Patent EP0322311A2 successfully refreshed from Google Patents.
EP0322311A2
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EP
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Method and kit for detecting a nucleic acid sequence
Assignee: Applied Biosystems Inc
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Filing: 1988-12-21
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Granted: —
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Expires: 2008-12-21
Abstract
Abstract Nucleic acid sequences are detected by a multi-step process, involving labeling sample nucleic acid sequences, duplexing the labeled sample with a probe having a coupling element, immobilizing all of the duplexed probe and target sequence and unduplexed probe, separating specifically immobilized nucleic acid from free and non-specifically immobilized nucleic acid, releasing specifically immobilized nucleic acid, and detecting the presence of the sequence of interest by means of the label. The labeled sequence may be characterized by sizing, e.g. electrophoresis. The method provide for a sensitive and rapid means for accurate detection of sequence of interest in a wide variety of situations.
Inventors
- 👤 Mel N. Kronick
- 👤 Douglas H. Keith
- 👤 Lincoln J. Mcbride
- 👤 Norman M. Whiteley
- 👤 Michael W. Hunkapiller
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-02 14:43
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6823; C12Q1/6804
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
133 words
A method for detecting a nucleic acid sequence comprising: labeling nucleic acid suspected of having a sequence of interest with a label; combining said nucleic acid with a probe comprising a sequence capable of specifically binding to said sequence of interest and a coupling entity for linking to a separation means, said combining under conditions of predetermined stringency to form complexes between said sequence of interest and said complementary sequence, with the proviso that when said nucleic acid is in double stranded form, a nucleic acid probe is complexed with a rec A protein; separating said probe and complexes from non-complementary sequences with said separation means; releasing said labeled nucleic acid from said separation means to provide unbound labeled nucleic acid; and detecting said unbound labeled nucleic acid by means of said label.
Claim 2
Independent Claim
133 words
A method for detecting a nucleic acid sequence comprising: labeling nucleic acid suspected of having a sequence of interest with a label; combining said nucleic acid with a probe comprising a sequence capable of specifically binding to said sequence of interest and a coupling entity for linking to a separation means, said combining under conditions of predetermined stringency to form complexes between said sequence of interest and said complementary sequence, with the proviso that when said nucleic acid is in double stranded form, a nucleic acid probe is complexed with a rec A protein; separating said probe and complexes from non-complementary sequences with said separation means; releasing said labeled nucleic acid from said separation means to provide unbound labeled nucleic acid; and detecting said unbound labeled nucleic acid by means of said label.
Claim 3
Dependent Claim
22 words
A method according to Claim 1, including the additional step of size separating said unbound labeled nucleic acid which preferably employ electrophoresis.
Claim 4
Dependent Claim
140 words
A method for detecting a nucleic acid sequence of interest in a sample comprising genomic DNA, according to claim 1, said method comprising : fragmenting said genomic DNA to provide nucleic fragments of less than about 50kbp; labeling said nucleic acid fragments with a label; combining said nucleic acid fragments in single stranded form with a probe comprising a nucleic acid sequence complementary to said sequence of interest and a coupling entity for linking to particles capable of binding to said coupling entity, said combining under conditions of predetermined stringency to form duplexes between said sequence of interest and said complemenentary sequence; separating said probe and duplexes from non-complementary sequences with said particles; releasing said labeled nucleic acid from said separation means by chemical and/or thermal denaturation; size separating said released labeled nucleic acid; and detecting said size-separated nucleic acid.
Claim 5
Dependent Claim
12 words
A method according to Claim 3, wherein said size separating employs electrophoresis.
Claim 6
Independent Claim
33 words
A method according to one of claims 3 and 4, wherein said labeling comprises ligating said DNA fragments with dsDNA labeled molecules having a terminus complementary to a terminus of said DNA fragments.
Claim 7
Dependent Claim
44 words
A method according to Claim 5, wherein said complementary terminus of said DNA fragments is produced by restriction enzyme digestion of a restriction site and said ligating of said complementary terminus results in a sequence other than the sequence cleaved by said restriction enzyme.
Claim 8
Dependent Claim
18 words
A method according to Claim 6 , wherein said complementary terminus of said DNA fragments is blunt ended.
Claim 9
Dependent Claim
35 words
A method according to Claim 5 , wherein said labeling comprises extending the chains of said DNA fragments with a labeled nucleotide preferably a radionuclide, fluorescer, chemiluminescer, ligand of a specific binding pair or enzyme.
Claim 10
Dependent Claim
42 words
A method according to Claim 5 , wherein said coupling entity comprises a receptor and ligand combination,preferably avidin or streptavidin and biotin; antibody and hapten or antigen; complementary strands of nucleic acid; receptor and hormone; lectin and sugar; or chelate and ion.
Claim 11
Dependent Claim
36 words
A kit comprising a nucleic acid sequence joined to a linking entity, a solid component capable of specifically binding said linking entity, reagents for labeling a nucleic acid sequence, and means for sizing nucleic acid fragments.
Claim 12
Dependent Claim
36 words
A kit comprising a nucleic acid sequence joined to a linking entity, a solid component capable of specifically binding said linking entity, reagents for labeling a nucleic acid sequence, and means for sizing nucleic acid fragments.
Claim 13
Dependent Claim
46 words
. 11. A kit according to Claim 10, wherein said linking entity is a member of a specific binding pair, said solid component comprises the complementary member of said specific binding pair, said reagents comprise a labeled nucleotide, and said sizing means comprises an electrophoretic gel.
Claim 14
Dependent Claim
47 words
A kit according to Claim 10, wherein said specific binding pair consists of a receptor and ligand combination which consists essentially of avidin or streptavidin and biotin; antibody and hapten or antigen; complementary strands of nucleic acid; receptor and hormone; lectin and sugar; or chelate and ion.
References Cited (Backward Citations)
No citation records indexed.
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