleotide of interest as compared to said single-stranded form, said binding causing a greater efficiency of fluorescence as compared with the unbound state forming a complex of said double-stranded polynucleotide and said fluorochrome dye; propagating an evanescent wave within said cell radiation which excites fluorescent radiation upon interaction with said fluorochrome dye, and detecting said fluorescence from said fluorochrome dye wherein said fluorescence is indicative of hybridization of the single stranded complementary polynucleotide and the single stranded polynucleotide of interest, thereby indicating the presence of the single stranded polynucleotide of interest.