US4599303A
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Nucleic acid hybridization assay employing probes crosslinkable to target sequences
Assignee: HRI ASSOCIATES Inc A CA CORP
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Filing: 1983-12-12
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Granted: 1986-07-08
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Expires: 2003-12-12
Abstract
Abstract Novel methods and reagents for determining the presence of specific nucleic acid base sequences by employing crosslinking reactions of unique molecules capable of forming covalent bonds which are bonded with various labels or ligands for amplication. Single stranded nucleic acid probes are employed which contain complementary base sequences to nucleic acid target molecules. By first hybridizing and then forming covalent bonds between the probe and target the amount of label in the crosslinked hybrid can be measured as an extremely sensitive method for assaying for specific nucleic acid sequences.
Inventors
- 👤 Kenichi K. Yabusaki
- 👤 Stephen T. Isaacs
- 👤 Howard B. Gamper, Jr.
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 07:31
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 43
Claim 1
Independent Claim
86 words
A probe for determining the presence of specific nucleic acid base sequences in nucleic acid target molecules comprising: single-stranded nucleic acid molecules having essentially complementary base sequences to defined regions in the nucleic acid target molecules; and labeled crosslinking molecules attached to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said single stranded nucleic acid molecules and said target molecules, whereby the presence of said specific nucleic acid base sequence is determined by measuring the amount of labeled probe covalently bound.
Claim 2
Dependent Claim
86 words
A probe for determining the presence of specific nucleic acid base sequences in nucleic acid target molecules comprising: single-stranded nucleic acid molecules having essentially complementary base sequences to defined regions in the nucleic acid target molecules; and labeled crosslinking molecules attached to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said single stranded nucleic acid molecules and said target molecules, whereby the presence of said specific nucleic acid base sequence is determined by measuring the amount of labeled probe covalently bound.
Claim 3
Dependent Claim
10 words
The probe of claim 1 wherein said label is radioactive.
Claim 4
Dependent Claim
32 words
A probe as recited in claim 1 wherein the crosslinking molecule is labeled with a member selected from the group consisting of radioactive nuclides, chromogenic labels, fluorogenic labels, chemiluminescent dyes and ligands.
Claim 5
Dependent Claim
12 words
The probe of claim 1 wherein said covalent crosslinks are formed chemically.
Claim 6
Dependent Claim
12 words
The probe of claim 1 wherein said covalent crosslinks are formed photochemically.
Claim 7
Dependent Claim
23 words
The probe of claim 1 wherein, said crosslinking molecule comprises a member selected from the group consisting of furocoumarins, benzodipyrones and bis azides.
Claim 8
Dependent Claim
14 words
The probe of claim 6 wherein said crosslinking molecule comprises bis azido ethidium bromide.
Claim 9
Dependent Claim
12 words
The probe of claim 1 wherein said crosslinking molecule comprises a psoralen.
Claim 10
Dependent Claim
13 words
The probe of claim 6 wherein said crosslinking molecule comprises a haloalkyl furocoumarin.
Claim 11
Dependent Claim
13 words
The probe of claim 6 wherein said crosslinking molecule comprises a haloalkyl benzodipyrone.
Claim 12
Dependent Claim
13 words
The probe of claim 6 wherein said crosslinking molecule comprises a haloalkyl courmarin.
Claim 13
Dependent Claim
14 words
The probe of claim 6 wherein said crosslinking molecule comprises an azido nucleoside triphosphate.
Claim 14
Dependent Claim
104 words
The probe of claim 6 wherein said crosslinking molecule comprises a member selected from the group consisting of: ##STR12## wherein R1 through R6 are the same or different and are each a member selected from the group consisting of H, CH 3 , CH 2 Cl, CH 2 Br, CH 2 I, CH 2 OH, CH 2 OCH 3 , CH 2 NH 2 , N 3 , COOH, COOCH 3 , COOCH 2 CH 3 , NH 2 , NO 2 , CF 3 , CCl 3 , CH(CH 3 ) 2 , C(CH 3 ) 3 , Cl, Br, I and F.
Claim 15
Dependent Claim
28 words
A probe as recited in claim 8 wherein the crosslinking molecule comprises a member selected from the group consisting of psoralen, 8-methoxypsoralen, 5-methoxypsoralen, 4,5',8-trimethylpsoralen, 4'-hydroxymethyl -4,5',8-trimethylpsoralen and 4'-aminomethyl-4,5',8-trimethylpsoralen.
Claim 16
Dependent Claim
16 words
A probe as recited in claim 3 wherein said label is 3 H or 14 C.
