One step in situ hybridization assay
AssigneeResearch Development Foundation
InventorsJoel Bresser, Mary J. Evinger-Hodges
Life of this patent
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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.
Claims39
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.
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.
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 .mu.g/ml of ribosomal RNA sheared and sized to about 50 bases, and 2.5 .mu.g/ml of a single stranded probe directly labeled with a fluorescent reporter molecule.
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.
Dependencies are inferred from the claim text.