Patent US5225326A successfully refreshed from Google Patents.
US5225326A
Expired / Lapsed
US
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One step in situ hybridization assay
Assignee: Abbott Molecular Inc
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Filing: 1991-10-28
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Granted: 1993-07-06
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Expires: 2011-10-28
Abstract
Abstract A quantitative, sensitive, One-Step In Situ hybridization assay is provided which will detect as few as 1-5 copies of target biopolymer per cell and may be accomplished in 5 minutes to 4 hours. There is provided a simultaneous assay for detecting multiple biopolymers within the same cell.
Inventors
- 👤 Joel Bresser
- 👤 Mary J. Evinger-Hodges
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 23:04
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6841; C12Q1/6876; C12Q1/6883; C12Q1/6886; C12Q1/70; C12Q1/701; C12Q1/702; C12Q1/703; C12Q1/705; C12Q2600/00; C12Q2600/158; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/80; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 43
Claim 1
Independent Claim
109 words
A method for assaying biopolymers in a sample of cells having substantially intact membranes comprising the steps of: contacting said sample with a medium comprising a fixative agent, a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least one probe, said contacting being under hybridizing conditions, incubating said sample with said medium in the presence of at least one detectable label, and detecting duplex formation by said probe(s) and target biopolymer(s) in said sample by means of said label, without performing a prehybridization step for blocking nonspecific binding of said probe(s) and facilitating probe entry before contacting said sample with said medium.
Claim 2
Independent Claim
109 words
A method for assaying biopolymers in a sample of cells having substantially intact membranes comprising the steps of: contacting said sample with a medium comprising a fixative agent, a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least one probe, said contacting being under hybridizing conditions, incubating said sample with said medium in the presence of at least one detectable label, and detecting duplex formation by said probe(s) and target biopolymer(s) in said sample by means of said label, without performing a prehybridization step for blocking nonspecific binding of said probe(s) and facilitating probe entry before contacting said sample with said medium.
Claim 3
Dependent Claim
13 words
The method of claim 1 wherein said label is attached to said probe.
Claim 4
Dependent Claim
16 words
The method of claim 1 wherein said label is added after the duplex formation is complete.
Claim 5
Dependent Claim
21 words
The method of claim 1 wherein said label is selected from the group consisting of fluorescers, chemiluminescers, enzyme labels, and radiolabels.
Claim 6
Dependent Claim
18 words
The method of claim 3 wherein said label is selected from the group consisting of avidin and streptavidin.
Claim 7
Dependent Claim
23 words
The method of claim 1 wherein said fixative agent is selected from the group consisting of ethanol, methanol, acetone, formaldehyde and combinations thereof.
Claim 8
Dependent Claim
26 words
The method of claim 1 wherein said denaturing agent is selected from the group consisting of formamide, urea, sodium iodide, thiocyanate, guanidine, perchlorate, trichloroacetate, and tetramethylamine.
Claim 9
Dependent Claim
28 words
The method of claim 1 wherein said hybrid stabilizing agent is selected from the group consisting of sodium chloride, lithium chloride, magnesium chloride, ferric sulfate and ammonium acetate.
Claim 10
Dependent Claim
30 words
The method of claim 1 wherein said pore forming agent is selected from the group consisting of polyoxyethylene 23 lauryl ether, polyoxyethylene 20 cetyl ether, polyoxyethylene ether, 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, desoxycholateandsodiumdodecyl sulfate.
Claim 11
Dependent Claim
10 words
The method of claim 1 wherein said biopolymer is RNA.
Claim 12
Dependent Claim
10 words
The method of claim 1 wherein said biopolymer is DNA.
Claim 13
Dependent Claim
11 words
The method of claim 1 wherein said biopolymer is an antigen.
Claim 14
Dependent Claim
17 words
The method of claim 1 wherein at least two biopolymers are assayed simultaneously in the same sample.
Claim 15
Dependent Claim
20 words
The method of claim 13 wherein at least one biopolymer is a polynucleotide and a second biopolymer is an antigen.
Claim 16
Dependent Claim
12 words
The method of claim 1 wherein said temperature is 15° C.-80° C.
Claim 17
Dependent Claim
14 words
The method of claim 15 wherein said temperature is 50° C. to 55° C.
Claim 18
Dependent Claim
14 words
The method of claim 1 wherein said method is accomplished within about 4 hours.
Claim 19
Dependent Claim
27 words
The method of claim 1 wherein said biopolymer is selected from the group consisting of a RNA, a DNA, a viral gene, an oncogene, and an antigen.
Claim 20
Dependent Claim
11 words
The method of claim 1, wherein said biopolymer is an oncogene.
Claim 21
Dependent Claim
11 words
The method of claim 1, wherein said biopolymer is a virus.
