US4959309A
Expired / Lapsed
US
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Fast photochemical method of labelling nucleic acids for detection purposes in hybridization assays
Assignee: MILES INC. A CORP. OF INDIANA
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Filing: 1987-10-09
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Granted: 1990-09-25
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Expires: 2007-10-09
Abstract
Abstract A labeled nucleic acid probe comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to the nucleic acid-binding ligand. The label can be a specifically bindable ligand such as a hapten or biotin, an enzyme such as a beta -galactosidase or horse radish peroxidase, a fluorescent radical, a phycobiliprotein, a luminescent radical, or a radioisotope. The probe can be used in assays of nucleic acids, taking advantage of the ability of the nucleic acid component to hydridize.
Inventors
- 👤 Nanibhushan Dattagupta
- 👤 Donald M. Crothers
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 00:27
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/533; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 47
Claim 1
Independent Claim
72 words
A labeled hydribizable nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), said label selected from the group consisting of fluorescein and phycobiliprotein.
Claim 2
Dependent Claim
72 words
A labeled hydribizable nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), said label selected from the group consisting of fluorescein and phycobiliprotein.
Claim 3
Dependent Claim
32 words
A hydridizable labeled nucleic acid according to claim 1, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.
Claim 4
Dependent Claim
19 words
A hybridizable labeled nucleic acid according to claim 2, wherein the intercalator compound is a furocoumarin or a phenanthridine.
Claim 5
Dependent Claim
14 words
A hybridizable labeled nucleic acid according to claim 1, wherein the label is fluorescein.
Claim 6
Dependent Claim
15 words
A hybridizable labeled nucleic acid according to claim 1, wherein the label is a phycobiliprotein.
Claim 7
Dependent Claim
20 words
A hybridizable labeled nucleic acid according to claim 1, wherein (b) is a radical of an amino-substituted angelicin or psoralen.
Claim 8
Dependent Claim
19 words
A hybridizable labeled nucleic acid according to claim 1, wherein the nucleic acid component is in single stranded form.
Claim 9
Dependent Claim
88 words
An adduct suitable for photochemical attachment to a hybridizable nucleic acid probe, comprising a nucleic acid-binding ligand and a label chemically linked thereto, whereby linkage of the adduct to the nucleic acid modifies the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, wherein the adduct is capable of being photochemically linked to the nucleic acid and wherein the label is selected from the group consisting of fluorescein and phycoiliprotein.
Claim 10
Dependent Claim
88 words
An adduct suitable for photochemical attachment to a hybridizable nucleic acid probe, comprising a nucleic acid-binding ligand and a label chemically linked thereto, whereby linkage of the adduct to the nucleic acid modifies the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, wherein the adduct is capable of being photochemically linked to the nucleic acid and wherein the label is selected from the group consisting of fluorescein and phycoiliprotein.
Claim 11
Dependent Claim
29 words
An adduct according to claim 8, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.
Claim 12
Dependent Claim
16 words
An adduct according to claim 9, wherein the intercalator compound is a furocoumarin or a phenanthridine.
Claim 13
Dependent Claim
11 words
An adduct according to claim 8, wherein the label is fluorescein.
Claim 14
Dependent Claim
12 words
An adduct according to claim 8, wherein the label is a phycobiliprotein.
Claim 15
Dependent Claim
20 words
An adduct according to claim 8, wherein the nucleic acid-binding ligand is an angelicin or psoralen carrying an amino substituent.
Claim 16
Independent Claim
112 words
A method for determining a particular polynucleotide sequence in a test sample, comprising the step of: (a) combining the test sample with a polynucleotide probe having a base sequence substantially complementary to the sequence to be determined, wherein a mono-adduct forming nucleic acid-binding ligand is photochemically linked to a sequence selected from the group consisting of the sample sequence and the probe sequence, the nucleic acid-binding ligand being chemically linked to a detectable label moiety, wherein the label is selected from the group consisting of fluorescein and phycobiliprotein, and (b) detecting the formation of hybrids between the sample sequence to be determined and the probe sequence by measuring said detectable label moiety.
Claim 17
Independent Claim
112 words
A method for determining a particular polynucleotide sequence in a test sample, comprising the step of: (a) combining the test sample with a polynucleotide probe having a base sequence substantially complementary to the sequence to be determined, wherein a mono-adduct forming nucleic acid-binding ligand is photochemically linked to a sequence selected from the group consisting of the sample sequence and the probe sequence, the nucleic acid-binding ligand being chemically linked to a detectable label moiety, wherein the label is selected from the group consisting of fluorescein and phycobiliprotein, and (b) detecting the formation of hybrids between the sample sequence to be determined and the probe sequence by measuring said detectable label moiety.
