Immobilized nucleic acid hybridization reagent and method
First Claim
1. A method for detecting the presence of a nucleic acid in a test sample, comprising:
- (a) providing a solid phase having an oligonucleotide bound thereon wherein the oligonucleotide comprises a fluorophore, the fluorophore being covalently attached to one end of the oligonucleotide and the solid phase being linked to the opposite end of the oligonucleotide, the oligonucleotide further comprising at least one hairpin structure between the two ends;
(b) incubating the test sample with the solid phase under conditions suitable for complementary binding between the oligonucleotide and nucleic acid from the test sample; and
(c) detecting the presence of the nucleic acid in the test sample by detecting fluorescence from the fluorophore.
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Abstract
A fluorescently labeled nucleic acid having a hairpin structure between the fluorophore label and a point of attachment to a solid phase is useful as a probe to detect nucleic acid from a sample. The solid phase quenches the fluorophore label when the hairpin structure exists but this quenching is relieved by duplex formation between probe and a sample oligonucleotide. Probes for specific nucleic acid sequences can be immobilized as arrays on solid phase surfaces for detection of multiple nucleic acid sequences simultaneously from electrophoresis gels and from aqueous solutions. These probes and methods for their use can be combined with known solid phases, particularly those used for plasmon surface detection and electron transfer detection of nucleic acid. The probes can be washed and reused, and have other advantageous features over known probe methods.
106 Citations
28 Claims
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1. A method for detecting the presence of a nucleic acid in a test sample, comprising:
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(a) providing a solid phase having an oligonucleotide bound thereon wherein the oligonucleotide comprises a fluorophore, the fluorophore being covalently attached to one end of the oligonucleotide and the solid phase being linked to the opposite end of the oligonucleotide, the oligonucleotide further comprising at least one hairpin structure between the two ends;
(b) incubating the test sample with the solid phase under conditions suitable for complementary binding between the oligonucleotide and nucleic acid from the test sample; and
(c) detecting the presence of the nucleic acid in the test sample by detecting fluorescence from the fluorophore. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for detecting the presence of two or more nucleic acids in a test sample, comprising:
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(a) providing a solid phase having two or more oligonucleotides bound thereon at separate regions wherein each oligonucleotide comprises a fluorophore, the fluorophore being covalently attached to one end of the oligonucleotide and the solid phase being linked to the opposite end of the oligonucleotide, each oligonucleotide further comprising at least one hairpin structure between the two ends;
(b) incubating the test sample with the solid phase under conditions suitable for complementary binding between the oligonucleotides and nucleic acid from the test sample; and
(c) detecting the presence of the nucleic acids in the test sample by detecting fluorescence from each of the separate regions of the solid phase. - View Dependent Claims (14, 15, 16)
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17. An analytical test device for detecting the presence of a nucleic acid in a test sample comprising:
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a solid phase surface comprising a material that quenches fluorescence; and
a self-complementary single stranded oligonucleotide probe linked to the solid phase surface at one of its ends, the probe comprising a fluorophore attached to its other end and a hairpin structure between the two ends. - View Dependent Claims (18)
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19. An analytical test device for detecting the presence of two or more nucleic acids in a test sample comprising:
a solid phase surface comprising a material that quenches fluorescence and having two or more oligonucleotides bound thereon at separate regions wherein each oligonucleotide comprises a fluorophore, the fluorophore being covalently attached to one end of the oligonucleotide and the solid phase being linked to the opposite end of the oligonucleotide, and wherein each oligonucleotide further comprises a hairpin structure between the two ends. - View Dependent Claims (20, 21, 22, 23, 24, 25)
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26. A kit for detecting the presence of a nucleic acid in a test sample, comprising:
a solid phase having an oligonucleotide bound thereon wherein the oligonucleotide comprises a fluorophore, the fluorophore being covalently attached to one end of the oligonucleotide and the solid phase being linked to the opposite end of the oligonucleotide, the oligonucleotide further comprising a hairpin structure between the two ends packaged in a container; and
instructions.
- 27. A fluorescence quenching surface having at least one type of oligonucleotide bound thereon wherein the oligonucleotide comprises a fluorophore, the fluorophore being covalently attached to one end of the oligonucleotide and the solid phase being linked to the opposite end of the oligonucleotide, the oligonucleotide further comprising at least one hairpin structure between the two ends.
Specification