EP0853129A2
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EP
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Devices and methods for detecting nucleic acid analytes in samples
Assignee: Abbott Molecular Inc
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Filing: 1997-12-16
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Granted: —
•
Expires: 2017-12-16
Abstract
Abstract The invention provides devices and methods for use in detecting nucleic acid analytes in samples. The devices each include a solid support to which is bound a two-dimensional distribution or field of nucleic acid probes that each bind to a nucleic acid analyte, which is detected by use of amplification methods. <IMAGE>
Inventors
- 👤 David J. Lane
- 👤 Michael P. Farrell
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 06:59
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682; C12Q1/6834; C12Q1/6837
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
64 words
An assay device for detecting the presence of a
nucleic acid analyte in a sample, said device comprising a
non-particulate, solid support having covalently bound
thereto a two-dimensional field of analyte-specific nucleic
acid probes, each probe comprising a first end covalently
bound to said support and including a 5' portion of an
autocatalytically replicable nucleic acid, and a second end
including an analyte-binding segment.
Claim 2
Dependent Claim
16 words
The device of claim 1, wherein said
autocatalytically replicable nucleic acid is replicable by
Q-beta replicase.
Claim 3
Dependent Claim
16 words
The device of claim 2, wherein said
autocatalytically replicable nucleic acid comprises a
midivariant nucleic acid.
Claim 4
Independent Claim
215 words
A method for detecting the presence of a nucleic
acid analyte in a sample, said method comprising the steps
of: (a) applying the sample to a solid support having
covalently bound thereto a two-dimensional field of first
analyte-specific nucleic acid probes, each of said first
probes comprising a first end bound to said support and
including a 5' portion of an autocatalytically replicable
nucleic acid, and a second end including a first analyte-binding
segment, wherein said first analyte-binding segment
of each of said first probes hybridizes to a first region of
the analyte; (b) applying a second nucleic acid probe to the
solid support, said second probe comprising a second
analyte-binding segment that hybridizes to a second region
of the analyte, said second probe further comprising a
remainder of said autocatalytically replicable nucleic acid,
wherein the first and second regions of the analyte are
adjacent nucleotide segments, and wherein the analyte, said
first probe, and said second probe hybridize together on the
support to form a complex including a complete
autocatalytically replicable nucleic acid; (c) applying a diffusion limiting matrix to said
solid support; (d) amplifying said complete autocatalytically
replicable nucleic acid to generate an amplified product;
and (e) detecting said amplified product as a measure of
the presence of the analyte in the sample.
Claim 5
Dependent Claim
28 words
The method of claim 4, further comprising the
step of ligating said first and second probes in said
complex together to form said complete autocatalytically
replicable nucleic acid.
Claim 6
Dependent Claim
38 words
The method of claim 4, wherein said
amplification step is carried out in the presence of a
fluorescent intercalating dye and said detection step is
carried out by monitoring said amplified product for the
presence of said dye.
Claim 7
Dependent Claim
17 words
The method of claim 4, wherein said detection
step is carried out by employing a fluor-quencher pair.
Claim 8
Dependent Claim
31 words
The method of claim 4, wherein multiple analytes
are simultaneously detected in the sample and said detecting
step is carried out by employing multiple fluor-quencher
pairs that comprise distinguishable fluorescent moieties.
Claim 9
Dependent Claim
19 words
The method of claim 4, wherein said
autocatalytically replicable nucleic acid comprises a
nucleic acid replicable by Q-beta replicase.
Claim 10
Dependent Claim
16 words
The method of claim 9, wherein said
autocatalytically replicable nucleic acid comprises a
midivariant nucleic acid.
Claim 11
Dependent Claim
18 words
The method of claim 4, further comprising
washing said complex to remove any unbound first or second
probes.
Claim 12
Dependent Claim
26 words
The method of claim 4, wherein said diffusion
limiting matrix is selected from the group consisting of
gelatin, agarose, polyacrylamide, polyethylene glycol, and
any combination thereof.
Claim 13
Dependent Claim
53 words
The method of claim 4, further comprising
determining the concentration of the analyte in the sample,
wherein the concentration of the analyte in the sample is
determined by counting colonies of amplified product formed
on said solid support, and the number of colonies
corresponds to the concentration of the analyte in the
sample.
Claim 14
Dependent Claim
153 words
An assay system for detecting a nucleic acid
analyte in a sample, said assay system comprising: (a) a non-particulate solid support having
covalently bound thereto a two-dimensional field of first
analyte-specific nucleic acid probes, each of said first
probes comprising a first end covalently bound to said
support and including a 5' portion of an autocatalytically
replicable nucleic acid, and a second end including a first
analyte-binding segment, wherein said first analyte-binding
segment of each of said first probes hybridizes to a first
region of the analyte; and (b) a second nucleic acid probe comprising a second
analyte-binding segment that hybridizes to a second region
of the analyte, said second probe further comprising a
remainder of said autocatalytically replicable nucleic acid,
and said first and second regions of the analyte comprising
adjacent nucleotide segments; wherein binding of said first and second probes to
the analyte permits amplification of said autocatalytically
replicable nucleic acid.
Claim 15
Dependent Claim
124 words
An assay system for detecting a nucleic acid
analyte in a sample, said assay system comprising: (a) a non-particulate solid support having bound
thereto a two-dimensional field of capture probes, each of
said capture probes comprising a capture segment that
hybridizes to a capture region of the analyte; (b) a first nucleic acid probe comprising a first
segment that hybridizes to a first region of the analyte; (c) a second nucleic acid probe comprising a second
segment that hybridizes to a second region of the analyte;
and (d) a diffusion limiting matrix for application to
said solid support; wherein binding of said capture probes and said
first and second probes to the analyte permits amplification
of a detectable product within the diffusion limiting
matrix.