Claim 18
Dependent Claim
29 words
A method according to claim 14, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.
Claim 19
Dependent Claim
16 words
A method according to claim 15, wherein the intercalator compound is a furocoumarin or a phenanthridine.
Claim 20
Dependent Claim
11 words
A method according to claim 14, wherein the label is fluorescein.
Claim 21
Dependent Claim
52 words
A method according to claim 14, wherein said probe sequence is a first probe sequence and is labeled and an immobilized form of a second probe sequence is combined with the test sample, the first and second probe sequences being complementary to mutually exclusive portions of the sample sequence to be determined.
Claim 22
Dependent Claim
27 words
A labeled hybridizale nucleic acid according to claim 11, wherein the linker group is selected from the group consisting of dithiobis succinimidyl propionate and 1,4-butanediol diglycidyl ether.
Claim 23
Dependent Claim
106 words
An adduct suitable for photochemical attachment to a nucleic acid comprising (a) a nucleic acid-binding ligand, (b) a linker group coupled to the nucleic acid-binding ligand and (c) a label chemically linked through the linker group to the nucleic acid-binding ligand, whereby linkage of the adduct to the nucleic acid modifies the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, wherein the adduct is capable of being photochemically linked to the nucleic acid and wherein the label is selected from the group consisting of fluorescein and phycobiliprotein.
Claim 24
Dependent Claim
106 words
An adduct suitable for photochemical attachment to a nucleic acid comprising (a) a nucleic acid-binding ligand, (b) a linker group coupled to the nucleic acid-binding ligand and (c) a label chemically linked through the linker group to the nucleic acid-binding ligand, whereby linkage of the adduct to the nucleic acid modifies the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, wherein the adduct is capable of being photochemically linked to the nucleic acid and wherein the label is selected from the group consisting of fluorescein and phycobiliprotein.
Claim 25
Dependent Claim
24 words
An adduct according to claim 20, wherein the linker group is selected from the group consisting of dithiobis succinimidyl propionate and 1,4-butanediol diglycidyl ether.
Claim 26
Dependent Claim
77 words
A hybridizable labeled nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hydridization, and (c) a label chemically linked to (b), wherein the nucleic acid is modified at not more than one site per 50 nucleotide bases.
Claim 27
Dependent Claim
77 words
A hybridizable labeled nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hydridization, and (c) a label chemically linked to (b), wherein the nucleic acid is modified at not more than one site per 50 nucleotide bases.
Claim 28
Dependent Claim
77 words
A hybridizable labeled nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), wherein the nucleic acid is modified at not more than one site per 100 nucleotide bases.
Claim 29
Dependent Claim
77 words
A hybridizable labeled nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), wherein the nucleic acid is modified at not more than one site per 100 nucleotide bases.
Claim 30
Dependent Claim
46 words
A method of making a labeled nucleic acid probe, which comprises contacting such probe with an adduct according to claim 8 and subjecting the probe and adduct to photochemical irradiation, wherein the nucleic acid is modified at not more than one site per 50 nucleotide bases.
Claim 31
Dependent Claim
46 words
A method of making a labeled nucleic acid probe, which comprises contacting such probe with an adduct according to claim 8 and subjected the probe and adduct to photochemical irradiation, wherein the nucleic acid is modified at not more than one site per 100 nucleotide bases.
Claim 32
Dependent Claim
95 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not to be so great as to substantially prevent hybridization, (c) a linker group coupled to the nucleic acid-binding ligand and (d) a label chemically linked through the linker group to the nucleic acid-binding ligand, wherein the nucleic acid is modified at not more than one site per 50 nucleotide bases.
Claim 33
Dependent Claim
95 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not to be so great as to substantially prevent hybridization, (c) a linker group coupled to the nucleic acid-binding ligand and (d) a label chemically linked through the linker group to the nucleic acid-binding ligand, wherein the nucleic acid is modified at not more than one site per 50 nucleotide bases.
Claim 34
Dependent Claim
96 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not to be so great as to substantially prevent hybridization, (c) a linker group coupled to the nucleic acid-binding ligand and (d) a label chemically linked through the linker group to the nucleic acid binding ligand, wherein the nucleic acid is modified at not more than one site per 100 nucleotide bases.
