US5354657A
Expired / Lapsed
US
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Process for the highly specific detection of nucleic acids in solid
Assignee: Roche Diagnostics GmbH
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Filing: 1992-12-18
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Granted: 1994-10-11
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Expires: 2012-12-18
Abstract
Abstract The present invention provides a process for the detection of nucleic acids of definite sequence by hybridisation with two single-stranded nucleic acid probes present in the same solution phase complementary to different regions of the nucleic acid to be detected, one nucleic acid probe serving as detector probe and containing as labelling at least one hapten bound via a chemical linkage and the other nucleic acid probe serving as capturing probe and being bound to a solid matrix, wherein, as hapten, a steroid is used which on at least one position of the detector probe, which does not participate in the hydrogen bridge formation with the nucleic acid to be detected, is covalently bound via a bridge of at least four atoms, the nucleic acid to be detected, present in solution, is incubated with the detector probe and the capturing probe in any desired sequence, whereby the binding of the capturing probe on the matrix with the nucleic acid to be detected is brought about before, during or after the incubation, the remaining solution is separated off from the matrix-bound complex of the capturing probe, nucleic acid to be detected and detector probe and the complex detected via an anti-hapten antibody which is labelled.
Inventors
- 👤 Hans J. Holtke
- 👤 Rudolf Seibl
- 👤 Christoph Kessler
- 👤 Ralf Mattes
- 👤 Hermann Graf
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 09:36
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6804
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
295 words
A process for the detection of a target nucleic acid in a solution by hybridization with a labelled, single stranded nucleic acid probe present in the same solution, comprising: (a) incubating the solution with a labelled single stranded nucleic acid probe, wherein said probe has at least one digoxigenin or digoxin steroid hapten molecule covalently bound thereto, via a bridge of at least four atoms in length, wherein said bridge is bound to a site on said probe which does not participate in hydrogen bridge formation with any target nucleic acid in said solution, wherein said site is selected from the group consisting of: position C5 of uracil, position C8 of adenine, position C8 of guanine, and position 2' of ribose; incubating said solution with a nucleic acid capturing probe, wherein said capturing probe is bound to a solid matrix before, during, or after incubation with said solution, said nucleic acid capturing probe being complementary to said target nucleic acid at a region different from the region of said target nucleic acid to which said probe is complementary, wherein said capturing probe is bound to said solid matrix (1) adsorptively, (2) coordinately, (3) covalently, (4) via a binding system which comprises biotin and avidin or (5) via a binding system which comprises biotin and streptavidin so as to form a solid phase bound complex of (i) matrix bound capture probe, (ii) target nucleic acid present in said solution, and (iii) said labelled nucleic acid probe; (b) separating solid phase bound complex from said solution, (c) contacting solid phase bound complex with a labelled antibody which specifically binds to said digoxigenin or digoxin steroid hapten molecule, and (d) determining target nucleic acid by determining any binding of said labelled antibody to said solid phase bound complex.
Claim 2
Dependent Claim
295 words
A process for the detection of a target nucleic acid in a solution by hybridization with a labelled, single stranded nucleic acid probe present in the same solution, comprising: (a) incubating the solution with a labelled single stranded nucleic acid probe, wherein said probe has at least one digoxigenin or digoxin steroid hapten molecule covalently bound thereto, via a bridge of at least four atoms in length, wherein said bridge is bound to a site on said probe which does not participate in hydrogen bridge formation with any target nucleic acid in said solution, wherein said site is selected from the group consisting of: position C5 of uracil, position C8 of adenine, position C8 of guanine, and position 2' of ribose; incubating said solution with a nucleic acid capturing probe, wherein said capturing probe is bound to a solid matrix before, during, or after incubation with said solution, said nucleic acid capturing probe being complementary to said target nucleic acid at a region different from the region of said target nucleic acid to which said probe is complementary, wherein said capturing probe is bound to said solid matrix (1) adsorptively, (2) coordinately, (3) covalently, (4) via a binding system which comprises biotin and avidin or (5) via a binding system which comprises biotin and streptavidin so as to form a solid phase bound complex of (i) matrix bound capture probe, (ii) target nucleic acid present in said solution, and (iii) said labelled nucleic acid probe; (b) separating solid phase bound complex from said solution, (c) contacting solid phase bound complex with a labelled antibody which specifically binds to said digoxigenin or digoxin steroid hapten molecule, and (d) determining target nucleic acid by determining any binding of said labelled antibody to said solid phase bound complex.
