EP0225807A2
Expired / Lapsed
EP
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Solution phase nucleic acid sandwich assay and polynucleotide probes useful therein
Assignee: Bayer Corp
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Filing: 1986-12-10
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Granted: —
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Expires: 2006-12-10
Abstract
Abstract Methods and compositions are provided for rapid detection of nucleic acid sequences. The method employs two reagent sets. The first set is a labeling set comprising: (1) a first nucleic acid sequence probe having an analyte complementary region and a first recognition sequence region and (2) a labeled sequence complementary to the first recognition sequence region. The second set is a capturing set comprising: (1) a second nucleic acid sequence probe having an analyte complementary region and a second recognition sequence region, (2) a specific binding pair member conjugated to a sequence complementary to the second recognition sequence, and (3) a separating means to which is bound a complementary specific binding pair member. The sample and probes are combined under annealing conditions, followed by addition of the other reagents, separation of the bound label from the supernatant and detection of the label in either phase. The invention also encompasses nucleic acid probes formed from one or more modified derivatizable nucleotides.
Inventors
- 👤 Michael Steven Urdea
- 👤 Brian Warner
- 👤 Thomas Horn
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 16:31
CPC Classifications
C; C07; C07H; C07H21/00; C07H19/00; C07H19/02; C07H19/04; C07H19/06; C07H19/10; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682; C12Q1/70; C12Q1/701; C12Q1/706; Y; Y02; Y02P; Y02P20/00; Y02P20/50; Y02P20/55; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
228 words
An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising: combining in the liquid medium under binding conditions for complementary pairs, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising: (a) one or a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and members of said second capturing set of reagents comprising: (c) one or a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate; and separation means; with the proviso that, said nucleic acid sequence complementary to said second recognition sequence may be bound to said separation means eliminating said specific binding pair, so that (d) is not employed; separating label into a bound phase and an unbound phase by means of said separation means; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.
Claim 2
Dependent Claim
228 words
An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising: combining in the liquid medium under binding conditions for complementary pairs, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising: (a) one or a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and members of said second capturing set of reagents comprising: (c) one or a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate; and separation means; with the proviso that, said nucleic acid sequence complementary to said second recognition sequence may be bound to said separation means eliminating said specific binding pair, so that (d) is not employed; separating label into a bound phase and an unbound phase by means of said separation means; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.
Claim 3
Dependent Claim
64 words
A method according to Claim 1, wherein said nucleic acid sequence complementary to said second recognition sequence is bound directly to said separation means, and said analyte, (a), (b), and (c) are combined together for a time sufficient for nucleic acid complexes to form, followed by the addition of said nucleic acid sequence complementary to said second recognition sequence bound to said separation means.
Claim 4
Dependent Claim
239 words
An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising: combining in the liquid medium under binding conditions for complementary pairs, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising: (a) one or a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and members of said second capturing set of reagents comprising: (c) one or a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate, for a time sufficient for nucleic acid complexes containing analyte, label and said specific binding member to form; combining any of said complexes with a separation means conjugated to a second complementary member of said specific binding pair under conditions resulting in specific binding pair under conditions of complex formation and separation of label into a bound phase and an unbound phase; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.
Claim 5
Dependent Claim
239 words
An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising: combining in the liquid medium under binding conditions for complementary pairs, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising: (a) one or a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and members of said second capturing set of reagents comprising: (c) one or a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate, for a time sufficient for nucleic acid complexes containing analyte, label and said specific binding member to form; combining any of said complexes with a separation means conjugated to a second complementary member of said specific binding pair under conditions resulting in specific binding pair under conditions of complex formation and separation of label into a bound phase and an unbound phase; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.
Claim 6
Dependent Claim
211 words
An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising: combining in a liquid medium under annealing conditions for complementary nucleic acid sequences, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising: (a) a set of labeling nucleic acid probes, each subset having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-fluorescer or enzyme label conjugate; and members of said second capturing set of reagents comprising: (c) a set of capturing nucleic acid probes, each subset having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-hapten conjugate, for a time sufficient for nucleic acid complexes containing said analyte, label and hapten to form; combining any of said complexes to a particle conjugated to a receptor for said hapten under conditions resulting in hapten-receptor complex formation and separation of label into a bound phase and an unbound phase; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.
Claim 7
Dependent Claim
211 words
An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising: combining in a liquid medium under annealing conditions for complementary nucleic acid sequences, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising: (a) a set of labeling nucleic acid probes, each subset having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-fluorescer or enzyme label conjugate; and members of said second capturing set of reagents comprising: (c) a set of capturing nucleic acid probes, each subset having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-hapten conjugate, for a time sufficient for nucleic acid complexes containing said analyte, label and hapten to form; combining any of said complexes to a particle conjugated to a receptor for said hapten under conditions resulting in hapten-receptor complex formation and separation of label into a bound phase and an unbound phase; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.
Claim 8
Dependent Claim
124 words
A kit for detecting a nucleic acid analyte comprising: (1) members of a labelling set of reagents comprising: (a) a labeling nucleic acid probe having a first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and (2) members of a second capturing set of reagents comprising: (c) a capturing nucleic acid probe having a second analyte complementary sequence and a second capturing reagent recognition sequence; (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate; and (e) support means conjugated to a second complementary member of said specific binding pair.
Claim 9
Dependent Claim
124 words
A kit for detecting a nucleic acid analyte comprising: (1) members of a labelling set of reagents comprising: (a) a labeling nucleic acid probe having a first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and (2) members of a second capturing set of reagents comprising: (c) a capturing nucleic acid probe having a second analyte complementary sequence and a second capturing reagent recognition sequence; (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate; and (e) support means conjugated to a second complementary member of said specific binding pair.