Claim 17
Independent Claim
117 words
A method for determining the presence of specific nucleic acid base sequences in nucleic acid target molecules comprising: A. providing single stranded nucleic acid molecules having an essentially complementary base sequence to a defined region in the target molecules; B. attaching labeled crosslinking molecules to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said single stranded nucleic acid molecules and said target molecules; C. hybridizing said single stranded nucleic acid molecules to said defined region in said target molecules; D. forming covalent bonds between said labelled crosslinking molecules and target molecules; and E. measuring the amount of said labelled crosslinking molecules and single stranded nucleic acid molecules present on said target molecules.
Claim 18
Independent Claim
117 words
A method for determining the presence of specific nucleic acid base sequences in nucleic acid target molecules comprising: A. providing single stranded nucleic acid molecules having an essentially complementary base sequence to a defined region in the target molecules; B. attaching labeled crosslinking molecules to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said single stranded nucleic acid molecules and said target molecules; C. hybridizing said single stranded nucleic acid molecules to said defined region in said target molecules; D. forming covalent bonds between said labelled crosslinking molecules and target molecules; and E. measuring the amount of said labelled crosslinking molecules and single stranded nucleic acid molecules present on said target molecules.
Claim 19
Dependent Claim
43 words
The method of claim 16 further comprising, prior to the measuring step of sub-part E, separating the crosslinking molecules which have not formed covalent bonds with the target from those which have by techniques that differentiate between single stranded and double stranded complexes.
Claim 20
Dependent Claim
29 words
The method of claim 17, wherein said techniques are selected from the group consisting of gel filtration, hydroxylapatite chromatography, enzyme digestion, and photoreversal or chemical reversal of uncrosslinked molecules.
Claim 21
Dependent Claim
25 words
The method as recited in claim 16 wherein the crosslinking molecule is chosen from the group consisting of psoralen, 8-methoxypsoralen, 5-methoxypsoralen, 4,5',8-trimethylpsoralen, 4'-hydroxymethyl-4,5',8-trimethylpsoralen and 4'-aminomethyl-4,5',8-trimethylpsoralen.
Claim 22
Dependent Claim
32 words
The method as recited in claim 16 wherein the crosslinking molecule is labeled with a label chosen from the group consisting of radioactive nuclides, chromogenic labels, fluorogenic labels, chemiluminescent dyes and ligands.
Claim 23
Dependent Claim
14 words
The method as recited in claim 20 wherein said label is 3H or 14C.
Claim 24
Dependent Claim
25 words
The method as recited in claim 16 wherein the crosslinking molecule is a member chosen from the group consisting of furocoumarins, benzodipyrones and bis azides.
Claim 25
Dependent Claim
14 words
The method as recited in claim 16 wherein said covalent bonds are formed chemically.
Claim 26
Dependent Claim
14 words
The method as recited in claim 16 wherein said covalent bonds are formed photochemically.
Claim 27
Dependent Claim
30 words
The method of claim 17 wherein said single stranded nucleic acid molecule comprises an RNA probe and said target comprises a DNA molecule and wherein said technique comprises alkaline hydrolysis.
Claim 28
Independent Claim
112 words
A method for determining the presence of specific nucleic acid sequences in nucleic acid target molecules comprising: A. providing labeled single-stranded nucleic acid molecules having an essentially complementary base sequence to a defined region in the target molecules; B. attaching crosslinking molecules to said labelled single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said labelled single-stranded nucleic acid molecules and said target molecules; C. hybridizing said labelled single-stranded nucleic acid molecules to said defined region in said target molecules; D. forming covalent bonds between said crosslinking molecules and target molecules; and E. measuring the amount of said labelled single-stranded nucleic acid molecules covalently bonded to said target molecules.
Claim 29
Independent Claim
112 words
A method for determining the presence of specific nucleic acid sequences in nucleic acid target molecules comprising: A. providing labeled single-stranded nucleic acid molecules having an essentially complementary base sequence to a defined region in the target molecules; B. attaching crosslinking molecules to said labelled single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said labelled single-stranded nucleic acid molecules and said target molecules; C. hybridizing said labelled single-stranded nucleic acid molecules to said defined region in said target molecules; D. forming covalent bonds between said crosslinking molecules and target molecules; and E. measuring the amount of said labelled single-stranded nucleic acid molecules covalently bonded to said target molecules.
Claim 30
Independent Claim
113 words
A method for determining the presence of specific nucleic acid base sequences in labelled nucleic acid target molecules comprising: A. providing single-stranded nucleic acid molecules having an essentially complementary base sequence to a defined region in the labelled target molecules; B. attaching crosslinking molecules to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said single-stranded nucleic acid molecules and said labelled target molecules; C. hybridizing said single-stranded nucleic acid molecules to said defined region in said labelled target molecules; D. forming covalent bonds between said crosslinking molecules and target molecules; and E. measuring the amount of said labelled target molecules covalently crosslinked to said single-stranded nucleic acid molecules.
