Patent US2238550A successfully refreshed from Google Patents.
US6465175B2
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US
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Oligonucleotide probes bearing quenchable fluorescent labels, and methods of use thereof
Assignee: Bayer Corp
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Filing: 1998-09-03
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Granted: 2002-10-15
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Expires: 2018-09-03
Abstract
Abstract Methods are provided for reducing background signals encountered in nucleic acid hybridization assays and other assays that involve hybridization of a labeled oligomer to its complement. The method is premised on the significant reduction of signal generation that occurs when a quenchable dye-labeled oligomer forms a hybrid complex. In addition, a method is provided for enhancing the detectable signal emitted from an amplification multimer hybridized to an oligomer probe to which a quenchable dye has been conjugated through a linker such that the emission from the dye is not quenched upon hybrid complex formation. Novel oligonucleotide probes are also provided that comprise an oligomer to which has been directly or indirectly through a linker a quenchable dye.
Inventors
- 👤 Thomas Horn
- 👤 Hartmut R. Schroeder
- 👤 Brian D. Warner
- 👤 Ellen Fiss
- 👤 Todd Sells
- 👤 Say-Jong Law
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 21:21
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818
Claims Analysis
Total Claims: 36
Claim 1
Independent Claim
138 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a nucleic acid sequence in the oligonucleotide of interest, and (ii) a label wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound that, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that, when the probe hybridizes to a complementary nucleic acid strand, is substantially quenched; (b) combining the first oligonucleotide probe with the sample suspected of containing the oligonucleotide of interest under hybridizing conditions to form a probe-oligonucleotide of interest hybrid complex, while monitoring emitted radiation from the first oligonucleotide probe; and (c) subsequent to step (b), correlating any change in emitted radiation with the presence or quantity of the oligonucleotide of interest.
Claim 2
Independent Claim
138 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a nucleic acid sequence in the oligonucleotide of interest, and (ii) a label wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound that, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that, when the probe hybridizes to a complementary nucleic acid strand, is substantially quenched; (b) combining the first oligonucleotide probe with the sample suspected of containing the oligonucleotide of interest under hybridizing conditions to form a probe-oligonucleotide of interest hybrid complex, while monitoring emitted radiation from the first oligonucleotide probe; and (c) subsequent to step (b), correlating any change in emitted radiation with the presence or quantity of the oligonucleotide of interest.
Claim 3
Dependent Claim
213 words
The method of claim 1 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryloxy, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 4
Dependent Claim
10 words
The method of claim 2 , wherein the dye is
Claim 5
Dependent Claim
15 words
The method of claim 1 , wherein the label is coupled directly to the probe.
Claim 6
Dependent Claim
15 words
The method of claim 1 , wherein the label is coupled indirectly to the probe.
Claim 7
Dependent Claim
32 words
The method of claim 5 , wherein the label is coupled to the probe through a linker substantially incapable of specifically hybridizing with a nucleic acid sequence in the oligonucleotide of interest.
Claim 8
Dependent Claim
14 words
The method of claim 6 , wherein the linker is a nucleic acid sequence.
Claim 9
Dependent Claim
198 words
In a solution phase sandwich hybridization assay for detecting a nucleic acid analyte in a sample, comprising (a) binding the analyte indirectly to a solid support, (b) labeling the analyte and (c) detecting the presence of label on the support, the improvement which comprises: incorporating a label probe system comprising (i) a label extender molecule having a first segment L-1 capable of hybridizing to a nucleic acid sequence in the analyte and a second segment L-2, (ii) an amplification multimer containing a nucleic acid sequence M-1 capable of hybridizing to nucleic acid sequence L-2; and a plurality of identical oligonucleotide subunits containing nucleic acid sequences M-2 capable of hybridizing to label probe, (iii) a single-stranded label probe comprising a nucleic acid sequence L-3 capable of hybridizing to M-2 and a quenchable fluorescent dye coupled to the probe through a linker incapable of specifically hybridizing with a nucleic acid sequence in the analyte, the label extender, the amplification multimer or a target nucleotide sequence, wherein the quenchable fluorescent dye provides detectable emission radiation that is substantially quenched upon hybridization of the label probe to a complementary nucleic acid strand and the quenchable fluorescent dye comprises a dipyrrometheneboron difluoride compound.
Claim 10
Dependent Claim
198 words
In a solution phase sandwich hybridization assay for detecting a nucleic acid analyte in a sample, comprising (a) binding the analyte indirectly to a solid support, (b) labeling the analyte and (c) detecting the presence of label on the support, the improvement which comprises: incorporating a label probe system comprising (i) a label extender molecule having a first segment L-1 capable of hybridizing to a nucleic acid sequence in the analyte and a second segment L-2, (ii) an amplification multimer containing a nucleic acid sequence M-1 capable of hybridizing to nucleic acid sequence L-2; and a plurality of identical oligonucleotide subunits containing nucleic acid sequences M-2 capable of hybridizing to label probe, (iii) a single-stranded label probe comprising a nucleic acid sequence L-3 capable of hybridizing to M-2 and a quenchable fluorescent dye coupled to the probe through a linker incapable of specifically hybridizing with a nucleic acid sequence in the analyte, the label extender, the amplification multimer or a target nucleotide sequence, wherein the quenchable fluorescent dye provides detectable emission radiation that is substantially quenched upon hybridization of the label probe to a complementary nucleic acid strand and the quenchable fluorescent dye comprises a dipyrrometheneboron difluoride compound.
