US5728527A
Expired / Lapsed
US
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Detection of hybridized oligonocleotide probes in living cells
Assignee: UMass Memorial Medical Center
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Filing: 1995-06-07
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Granted: 1998-03-17
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Expires: 2015-06-07
Abstract
Abstract A method of nucleic acid hybridization in living cells is described, which is useful for detecting, quantitating and locating a specific nucleic acid in a cell or tissue, for selecting cells based on the expression or presence of a specific nucleic acid, and for monitoring the amount and location of a specific nucleic acid over time or under various inducing or inhibiting conditions.
Inventors
- 👤 Robert H. Singer
- 👤 Joan C. Politz
- 👤 Krishan Taneja
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 21:55
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/6841
Claims Analysis
Total Claims: 36
Claim 1
Independent Claim
122 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a labelled single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting presence of the labelled probe in the cell or tissue after step b) is performed, wherein detection of the labelled probe in the cell or tissue indicates the presence of the target nucleic acids in the cell or tissue.
Claim 2
Independent Claim
122 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a labelled single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting presence of the labelled probe in the cell or tissue after step b) is performed, wherein detection of the labelled probe in the cell or tissue indicates the presence of the target nucleic acids in the cell or tissue.
Claim 3
Dependent Claim
10 words
The method of claim 1, wherein the oligonucleotide is unmodified.
Claim 4
Dependent Claim
25 words
The method of claim 1, wherein the oligonucleotide is modified to increase its ability to penetrate cellular membranes, thereby producing a modified single-stranded oligonucleotide probe.
Claim 5
Dependent Claim
31 words
The method of claim 3, wherein the oligonucleotide has at least one substitution of a phosphate backbone oxygen that decreases the hydrophilicity of the modified oligonucleotide compared to an unmodified oligonucleotide.
Claim 6
Dependent Claim
23 words
The method of claim 4, wherein the modification of the oligonucleotide is selected from the group consisting of: a) phosphorothioate; and b) phosphorodithioate.
Claim 7
Dependent Claim
16 words
The method of claim 3, wherein the probe is an oligonucleotide with a 3'-end blocking group.
Claim 8
Dependent Claim
37 words
The method of claim 6, wherein the 3'-end blocking group is selected from the group consisting of: a) amino-modified nucleotide; b) methyl group; c) fluorochrome-conjugated group; d) 3' to 5' nucleotide linkage; and e) succinyl ester group.
Claim 9
Dependent Claim
13 words
The method of claim 1, wherein the probe is labelled with a fluorochrome.
Claim 10
Dependent Claim
32 words
The method of claim 1, wherein in step a), two or more differently labelled probes are used, and wherein each said probe has a nucleotide sequence complementary to different target nucleic acids.
Claim 11
Dependent Claim
48 words
The method of claim 3, wherein in step a), the cell or tissue is incubated with a labelled probe and an unlabelled oligonucleotide with a nucleotide sequence different from the nucleotide sequence of the labelled probe, wherein the unlabelled oligonucleotide has the same modification as the labelled oligonucleotide.
Claim 12
Dependent Claim
34 words
The method of claim 1, wherein in step b), the cell or tissue is washed in medium containing unlabelled probe which has a nucleotide sequence that is not complementary to the target nucleic acids.
Claim 13
Dependent Claim
12 words
The method of claim 1, wherein the target nucleic acid are RNAs.
Claim 14
Dependent Claim
15 words
The method of claim 1, wherein the target nucleic acids are from an infectious agent.
Claim 15
Dependent Claim
12 words
The method of claim 13, wherein the infectious agent is a virus.
Claim 16
Dependent Claim
13 words
The method of claim 1, wherein the target nucleic acids are antisense oligonucleotides.
Claim 17
Dependent Claim
39 words
The method of claim 1, wherein in step c), detecting presence of the labelled probe in the cell or tissue comprises a method selected from the group consisting of: a) fluorescence microscopy; b) flow cytometry; and c) image analysis.