Claim 31
Independent Claim
113 words
A method for determining the presence of specific nucleic acid base sequences in labelled nucleic acid target molecules comprising: A. providing single-stranded nucleic acid molecules having an essentially complementary base sequence to a defined region in the labelled target molecules; B. attaching crosslinking molecules to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinks between said single-stranded nucleic acid molecules and said labelled target molecules; C. hybridizing said single-stranded nucleic acid molecules to said defined region in said labelled target molecules; D. forming covalent bonds between said crosslinking molecules and target molecules; and E. measuring the amount of said labelled target molecules covalently crosslinked to said single-stranded nucleic acid molecules.
Claim 32
Independent Claim
21 words
The method of claims 16, 26 or 27 wherein said single-stranded nucleic acid molecules are chemically linked to a solid support.
Claim 33
Dependent Claim
11 words
The method of claim 28 wherein said solid support comprises silicon.
Claim 34
Dependent Claim
13 words
The method of claim 29 wherein said solid support comprises controlled pore glass.
Claim 35
Dependent Claim
28 words
The probe of claim 1 wherein said crosslinking molecule comprises a member selected from the group consisting of furocoumarins and benzodipyrones monoadducted to pyrimidine via cyclobutane bridge linkage.
Claim 36
Independent Claim
47 words
The method of any one of claims 16, 26 or 27 wherein said single-stranded nucleic acid molecules are attached to a crosslinking molecule comprising a member selected from the group consisting of furocoumarins and benzodipyrones by direct incorporation during chemical synthesis of said single-stranded nucleic acid molecule.
Claim 37
Dependent Claim
84 words
A probe for determining the presence of specific nucleic acid based sequences in nucleic acid target molecules comprising: labeled single-stranded nucleic acid molecules having essentially complementary based sequences to defined regions in the nucleic acid target molecules; and crosslinking molecules attached to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinking between said single-stranded nucleic acid molecules and said target molecules, whereby the presence of said specific nucleic acid base sequences determined by measuring the amount of labeled probe covalently bound.
Claim 38
Dependent Claim
84 words
A probe for determining the presence of specific nucleic acid based sequences in nucleic acid target molecules comprising: labeled single-stranded nucleic acid molecules having essentially complementary based sequences to defined regions in the nucleic acid target molecules; and crosslinking molecules attached to said single-stranded nucleic acid molecules which are capable of forming covalent crosslinking between said single-stranded nucleic acid molecules and said target molecules, whereby the presence of said specific nucleic acid base sequences determined by measuring the amount of labeled probe covalently bound.
Claim 39
Dependent Claim
10 words
The probe of claim 33 wherein said label is radioactive.
Claim 40
Dependent Claim
34 words
A probe as recited in claim 33 wherein the single-stranded nucleic acid molecule is labeled with a member selected from the group consisting of radioactive nuclides, chromogenic labels, fluorogenic labels, chemiluminescent dyes and ligands.
Claim 41
Dependent Claim
107 words
A reagent for DNA synthesis comprising a pyrimidine monophosphoramadite nucleoside furan-side monoadducted to a member selected from the group consisting of ##STR13## wherein R 1 -R 6 are the same or different and are each a member selected from the group consisting of H, CH 3 , Ch 2 Cl, CH 2 Br, CH 2 I, CH 2 OH, CH 2 OCH 3 , CH 2 NH 2 , N 3 , COOH, COOCH 3 , COOCH 2 CH 3 , NH 2 , NO 2 , CF 3 , CCl 3 ,CH(CH 3 ) 2 , C(CH 3 ) 3 , Cl, Br, I and F.
Claim 42
Dependent Claim
107 words
A reagent for DNA synthesis comprising a pyrimidine monophosphoramadite nucleoside furan-side monoadducted to a member selected from the group consisting of ##STR13## wherein R 1 -R 6 are the same or different and are each a member selected from the group consisting of H, CH 3 , Ch 2 Cl, CH 2 Br, CH 2 I, CH 2 OH, CH 2 OCH 3 , CH 2 NH 2 , N 3 , COOH, COOCH 3 , COOCH 2 CH 3 , NH 2 , NO 2 , CF 3 , CCl 3 ,CH(CH 3 ) 2 , C(CH 3 ) 3 , Cl, Br, I and F.
Claim 43
Dependent Claim
27 words
A reagent as recited in claim 36 wherein the molecule comprises a member selected from the group consisting of psoralen, 8-methoxypsoralen, 5-methoxypsoralen, 4,5',8-trimethylpsoralen, 4'-hydroxymethyl, 4,5',8-trimethylpsoralen, and 4'-aminomethyl-4,5'8-trimethylpsoralen.
References Cited (Backward Citations)
US3798131A
Cited
EP0070687B1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-07-07 | 1990-01-06 | 1990-07-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-07-07 | 1994-01-06 | 1994-07-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-07-07 | 1998-01-06 | 1998-07-08 | $7700.00 | Window Closed / Lapsed |