Nucleic acid assay method

• US 5,242,797 A
• Filed: 01/02/1992
• Issued: 09/07/1993
• Est. Priority Date: 03/21/1986
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Myron J. Block
• Inventors: Hirschfeld, Tomas B.
• Primary Examiner(s): Chambers, Scott A.
• Application Number:
  US07/817,942
• Time in Patent Office: 614 Days
• Field of Search: 435/6
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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.

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.

Claims

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.

2. The method as defined in claim 1 wherein said step of contacting said surface comprises the further step of adding a known quantity of said single stranded polynucleotide of interest with a fluorescent dye coupled thereto to said sample prior to contacting said surface with said sample, so that said single-stranded polynucleotide with said fluorescent dye will compete with single stranded polynucleotide of interest originally in said sample in said hybridization.

3. The method as defined in claim 1 wherein said bonding step includes binding an aromatic amine to said surface, and then diazo coupling said aromatic amine to said selected single-stranded polynucleotide.

4. The method as defined in claim 3 wherein said aromatic amine is coupled to said surface through a spacer molecule.

5. The method as defined in claim 4 wherein said spacer is a trimethoxysilane substituted with a chain of methylene groups.

6. The method as defined in claim 4 wherein said spacer is formed of a pair of epoxy groups connected by a methylene chain.

7. The method as defined in claim 4 wherein said spacer is an aliphatic amine.

8. The method as defined in claim 4 wherein said spacer is a trimethoxysilane having a chlorbenzyl group.

9. The method as defined in claim 3 wherein said diazo coupling is formed by first converting said amine to a diazo group, coupling said diazo group to a polythymidine, and conjugating said polythymidine with a polyadenine moiety present at the beginning of said selected single-stranded polynucleotide.

10. The method as defined in claim 9 wherein said amine is converted to a diazo group by treatment with a diazonium halide.

11. The method as defined in claim 9 including the step of cross-linking said polyadenine with said polythymidine.

12. The method as defined in claim 9 wherein the step of measuring comprises measurement of the intensity of said fluorescent radiation.

13. The method as defined in claim 9 wherein the step of measuring comprises measurement of the fluorescent decay lifetime of said fluorescent radiation.

14. The method as defined in claim 1 wherein said fluorochrome dye is selected form the group consisting of ethidium bromide, acridine orange, diethidium bromide, diacridine orange, and quinacrine.

15. The method as defined in claim 14 wherein said dye is ethidium bromide or its dimers.

16. The method as defined in claim 14 wherein said dye is acridine orange or its dimers.

17. 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.