Claim 35
Dependent Claim
96 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not to be so great as to substantially prevent hybridization, (c) a linker group coupled to the nucleic acid-binding ligand and (d) a label chemically linked through the linker group to the nucleic acid binding ligand, wherein the nucleic acid is modified at not more than one site per 100 nucleotide bases.
Claim 36
Dependent Claim
132 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) an intercalator compound photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), the labeled hybridizable nucleic acid produced by a process of (i) photochemically linking the nucleic acid component to the intercalator compound and chemically linking a label to a photochemically linked intercalator compound, or (ii) chemically linking a label to the intercalator compound to form a composite and photochemically linking said composite to the nucleic acid component, wherein the nucleic acid is modified at not more than one site per 50 nucleotide bases.
Claim 37
Dependent Claim
132 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) an intercalator compound photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), the labeled hybridizable nucleic acid produced by a process of (i) photochemically linking the nucleic acid component to the intercalator compound and chemically linking a label to a photochemically linked intercalator compound, or (ii) chemically linking a label to the intercalator compound to form a composite and photochemically linking said composite to the nucleic acid component, wherein the nucleic acid is modified at not more than one site per 50 nucleotide bases.
Claim 38
Dependent Claim
132 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) an intercalator compound photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), the labeled hybridizable nucleic acid produced by a process of (i) photochemically linking the nucleic acid component to the intercalator compound and chemically linking a label to a photochemically linked intercalator compound, or (ii) chemically linking a label to the intercalator compound to form a composite and photochemically linking said composite to the nucleic acid component, wherein the nucleic acid is modified at not more than one site per 100 nucleotide bases.
Claim 39
Dependent Claim
132 words
A labeled hybridizable nucleic acid comprising (a) a nucleic acid component, (b) an intercalator compound photochemically linked to the nucleic acid component thereby modifying the nucleic acid such that the frequency of modification along a hybridizable single stranded portion of the nucleic acid not be so great as to substantially prevent hybridization, and (c) a label chemically linked to (b), the labeled hybridizable nucleic acid produced by a process of (i) photochemically linking the nucleic acid component to the intercalator compound and chemically linking a label to a photochemically linked intercalator compound, or (ii) chemically linking a label to the intercalator compound to form a composite and photochemically linking said composite to the nucleic acid component, wherein the nucleic acid is modified at not more than one site per 100 nucleotide bases.
Claim 40
Dependent Claim
80 words
A kit for determining a particular polynucleotide sequence in a test sample, comprising in one or more containers (a) a polynucleotide probe having a base sequence complementary to the sequence to be determined and (b) a mono-adduct forming nucleic acid-binding ligand being chemically linked to a detectable label moiety, said ligand being photochemically linked to the probe or being capable of being photochemically linked to the sample, wherein the label is selected from the group consisting of fluorescein and phycobiliprotein.
Claim 41
Dependent Claim
80 words
A kit for determining a particular polynucleotide sequence in a test sample, comprising in one or more containers (a) a polynucleotide probe having a base sequence complementary to the sequence to be determined and (b) a mono-adduct forming nucleic acid-binding ligand being chemically linked to a detectable label moiety, said ligand being photochemically linked to the probe or being capable of being photochemically linked to the sample, wherein the label is selected from the group consisting of fluorescein and phycobiliprotein.
Claim 42
Dependent Claim
29 words
A kit according to claim 30, wherein the nucleic acid-binding ligand is an intercalator compound selected from the group consisting of acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.
Claim 43
Dependent Claim
16 words
A kit according to claim 31, wherein the intercalator compound is a furocoumarin or a phenanthridine.
Claim 44
Dependent Claim
11 words
A kit according to claim 30, wherein the label is fluorescein.
Claim 45
Dependent Claim
11 words
A kit according to claim 30, wherein the label is phycobiliprotein.
Claim 46
Dependent Claim
11 words
A method according to claim 14, wherein the label is phycobiliprotein.
Claim 47
Dependent Claim
27 words
A labeled hybridizable nucleic acid according to claim 27, wherein the linker group is selected from the group consisting of dithiobis succinimidyl propionate and 1,4-butanediol diglycidyl ether.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-09-24 | 1994-03-26 | 1994-09-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-09-24 | 1998-03-26 | 1998-09-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-09-24 | 2002-03-26 | 2002-09-25 | $7700.00 | Window Closed / Lapsed |