US4886741A
Expired / Lapsed
US
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Use of volume exclusion agents for the enhancement of in situ hybridization
Assignee: Becton Dickinson and Co
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Filing: 1987-12-09
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Granted: 1989-12-12
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Expires: 2007-12-09
Abstract
Abstract This invention relates to methods for using volume exclusion agents to enhance in situ hybridization rates between short oligonucleotide probes and their target polynucleotides where the cells containing the target polynucleotides are adhered onto a glass substrate. In one aspect, the invention specifically relates to the use of volume exclusion agents to facilitate assay and diagnostic procedures for the detection of a virus, such as human papillomavirus (HPV). In addition, diagnostic kits embracing the above methods are described herein.
Inventors
- 👤 Dennis E. Schwartz
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 06:00
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6841; C12Q1/6832; C12Q1/70; C12Q1/701; C12Q1/708; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
114 words
A method for the in situ detection of polynucleotide targets in cells, comprising: (a) fixing the cells to an inert, transparent, and solid support; (b) contacting the fixed cells with a hybridization mixture comprising labeled short probes or between 15 and 30 nucleotides having a nucleotide sequence capable of hybridizing to the polynucleotide targets and a volume exclusion agent at a concentration of 2% to 25% (w/v) of the hybridization mixture; (c) washing excess short probes from the solid support, wherein steps (b) and (c) are completed in 4 hours or less; and (d) detecting labeled probes hybridized to said cells, wherein said detecting of probe is correlated to detection of said polynucleotide targets.
Claim 2
Independent Claim
114 words
A method for the in situ detection of polynucleotide targets in cells, comprising: (a) fixing the cells to an inert, transparent, and solid support; (b) contacting the fixed cells with a hybridization mixture comprising labeled short probes or between 15 and 30 nucleotides having a nucleotide sequence capable of hybridizing to the polynucleotide targets and a volume exclusion agent at a concentration of 2% to 25% (w/v) of the hybridization mixture; (c) washing excess short probes from the solid support, wherein steps (b) and (c) are completed in 4 hours or less; and (d) detecting labeled probes hybridized to said cells, wherein said detecting of probe is correlated to detection of said polynucleotide targets.
Claim 3
Dependent Claim
28 words
The method of claim 1 wherein the volume exclusion agent is polyethylene glycol or an anionic polymer selected from the group consisting of polyacrylate, polymethylacrylate, and polysaccharidic polymers.
Claim 4
Dependent Claim
19 words
The method of claim 2 wherein the polysaccharidic polymer is dextran sulfate in a concentration of 5% to 20%.
Claim 5
Dependent Claim
27 words
A method of claim 1 wherein the labeled probe will bind to target polynucleotides arising from infection of the cells by a type of human papilloma virus.
Claim 6
Dependent Claim
11 words
The method of claim 4 in which the probes are biotinylated.
Claim 7
Dependent Claim
15 words
The method of claim 4 in which the probes are directly conjugated to an enzyme.
Claim 8
Dependent Claim
12 words
The method of claim 6 in which the enzyme is alkaline phosphatase.
Claim 9
Dependent Claim
24 words
The method of claim 4 wherein the human papilloma virus isolates are from cells originating from the cervix or tissue of the anogenital region.
Claim 10
Dependent Claim
10 words
The method of claim 1 wherein the support is glass.
Claim 11
Dependent Claim
118 words
A kit for the in situ detection of polynucleotide targets in cells by nucleic acid hybridization methodology comprising hybridization and washing steps wherein the hybridization and washing steps are completed in four hours or less, said kit comprising: (a) fixation reagents for the fixation of cells to an inert support; (b) hybridization reagents for assaying the fixed cells, comprising an in situ hybridization reaction mixture which comprises labeled short probes of between 15 and 30 nucleotides and a volume exclusion agent at a concentration of 2 to 25% of the reaction mixture, wherein said reaction mixture effects detectable hybridization of said probe to the polynucleotide target in said fixed cells in a hybridization period of about 1 hour.
Claim 12
Dependent Claim
118 words
A kit for the in situ detection of polynucleotide targets in cells by nucleic acid hybridization methodology comprising hybridization and washing steps wherein the hybridization and washing steps are completed in four hours or less, said kit comprising: (a) fixation reagents for the fixation of cells to an inert support; (b) hybridization reagents for assaying the fixed cells, comprising an in situ hybridization reaction mixture which comprises labeled short probes of between 15 and 30 nucleotides and a volume exclusion agent at a concentration of 2 to 25% of the reaction mixture, wherein said reaction mixture effects detectable hybridization of said probe to the polynucleotide target in said fixed cells in a hybridization period of about 1 hour.
Claim 13
Dependent Claim
25 words
The kit of claim 10 wherein the volume exclusion agent is an anionic polymer selected from the group consisting of polyacrylate, polymethylacrylate, and polysaccharidic polymers.
Claim 14
Dependent Claim
19 words
The kit of claim 11 wherein the polysaccharidic polymer is dextran sulfate in a concentration of 5% to 20%.
References Cited (Backward Citations)
EP0098373A2
Cited
WO1986005816A1
Cited
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