Claim 3
Dependent Claim
15 words
The process of claim 1, wherein said bridge consists of from 11 to 16 atoms.
Claim 4
Dependent Claim
14 words
The process of claim 1, wherein said bridge comprises at least one hydrophilic group.
Claim 5
Dependent Claim
10 words
The process of claim 1, wherein said bridge is linear.
Claim 6
Dependent Claim
12 words
The process of claim 1, wherein said bridge comprises a branched chain.
Claim 7
Dependent Claim
21 words
The process of claim 1, wherein said bridge is bound to C5 of uracil, C8 of adenine or C8 of guanine.
Claim 8
Dependent Claim
15 words
The process of claim 1, wherein said bridge is bound to 2' position of ribose.
Claim 9
Dependent Claim
28 words
The process of claim 1, wherein said digoxigenin or digoxin steroid hapten is bound to said bridge via an ether bond, an amide bond, or an ester bond.
Claim 10
Dependent Claim
20 words
The process of claim 1, wherein said capture probe is bound to said solid matrix via a biotin-avidin binding system.
Claim 11
Dependent Claim
20 words
The process of claim 1, wherein said capture probe is bound to said solid matrix via a biotin-streptavidin binding system.
Claim 12
Dependent Claim
25 words
The process of claim 9, wherein one of said biotin and avidin is directly bound to the 5'-end or the 3'end of said capturing probe.
Claim 13
Dependent Claim
24 words
The process of claim 10, wherein one of said biotin and streptavidin is directly bound to 5'-and or the 3'end of said capturing probe.
Claim 14
Dependent Claim
53 words
The process of claim 1 wherein said solid matrix is selected from the group consisting of a nitrocellulose filter, a nylon filter, a plastic, a plexiglass, a plexiglass coated with nitrocellulose, a plexiglass coated with nylon, a plastic coated with nitrocellulose, a plastic coated with nylon, and a wall of a reaction vessel.
Claim 15
Dependent Claim
43 words
The process of claim 1, wherein the digoxin or digoxigenin has been incorporated into the nucleic acid probe enzymatically with an RNA polymerase, a DNA polymerase, an exonuclease or a reverse transcriptases and a digoxin or digoxigenin modified deoxy- or ribonucleoside triphosphase substrate.
Claim 16
Dependent Claim
21 words
The process of claim 1, wherein the digoxin or digoxigenin has been incorporated into the nucleic acid probe photochemically via photohapten.
Claim 17
Dependent Claim
68 words
The process of claim 1, wherein digoxin or digoxigenin has been incorporated into the nucleic acid probe chemically via oligo-desoxyribonucleotide synthesis, by the incorporation of protected nucleoside phosphoamidites modified with substitutable amino functions and, after removal of protective groups thereon, by reaction of the modified oligodesoxyribonucleotide with a member selected from the group consisting of activated ester, an activated amide or an activated ether of digoxin or digoxigenin.
Claim 18
Dependent Claim
40 words
The process of claim 1 further comprising detecting the complex via a labelled antibody directed against digoxin or digoxigenin wherein the antibody is labelled via an enzyme label, a radioactive label, a fluorescent label, a luminiscent or a bioluminescent label.
Claim 19
Dependent Claim
15 words
The process of claim 17 wherein said enzyme is alkaline phosphatase, a peroxidase or beta-galactosidase.
Claim 20
Dependent Claim
15 words
The process of claim 18 further comprising determining activity of said enzyme via redox system.
Claim 21
Dependent Claim
19 words
The process of claim 19 further comprising determining the activity of the said enzyme via a color forming system.
Claim 22
Dependent Claim
25 words
The process of claim 19 wherein said enzyme is alkaline phosphatase, further comprising determining the alkaline phosphate via a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium redox system.
Claim 23
Dependent Claim
28 words
The process of claim 1, wherein at least one of said capture probe and said labelled nucleic acid probe comprises a poly d(C) or a poly d(G) sequence.
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