Claim 16
Dependent Claim
53 words
The assay system of claim 15, wherein the first
and second regions of the analyte are adjacent nucleotide
segments, said first nucleic acid probe further comprises a
portion of an autocatalytically replicable nucleic acid
molecule, and said second nucleic acid probe further
comprises a remaining portion of said autocatalytically
replicable nucleic acid molecule.
Claim 17
Dependent Claim
66 words
The assay system of claim 15, wherein the
capture and first regions of the analyte are adjacent
nucleotide segments and the capture and second regions of
the analyte are adjacent nucleotide segments, said first
nucleic acid probe further comprises a portion of an
autocatalytically replicable nucleic acid molecule, and said
second nucleic acid probe further comprises a remaining
portion of said autocatalytically replicable nucleic acid
molecule.
Claim 18
Dependent Claim
159 words
A method for detecting the presence of a
nucleic acid analyte in a sample, said method comprising the
steps of: (a) contacting the sample with said bound capture
probes and said first and second probes of the assay system
of claim 15, wherein said first and second regions of the
analyte are adjacent nucleotide segments, said first nucleic
acid probe further comprises a portion of an
autocatalytically replicable nucleic acid molecule, said
second nucleic acid probe further comprises a remaining
portion of said autocatalytically replicable nucleic acid
molecule, and said contacting permits the analyte, said
first probe, and said second probe to hybridize together on
the support to form a complex including a complete
autocatalytically replicable nucleic acid; (b) applying a diffusion limiting matrix to said
solid support; (c) amplifying said complete autocatalytically
replicable nucleic acid to generate an amplified product;
and (d) detecting said amplified product as a measure of
the presence of the analyte in the sample.
Claim 19
Dependent Claim
173 words
A method for detecting the presence of a
nucleic acid analyte in a sample, said method comprising the
steps of: (a) contacting the sample with said bound capture
probes and said first and second probes of the assay system
of claim 15, wherein the capture and first regions of the
analyte are adjacent nucleotide segments and the capture and
second regions of the analyte are adjacent nucleotide
segments, said first nucleic acid probe further comprises a
portion of an autocatalytically replicable nucleic acid
molecule, and said second nucleic acid probe further
comprises a remaining portion of said autocatalytically
replicable nucleic acid molecule, and said contacting
permits the analyte, said first probe, and said second probe
to hybridize together on the support to form a complex
including a complete autocatalytically replicable nucleic
acid; (b) applying a diffusion limiting matrix to said
solid support; (c) amplifying said complete autocatalytically
replicable nucleic acid to generate an amplified product;
and (d) detecting said amplified product as a measure of
the presence of the analyte in the sample.
Claim 20
Independent Claim
157 words
A method for detecting the presence of a
nucleic acid analyte in a sample, said method comprising the
steps of: (a) contacting the sample with a solid support
having covalently bound thereto a two-dimensional field of
first analyte-specific nucleic acid probes, each of said
first probes comprising a first end bound to said support
and a second end including a first analyte-binding segment; (b) applying a diffusion limiting matrix to said
support; (c) performing amplification by one of polymerase
chain reaction, ligase chain reaction, transcription-mediated
amplification, nucleic acid sequence-based
amplification, and strand displacement amplification in a
manner dependent upon the presence of both the first probe
and the specified analyte nucleic acid, and in such a manner
that the products of such amplification reaction are
restrained to form localized foci within the diffusion
limiting matrix on the solid support; and (d) detecting said amplified product as a measure of
the presence of said analyte in the sample.
Claim 21
Dependent Claim
27 words
The device of claim 1, wherein each of the
analyte-specific nucleic acid probes is an RNA synthesized
by an RNA polymerase in the presence of guanosine 5'-monophosphate.
Claim 22
Dependent Claim
13 words
The device of claim 21, wherein the RNA
polymerase is T7 RNA polymerase.
Claim 23
Dependent Claim
28 words
The method of claim 4, wherein each of the
first analyte-specific nucleic acid probes is an RNA
synthesized by an RNA polymerase in the presence of
guanosine 5'-monophosphate.
Claim 24
Independent Claim
26 words
A method of synthesizing an RNA, the method
comprising contacting a template for the RNA with T7
polymerase in the presence of 5' modified guanosine 5'-monophosphate.
Claim 25
Dependent Claim
25 words
The assay device of claim 1, wherein each of
the analyte-specific nucleic acid probes is covalently bound
to the solid support with bovine serum albumin.
Claim 26
Dependent Claim
25 words
The method of claim 4, wherein each of the
first analyte-specific nucleic acid probes is covalently
bound to the solid support with bovine serum albumin.
Claim 27
Dependent Claim
13 words
The method of claim 4, wherein the diffusion
limiting matrix comprises polyethylene glycol.
Claim 28
Dependent Claim
15 words
The method of claim 27, wherein the diffusion
limiting matrix further comprises a cationic polymer.
Claim 29
Dependent Claim
14 words
The assay system of claim 15, wherein the
diffusion limiting matrix comprises polyethylene glycol.
Claim 30
Dependent Claim
16 words
The assay system of claim 29, wherein the
diffusion limiting matrix further comprises a cationic
polymer.
References Cited (Backward Citations)
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