Patent US4908307A successfully refreshed from Google Patents.
US4908307A
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US
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Hybridization method and probe for detecting nucleic acid sequences
Assignee: RODLAN KARIN D
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Filing: 1986-12-19
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Granted: 1990-03-13
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Expires: 2006-12-19
Abstract
Abstract Improved hybridization probe compositions and method for detecting nucleic acids of interest are provided. A hybridization probe constructed to contain thionucleotides and a detectable label is hybridized to a nucleotide sequence of interest in the absence of nonpolar solvents, reducing agents and volume exclusion agents. The thionucleotides serve to increase the mass of bound probe molecules, thereby amplifying the signal available to detect the presence of the nucleic acid sequence of interest. Certain cations interact specifically with the thiol moiety of phosphorothioate groups of nucelic acid molecules in a manner that can be exploited to amplify probe binding or convey a detectable label. Thus, the mass of bound probe molecules can be further increased by the presence of specified multivalent cations from the transition metal group, such as Cu 2+ or Fe 3+ , during the annealing step.
Inventors
- 👤 Karin D. Rodland
- 👤 Peter J. Russell
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 13:32
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6832; C12Q1/6816
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
151 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; converting any double-stranded probe molecules to single-stranded form; annealing said single-stranded, thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations capable of promoting the formation of ionic interactions between phosphorothioate groups of said probe molecules; subsequent to said annealing, detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences; and prior to said detecting, providing said probe molecules with a detectable label to provide a means for later detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences.
Claim 2
Independent Claim
151 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; converting any double-stranded probe molecules to single-stranded form; annealing said single-stranded, thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations capable of promoting the formation of ionic interactions between phosphorothioate groups of said probe molecules; subsequent to said annealing, detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences; and prior to said detecting, providing said probe molecules with a detectable label to provide a means for later detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences.
Claim 3
Independent Claim
155 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; converting any double-stranded probe molecules to single-stranded form; annealing said single-stranded, thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations selected from the group consisting of those transition metals in groups VIII, IB and IIB of the periodic table; subsequent to said annealing, detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences; and prior to said detecting, providing said probe molecules with a detectable label to provide a means for later detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences.
Claim 4
Independent Claim
155 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; converting any double-stranded probe molecules to single-stranded form; annealing said single-stranded, thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations selected from the group consisting of those transition metals in groups VIII, IB and IIB of the periodic table; subsequent to said annealing, detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences; and prior to said detecting, providing said probe molecules with a detectable label to provide a means for later detecting the presence of any said probe molecules annealed to said suspected nucleic acid base sequences.
Claim 5
Dependent Claim
40 words
The method of claim 2 wherein said cation is selected from the group consisting of those group VIII and IB transition metals having a greater affinity for thiol groups than for oxyanions and a redox potential more electronegative than +0.4.
Claim 6
Dependent Claim
24 words
The method of claim 2 wherein said cations are selected from the group consisting of those transition metals in the iron and copper families.
Claim 7
Dependent Claim
21 words
The method of claim 2 wherein said cations are selected from the group consisting of Cu 2+ and Fe 3+ .
Claim 8
Dependent Claim
12 words
The method of claim 3 wherein said cations are Cu 2+ .
Claim 9
Independent Claim
37 words
The method of claims 2, 3, 4, 5, or 6 wherein said cation is present in an amount ranging from about equimolar cation and thiol groups to about a 10-fold molar excess of cations to thiol groups.
Claim 10
Independent Claim
17 words
The method of claims 2, 3 or 4 wherein said annealing occurs in the absence of EDTA.
Claim 11
Independent Claim
22 words
The method of claims 2, 4 or 5 wherein said probe molecules are provided with said detectable label prior to said annealing.
Claim 12
Independent Claim
21 words
The method of claims 2, 4 or 5 wherein said probe molecules are provided with said detectable label after said annealing.
Claim 13
Dependent Claim
13 words
The method of claim 2 wherein said detectable label is a chemical modification.
Claim 14
Dependent Claim
34 words
The method of claim 11 wherein said single-stranded thionucleotide-containing probe molecules are provided with poly T-tails and said detectable label is attached to a polymer of adenosine which is annealed to said poly T-tails.
Claim 15
Dependent Claim
12 words
The method of claim 12 wherein said detectable label is biotinylated adenosine.
Claim 16
Independent Claim
21 words
The method of claims 2, 4 or 5 wherein racemically pure Sp thionucleotides are used to construct said thionucleotide-containing probe molecules.