Claim 18
Dependent Claim
48 words
The method of claim 1, wherein in step c), detecting presence of the labelled probe in the cell or tissue comprises quantitating the amount of labelled probe detected, wherein the amount of labelled probe detected indicates the amount of the target nucleic acids in the cell or tissue.
Claim 19
Independent Claim
126 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the cell or tissue in a physiologically acceptable medium in which cells are able to grow and/or divide and containing a fluorochrome-labelled, single-stranded oligonucleotide probe, under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) removing the medium containing the probe and washing the cell or tissue in medium without probe; and c) detecting fluorescence of the probe in the cell or tissue after step b) is performed, wherein fluorescence indicates the presence of the target nucleic acids in the cell or tissue.
Claim 20
Independent Claim
126 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the cell or tissue in a physiologically acceptable medium in which cells are able to grow and/or divide and containing a fluorochrome-labelled, single-stranded oligonucleotide probe, under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) removing the medium containing the probe and washing the cell or tissue in medium without probe; and c) detecting fluorescence of the probe in the cell or tissue after step b) is performed, wherein fluorescence indicates the presence of the target nucleic acids in the cell or tissue.
Claim 21
Independent Claim
129 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids and modified to increase its ability to penetrate cellular membranes, wherein said probe is hydrolyzed by nucleases when not hybridized and is not hydrolyzed by nucleases when hybridized to complementary nucleic acids; and b) detecting presence of the probe in the cell or tissue, wherein detection of the probe in the cell or tissue indicates the presence of the target nucleic acids in the cell or tissue.
Claim 22
Independent Claim
129 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids and modified to increase its ability to penetrate cellular membranes, wherein said probe is hydrolyzed by nucleases when not hybridized and is not hydrolyzed by nucleases when hybridized to complementary nucleic acids; and b) detecting presence of the probe in the cell or tissue, wherein detection of the probe in the cell or tissue indicates the presence of the target nucleic acids in the cell or tissue.
Claim 23
Independent Claim
202 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with probes comprising a first and a second single-stranded oligonucleotide under conditions suitable for uptake of said probes and their specific hybridization to complementary nucleic acids in the cell or tissue, wherein the first and the second oligonucleotides are complementary to immediately adjacent sites on the target nucleic acid sequence, the first oligonucleotide with its 5' end closest to the 3' end of the second oligonucleotide when hybridized to immediately adjacent sites on the target nucleic acid sequence being labelled on the 5' end with either an energy donor group or an energy acceptor group and the second oligonucleotide being labelled on the 3' end with whichever group, either an energy donor group or an energy acceptor group, not being used as a label on the first oligonucleotide; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting energy transfer between the first and the second oligonucleotides after step b) is performed, wherein energy transfer indicates the presence of the target nucleic acids in the cell or tissue.
Claim 24
Independent Claim
202 words
A method for detecting target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with probes comprising a first and a second single-stranded oligonucleotide under conditions suitable for uptake of said probes and their specific hybridization to complementary nucleic acids in the cell or tissue, wherein the first and the second oligonucleotides are complementary to immediately adjacent sites on the target nucleic acid sequence, the first oligonucleotide with its 5' end closest to the 3' end of the second oligonucleotide when hybridized to immediately adjacent sites on the target nucleic acid sequence being labelled on the 5' end with either an energy donor group or an energy acceptor group and the second oligonucleotide being labelled on the 3' end with whichever group, either an energy donor group or an energy acceptor group, not being used as a label on the first oligonucleotide; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting energy transfer between the first and the second oligonucleotides after step b) is performed, wherein energy transfer indicates the presence of the target nucleic acids in the cell or tissue.
Claim 25
Independent Claim
122 words
A method for detecting expression of target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a labelled single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary RNA nucleotides in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting presence of the labelled probe in the cell or tissue after step b) is performed, wherein detection of the labelled probe in the cell or tissue indicates that the cell or tissue expresses the target nucleic acids.
Claim 26
Independent Claim
122 words
A method for detecting expression of target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a labelled single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary RNA nucleotides in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting presence of the labelled probe in the cell or tissue after step b) is performed, wherein detection of the labelled probe in the cell or tissue indicates that the cell or tissue expresses the target nucleic acids.