Claim 22
Dependent Claim
37 words
A kit for assaying the presence of a suspect biopolymer in a cell sample comprising, a hybridization solution comprising a precipitating agent, a denaturing agent, a hybrid stabilizing agent, a buffering agent, and a membrane pore-forming agent.
Claim 23
Dependent Claim
37 words
A kit for assaying the presence of a suspect biopolymer in a cell sample comprising, a hybridization solution comprising a precipitating agent, a denaturing agent, a hybrid stabilizing agent, a buffering agent, and a membrane pore-forming agent.
Claim 24
Dependent Claim
31 words
The kit of claim 21 also comprising, a supply of a probe selected so that it will hybridize with said suspect biopolymer if it is present, to form a hybridized complex.
Claim 25
Dependent Claim
37 words
The kit of claim 22 also comprising, means for contacting said suspect sample with said probe to form said hybridized complex, and means for measuring for the presence and/or extent of the presence of such labeled probe.
Claim 26
Dependent Claim
12 words
The kit of claim 22 where in said probe is detectably labelled.
Claim 27
Dependent Claim
15 words
The kit of claim 22 also comprising, a detectable label capable of detecting hybrid formation.
Claim 28
Dependent Claim
61 words
A kit for assaying the presence of a biopolymer in a suspect cell sample comprising, a hybridization solution comprising 30% ethanol, 30% formamide, 5% formaldehyde, 0.8M LiCl, 0.1M Tris-acetate (pH 7.4), 0.1% polyoxyethylene ether, 50 μg/ml of ribosomal RNA sheared and sized to about 50 bases, and 2.5 μg/ml of a single stranded probe directly labeled with a fluorescent reporter molecule.
Claim 29
Dependent Claim
61 words
A kit for assaying the presence of a biopolymer in a suspect cell sample comprising, a hybridization solution comprising 30% ethanol, 30% formamide, 5% formaldehyde, 0.8M LiCl, 0.1M Tris-acetate (pH 7.4), 0.1% polyoxyethylene ether, 50 μg/ml of ribosomal RNA sheared and sized to about 50 bases, and 2.5 μg/ml of a single stranded probe directly labeled with a fluorescent reporter molecule.
Claim 30
Dependent Claim
31 words
The kit of claim 26 also comprising, a supply of a probe selected so that it will hybridize with said suspect biopolymer if it is present, to form a hybridized complex.
Claim 31
Dependent Claim
11 words
The kit of claim 27 wherein said probe is detectably labeled.
Claim 32
Dependent Claim
15 words
The kit of claim 27 also comprising, a detectable label capable of detecting hybrid formation.
Claim 33
Dependent Claim
13 words
The method of claim 1 wherein said detecting of hybrid formation is quantitative.
Claim 34
Dependent Claim
27 words
The method of claim 1 wherein said probe is a polynucleotide having a nucleotide sequence at least substantially complementary to a specific nucleotide sequence to be detected.
Claim 35
Dependent Claim
16 words
The method of claim 1 wherein said probe is an antibody directed toward a specific antigen.
Claim 36
Dependent Claim
14 words
The method of claim 1 wherein said detectable label is an energy emitting label.
Claim 37
Independent Claim
25 words
The method of any of claims 1 or 21 wherein said probe comprises a mixture of short probes to multiple regions of the target biopolymer.
Claim 38
Dependent Claim
30 words
The method of claim 1 wherein the target biopolymer is a nucleic acid and said method is capable of detecting fewer than five copies of said nucleic acid per cell.
Claim 39
Independent Claim
101 words
A method for assaying nucleic acids in a sample of cells having substantially intact membranes, consisting essentially of the steps of: contacting said sample with a medium comprising a fixative agent, a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least one nucleic acid probe, said contacting being under hybridizing conditions, incubating said sample with said medium in the presence of at least one detectable label, and detecting duplex formation by means of said label, wherein said method is capable of detecting as few as five copies of a target nucleic acid per cell.
Claim 40
Independent Claim
101 words
A method for assaying nucleic acids in a sample of cells having substantially intact membranes, consisting essentially of the steps of: contacting said sample with a medium comprising a fixative agent, a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least one nucleic acid probe, said contacting being under hybridizing conditions, incubating said sample with said medium in the presence of at least one detectable label, and detecting duplex formation by means of said label, wherein said method is capable of detecting as few as five copies of a target nucleic acid per cell.
Claim 41
Dependent Claim
20 words
The method of claim 36, in which the sample is treated with a fixative before being contacted with said medium.
Claim 42
Dependent Claim
14 words
The method of claim 36, in which the contacting is carried out in solution.
Claim 43
Dependent Claim
15 words
The method of claim 36, in which the detecting is carried out by flow cytometry.
References Cited (Backward Citations)
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| 3.5 Years (Year 4) | 1996-07-05 | 1997-01-04 | 1997-07-06 | $2000.00 | Window Closed / Lapsed |
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