EP0320308B1
Expired / Lapsed
EP
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Method for detecting a target nucleic acid sequence
Assignee: Abbott Laboratories
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Filing: 1988-12-12
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Granted: 1993-11-03
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Expires: 2008-12-12
Abstract
Abstract Target nucleic acid is detected by reorganizing an excess of two complementary pairs of single stranded probes, which hybridize to contiguous target sequences. Nucleic acid in the sample is annealed to the probes, and contiguous sequences are ligated to form complementary detectable fused probes complementary to the original target, and the fused probes serve as a template for further fusions. The reorganized species being detected is increased at a geometric rate by cycles of annealing probes to the target, ligating the annealed probes in a template-dependent manner, and separating the fused probes from the template to form new templates.
Inventors
- 👤 Keith C. Backman
- 👤 Chang-Ning J. Wang
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 03:41
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6862; C12Q1/6813
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
263 words
A method of detecting target nucleic acid in a sample comprising the steps of: (a) providing nucleic acid of the sample as single-stranded nucleic acid; (b) providing in the sample a stoichiometric excess of at least four nucleic acid probes, wherein: i) the first and second of said probes are primary probes, and the third and fourth of said probes are secondary nucleic acid probes; ii) the first probe is a single strand capable of hybridizing to a first segment of a primary strand of the target nucleic acid; iii) the second probe is a single strand capable of hybridizing to a second segment of said primary strand of the target nucleic acid sequence; iv) the 5' end of the first segment of said primary strand of the target is positioned relative to the 3' end of the second segment of said primary strand of the target to enable joining of the 3' end of the first probe to the 5' end of the second probe, when said probes are hybridized to said primary strand of said target nucleic acid; v) the third probe is capable of hybridizing to the first probe; and vi) the fourth probe is capable of hybridizing to the second probe; and (c) repeatedly performing the following cycle: i) hybridizing said probes with nucleic acid in said sample; ii) ligating hybridized probes to form reorganized fused probe sequences; and iii) denaturing DNA in said sample; and (d) detecting the reorganized fused probe sequences; whereby with successive cycles the quantity of reorganized fused primary and fused secondary probes is increased.
Claim 2
Dependent Claim
49 words
A method according to Claim 1, wherein the 5' end of the first segment of said primary strand of the target sequence abuts, and is joined by a covalent bond to, the 3' end of the second segment of said primary strand of the target sequence, without intervening bases.
Claim 3
Independent Claim
17 words
A method according to Claims 1 or 2, wherein the probes are joined by a ligating enzyme.
Claim 4
Dependent Claim
18 words
A method according to Claim 3, wherein the probes are joined by a ligase, preferably a bacterial ligase.
Claim 5
Dependent Claim
19 words
A method according to Claim 4, wherein the ligase is Escherichia coli DNA ligase or Thermus thermophilus DNA ligase.
Claim 6
Dependent Claim
16 words
A method according to Claim 3, wherein the probes are joined by a thermostable ligating enzyme.
Claim 7
Independent Claim
14 words
A method according to any preceding claim, wherein the nucleic acid probes are DNA.
Claim 8
Independent Claim
15 words
A method according to any preceding claim, wherein the target nucleic acid sequence is DNA.
Claim 9
Independent Claim
21 words
A method according to any preceding claim, wherein the fused nucleic acid is separated from the target sequence by heat denaturation.
Claim 10
Independent Claim
21 words
A method according to any preceding claim, wherein said cycle is repeated at least twice, preferably between 20 and 50 times.
Claim 11
Independent Claim
23 words
A method according to any preceding claim, wherein the 5' end of the second probe but not of the first probe is phosphorylated.
Claim 12
Independent Claim
16 words
A method according to any preceding claim, wherein the target sequence is double-stranded before step (a).
Claim 13
Independent Claim
20 words
A method according to any preceding claim, wherein at least one of said probes is labelled with a labelling entity.
Claim 14
Dependent Claim
18 words
A method according to Claim 13, wherein both of said primary probes are labelled with a labelling entity.
Claim 15
Independent Claim
20 words
A method according to Claims 13 or 14, wherein both of said secondary probes are labelled with a labelling entity.
Claim 16
Independent Claim
25 words
A method according to any of Claims 13, 14 or 15, wherein the or at least one said labelling entity comprises a chromophore or flurophore.
Claim 17
Independent Claim
29 words
A method according to any of Claims 13, 14 or 15, wherein the or at least one said labelling entity comprises a specific binding partner for an insoluble phase.
Claim 18
Dependent Claim
213 words
A composition for use in a method of detecting target nucleic acid in a sample, said composition comprising a plurality of at least four nucleic acid probes, wherein: i) the first and second of said probes are primary probes, and the third and fourth of said probes are secondary probes; ii) the first probe is a single strand capable of hybridizing to a first segment of a primary strand of the target nucleic acid; iii) the second probe is a single strand capable of hybridizing to a second segment of said primary strand of the target nucleic acid sequence, the 5' end of the first segment of said primary strand of the target being positioned relative to the 3' end of the second segment of said primary strand of the target to enable joining of the first probe to the second probe when said probes are hybridized to said primary strand of said target nucleic acid; iv) the third probe is capable of hybridizing to the first probe; and v) the fourth probe is capable of hybridizing to the second probe such that, when the first and second probes are joined, the third and fourth probes are positioned relative to one another to enable joining of the third probe to the fourth probe.
Claim 19
Dependent Claim
44 words
A kit for performing an assay in accordance with any of claims 1-17 comprising: a) the probe composition of Claim 18; b) a nucleic acid ligase; and c) means adapted to contain said probe composition and means adapted to contain said nucleic acid ligase.
References Cited (Backward Citations)
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