Claim 10
Dependent Claim
102 words
A modified nucleotide given by the structure wherein R¹ is a reactive group derivatizable with a detectable label, R² is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof. R³ is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R⁴ is selected from the group consisting of hydrogen, an acid-sensitive, base-stable blocking group or an acyl capping group, R⁵ is hydrogen or a phosphorus derivative, R⁶ is H, OH, or OR where R is an acid-sensitive, base-stable protecting group and x is an integer in the range of 1 and 8 inclusive.
Claim 11
Dependent Claim
102 words
A modified nucleotide given by the structure wherein R¹ is a reactive group derivatizable with a detectable label, R² is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof. R³ is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R⁴ is selected from the group consisting of hydrogen, an acid-sensitive, base-stable blocking group or an acyl capping group, R⁵ is hydrogen or a phosphorus derivative, R⁶ is H, OH, or OR where R is an acid-sensitive, base-stable protecting group and x is an integer in the range of 1 and 8 inclusive.
Claim 12
Independent Claim
168 words
A method of making a modified nucleotide, comprising the steps of: reacting a pyrimidine nucleotide having the structure wherein R³ is selected from the group consisting of hydrogen, methyl, fluorine, bromine and iodine, R⁴ and R⁵ are hydrogen, and R⁶ is hydrogen, hydroxyl, or blocked hydroxyl, with at least one protecting compound, thereby producing a 3ʹ- and 5ʹ-protected nucleotide; where said nucleotide is thymine, unsubstituted uracil, or uracil substituted at the 5-position with R³, reacting said protected nucleotide with an activating agent, thereby providing an amine moiety on said nucleotide; optionally reacting the amine moiety of said nucleotide with a linking agent, thereby providing a structure having extending from the 4-position of said pyrimidine an amide, thioether or disulfide linkage or a combination thereof, and further having a free amine, carboxylic acid, or sulfhydryl reactive moiety reacting said free amine, carboxylic acid, or sulfhydryl moiety with a caproic acid reagent selected from the group consisting of caproic acid, an activated caproic acid ester, 6-aminocaproic acid and combinations thereof.
Claim 13
Independent Claim
168 words
A method of making a modified nucleotide, comprising the steps of: reacting a pyrimidine nucleotide having the structure wherein R³ is selected from the group consisting of hydrogen, methyl, fluorine, bromine and iodine, R⁴ and R⁵ are hydrogen, and R⁶ is hydrogen, hydroxyl, or blocked hydroxyl, with at least one protecting compound, thereby producing a 3ʹ- and 5ʹ-protected nucleotide; where said nucleotide is thymine, unsubstituted uracil, or uracil substituted at the 5-position with R³, reacting said protected nucleotide with an activating agent, thereby providing an amine moiety on said nucleotide; optionally reacting the amine moiety of said nucleotide with a linking agent, thereby providing a structure having extending from the 4-position of said pyrimidine an amide, thioether or disulfide linkage or a combination thereof, and further having a free amine, carboxylic acid, or sulfhydryl reactive moiety reacting said free amine, carboxylic acid, or sulfhydryl moiety with a caproic acid reagent selected from the group consisting of caproic acid, an activated caproic acid ester, 6-aminocaproic acid and combinations thereof.
Claim 14
Dependent Claim
84 words
Polynucleotide probes having at least two nucleotides, at least one of which is given by the structure wherein R¹ is a reactive group derivatized with a detectable label, R² is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof, R³ is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R⁶ is H, OH, or OR where R is a protecting group and x is an integer in the range of 1 and 8 inclusive.
Claim 15
Dependent Claim
84 words
Polynucleotide probes having at least two nucleotides, at least one of which is given by the structure wherein R¹ is a reactive group derivatized with a detectable label, R² is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof, R³ is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R⁶ is H, OH, or OR where R is a protecting group and x is an integer in the range of 1 and 8 inclusive.
Claim 16
Independent Claim
147 words
A method of detecting a nucleotide sequence in a sample containing single-stranded or double-stranded DNA or RNA, comprising the steps of: providing an analyte having a nucleotide sequence; providing a labeled polynucleotide probe having a nucleotide sequence complementary to said analyte sequence, said polynucleotide probe including at least one nucleotide having the structure wherein R¹ is a reactive group derivatized with a detectable label, R² is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof, R³ is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R⁶ is H, OH, or OR where R is a protecting group and x is an integer in the range of 1 and 8 inclusive; contacting said analyte with said labeled probe for a time sufficient for nucleic acid complexes to form; and detecting the presence of any such nucleic acid complexes.
Claim 17
Independent Claim
147 words
A method of detecting a nucleotide sequence in a sample containing single-stranded or double-stranded DNA or RNA, comprising the steps of: providing an analyte having a nucleotide sequence; providing a labeled polynucleotide probe having a nucleotide sequence complementary to said analyte sequence, said polynucleotide probe including at least one nucleotide having the structure wherein R¹ is a reactive group derivatized with a detectable label, R² is an optional linking moiety including an amide, thioether or disulfide linkage or a combination thereof, R³ is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R⁶ is H, OH, or OR where R is a protecting group and x is an integer in the range of 1 and 8 inclusive; contacting said analyte with said labeled probe for a time sufficient for nucleic acid complexes to form; and detecting the presence of any such nucleic acid complexes.
Claim 18
Dependent Claim
16 words
The method of claim 9, wherein said labeled polynucleotide probe is bound to a solid support.
References Cited (Backward Citations)
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