Claim 27
Dependent Claim
50 words
The method of claim 21, wherein in step c), detecting presence of the labelled probe in the cell or tissue comprises quantitating the amount of labelled probe detected, wherein the amount of labelled probe detected indicates the level of expression of the target nucleic acids in the cell or tissue.
Claim 28
Dependent Claim
33 words
A method for monitoring the expression of target nucleic acids in a living cell or tissue comprising performing the method of claim 21 on the same cell or tissue sample more than once.
Claim 29
Independent Claim
108 words
A method for identifying a cell that expresses target nucleic acids, comprising the steps of: a) incubating a population of living cells with a labelled single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary RNA nucleotides in the cells, said oligonucleotide having a nucleotide sequence which is complementary to the target nucleic acids; b) washing the cells to allow unhybridized probe to exit from the cell; and c) detecting presence of the labelled probe in the cell after step b) is performed, wherein detection of the labelled probe in the cell indicates that the cell expresses the target nucleic acids.
Claim 30
Independent Claim
108 words
A method for identifying a cell that expresses target nucleic acids, comprising the steps of: a) incubating a population of living cells with a labelled single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary RNA nucleotides in the cells, said oligonucleotide having a nucleotide sequence which is complementary to the target nucleic acids; b) washing the cells to allow unhybridized probe to exit from the cell; and c) detecting presence of the labelled probe in the cell after step b) is performed, wherein detection of the labelled probe in the cell indicates that the cell expresses the target nucleic acids.
Claim 31
Dependent Claim
18 words
The method of claim 24, further comprising separating the expressing cell from nonexpressing cells in the cell population.
Claim 32
Dependent Claim
30 words
The method of claim 25, wherein the separating step comprises a method selected from the group consisting of: a) flow sorting; and b) microdissection followed by subcloning of microdissected cells.
Claim 33
Independent Claim
121 words
A method for locating target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence which is complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting presence of the probe in the cell or tissue after step b) is performed, wherein detection of the probe in the cell or tissue indicates the location of the target nucleic acids in the cell or tissue.
Claim 34
Independent Claim
121 words
A method for locating target nucleic acids in a living cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence which is complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue; and c) detecting presence of the probe in the cell or tissue after step b) is performed, wherein detection of the probe in the cell or tissue indicates the location of the target nucleic acids in the cell or tissue.
Claim 35
Independent Claim
145 words
A method for confirming hybridization of an oligonucleotide probe to its complementary target nucleic acids in a cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue, thereby obtaining a washed cell or tissue; c) fixing the washed cell or tissue; and d) detecting presence of the probe in the cell or tissue by in situ transcription after step c) is performed, wherein a signal generated by in situ transcription indicates hybridization of the probe to its complementary target nucleic acids in the cell or tissue.
Claim 36
Independent Claim
145 words
A method for confirming hybridization of an oligonucleotide probe to its complementary target nucleic acids in a cell or tissue, comprising the steps of: a) incubating the living cell or tissue with a single-stranded oligonucleotide probe under conditions suitable for uptake of said probe and its specific hybridization to complementary nucleic acids in the cell or tissue, said oligonucleotide having a nucleotide sequence complementary to the target nucleic acids; b) washing the cell or tissue to allow unhybridized probe to exit from the cell or tissue, thereby obtaining a washed cell or tissue; c) fixing the washed cell or tissue; and d) detecting presence of the probe in the cell or tissue by in situ transcription after step c) is performed, wherein a signal generated by in situ transcription indicates hybridization of the probe to its complementary target nucleic acids in the cell or tissue.
References Cited (Backward Citations)
EP0070685A2
Cited
EP0229943B1
Cited
US5225326A
Cited
WO1990010715A1
Cited
US5236844A
Cited
WO1993005177A1
Cited
WO1993024652A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-03-16 | 2001-09-15 | 2002-03-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-03-16 | 2005-09-15 | 2006-03-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-03-16 | 2009-09-15 | 2010-03-17 | $7700.00 | Window Closed / Lapsed |