Claim 17
Independent Claim
127 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; providing said probe molecules with a detectable label; converting any double-stranded probe molecules to single-stranded form; thereafter annealing said labelled, single-stranded thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations selected from the group consisting of those transition metals in groups VIII and IB of the periodic table; thereafter detecting the presence of any said labelled probe molecules annealed to said suspected nucleic acid base sequences.
Claim 18
Independent Claim
127 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; providing said probe molecules with a detectable label; converting any double-stranded probe molecules to single-stranded form; thereafter annealing said labelled, single-stranded thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations selected from the group consisting of those transition metals in groups VIII and IB of the periodic table; thereafter detecting the presence of any said labelled probe molecules annealed to said suspected nucleic acid base sequences.
Claim 19
Dependent Claim
18 words
The method of claim 15 wherein racemically pure Sp thionucleotides are used to construct said thionucleotide-containing probe molecules.
Claim 20
Dependent Claim
12 words
The method of claim 15 wherein said detectable label is a radionuclide.
Claim 21
Dependent Claim
13 words
The method of claim 15 wherein said detectable label is a chemical modification.
Claim 22
Independent Claim
126 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; converting any double-stranded probe molecules to single-stranded form; annealing said single-stranded, thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations selected from the group consisting of those transition metals in groups VIII and IB of the periodic table; thereafter providing said probe molecules with a detectable label; and detecting the presence of any said labelled probe molecules annealed to said suspected nucleic acid base sequences.
Claim 23
Independent Claim
126 words
A method for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences in single-stranded form comprising: providing nucleic acid probe molecules which in single-stranded form are capable of binding specifically to said suspected nucleic acid base sequences and contain at least one thionucleotide; converting any double-stranded probe molecules to single-stranded form; annealing said single-stranded, thionucleotide-containing probe molecules to said suspected nucleic acid base sequences in the presence of a concentration of multivalent cations selected from the group consisting of those transition metals in groups VIII and IB of the periodic table; thereafter providing said probe molecules with a detectable label; and detecting the presence of any said labelled probe molecules annealed to said suspected nucleic acid base sequences.
Claim 24
Dependent Claim
18 words
The method of claim 19 wherein racemically pure Sp thionucleotides are used to construct said thionucleotide-containing probe molecules.
Claim 25
Dependent Claim
13 words
The method of claim 19 wherein said detectable label is a chemical modification.
Claim 26
Dependent Claim
33 words
The method of claim 19 wherein said single-stranded probe molecules are provided with poly T-tails and said detectable label is attached to a polymer of adenosine which is annealed to said poly T-tails.
Claim 27
Dependent Claim
26 words
The method of claim 19 wherein said detectable label is added to said thionucleotide-containing probe molecules by means of an ionic interaction between phosphorothioates and cations.
Claim 28
Dependent Claim
12 words
The method of claim 19 wherein said detectable label is a radionuclide.
Claim 29
Dependent Claim
26 words
The method of claim 19 wherein said detectable label includes labelling molecules each containing thionucleotides capable of interacting specifically with the thionucleotides of said probe molecules.
Claim 30
Dependent Claim
33 words
The method of claim 19 wherein following annealing said probe molecules are contacted with non-complementary thionucleotide-containing molecules in the presence of a cation forming ionic interaction, said non-complementary molecules containing a detectable label.
Claim 31
Dependent Claim
85 words
A hybridization probe for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences comprising: labelled single-stranded nucleic acid probe molecules having essentially complementary base sequences to defined regions in said suspected nucleic acid base sequences; a detectable label attached to said probe molecules; and one or more phosphorothioate groups attached to said probe molecules which are capable of forming crosslinking ionic bonds with each other to bond together multiple labelled probe molecules, said phosphorothioate groups being non-radioactive.
Claim 32
Dependent Claim
85 words
A hybridization probe for detecting the presence of specific nucleic acid base sequences in a sample suspected of containing said sequences comprising: labelled single-stranded nucleic acid probe molecules having essentially complementary base sequences to defined regions in said suspected nucleic acid base sequences; a detectable label attached to said probe molecules; and one or more phosphorothioate groups attached to said probe molecules which are capable of forming crosslinking ionic bonds with each other to bond together multiple labelled probe molecules, said phosphorothioate groups being non-radioactive.
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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) | 1993-03-12 | 1993-09-11 | 1994-03-13 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-03-12 | 1997-09-11 | 1998-03-13 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-03-12 | 2001-09-11 | 2002-03-13 | $7700.00 | Window Closed / Lapsed |