Claim 11
Dependent Claim
213 words
The assay of claim 8 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryloxy, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 12
Dependent Claim
10 words
The assay of claim 9 , wherein the dye is
Claim 13
Dependent Claim
14 words
The assay of claim 8 , wherein the linker is a nucleic acid sequence.
Claim 14
Dependent Claim
99 words
An oligonucleotide probe comprising (i) a nucleic acid sequence complementary to a nucleic acid sequence in an oligonucleotide of interest, and (ii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound that, when the probe is in single-stranded, nonhybridized form, provides a detectable fluorescent signal that, when the probe hybridizes to a complementary nucleic acid strand, is substantially quenched wherein the label is coupled to the probe through a linker comprised of a nucleic acid sequence that is substantially incapable of specifically hybridizing with a nucleic acid sequence in the oligonucleotide of interest.
Claim 15
Dependent Claim
99 words
An oligonucleotide probe comprising (i) a nucleic acid sequence complementary to a nucleic acid sequence in an oligonucleotide of interest, and (ii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound that, when the probe is in single-stranded, nonhybridized form, provides a detectable fluorescent signal that, when the probe hybridizes to a complementary nucleic acid strand, is substantially quenched wherein the label is coupled to the probe through a linker comprised of a nucleic acid sequence that is substantially incapable of specifically hybridizing with a nucleic acid sequence in the oligonucleotide of interest.
Claim 16
Dependent Claim
214 words
The oligonucleotide probe of claim 12 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryloxy, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 17
Dependent Claim
11 words
The oligonucleotide probe of claim 13 , wherein the dye is
Claim 18
Dependent Claim
166 words
The method of claim 1 , wherein: the oligonucleotide of interest is a wild type gene and the nucleic acid sequence in the oligonucleotide probe is complementary to a target sequence in a wild type gene, wherein: (i) formation of a hybrid duplex between the oligonucleotide probe and the target sequence in the wild type gene creates a site cleavable by a specific restriction enzyme; (ii) formation of a hybrid duplex between the oligonucleotide probe and a point mutation-containing target sequence in a variant of the wild type gene does not create a site cleavable by the restriction enzyme; (iii) step (b) comprises incubating the oligonucleotide probe with a sample suspected of containing the variant of the wild type gene to form a variant-probe hybrid; adding to the sample an oligomer capable of hybridizing to the oligonucleotide probe to form a hybrid complex that does not contain the restriction enzyme cleavage site; and following the addition of the oligomer, adding the restriction enzyme to the sample.
Claim 19
Dependent Claim
213 words
The method of claim 15 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryloxy, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 20
Dependent Claim
10 words
The assay of claim 16 , wherein the dye is
Claim 21
Independent Claim
201 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a corresponding first nucleic acid sequence in the oligonucleotide of interest, and (ii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound bound directly or indirectly to the 5′ terminal nucleotide of the probe which, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that is substantially quenched when the probe hybridizes to a complementary nucleic acid strand; (a′) providing an oligonucleotide primer, wherein the primer is complementary to a second sequence in the oligonucleotide of interest and further wherein the corresponding first nucleic acid sequence of the oligonucleotide of interest is downstream from the second nucleic acid sequence in the oligonucleotide of interest; (b) incubating the first oligonucleotide probe and the primer with a sample containing the oligonucleotide of interest under conditions in which the first oligonucleotide probe and the primer hybridize to the oligonucleotide of interest; (b′) adding an enzyme to the sample under conditions wherein the enzyme has 3′ polymerase activity and 5′ nuclease activity, whereby the detectable emission radiation is increased.
Claim 22
Independent Claim
201 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a corresponding first nucleic acid sequence in the oligonucleotide of interest, and (ii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound bound directly or indirectly to the 5′ terminal nucleotide of the probe which, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that is substantially quenched when the probe hybridizes to a complementary nucleic acid strand; (a′) providing an oligonucleotide primer, wherein the primer is complementary to a second sequence in the oligonucleotide of interest and further wherein the corresponding first nucleic acid sequence of the oligonucleotide of interest is downstream from the second nucleic acid sequence in the oligonucleotide of interest; (b) incubating the first oligonucleotide probe and the primer with a sample containing the oligonucleotide of interest under conditions in which the first oligonucleotide probe and the primer hybridize to the oligonucleotide of interest; (b′) adding an enzyme to the sample under conditions wherein the enzyme has 3′ polymerase activity and 5′ nuclease activity, whereby the detectable emission radiation is increased.
Claim 23
Dependent Claim
213 words
The method of claim 18 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryloxy, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 24
Dependent Claim
10 words
The assay of claim 19 , wherein the dye is
Claim 25
Independent Claim
188 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a corresponding first nucleic acid sequence in the oligonucleotide of interest, and (ii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound which, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that is substantially quenched when the probe hybridizes to a complementary nucleic acid strand; (a′) providing a second oligonucleotide prove comprising a second nucleic acid sequence complementary to the corresponding first nucleic acid sequence in the oligonucleotide of interest; (b) combining the first oligonucleotide probe with the sample suspected of containing the oligonucleotide of interest under hybridizing conditions to form a first probe-oligonucleotide of interest hybrid complex, while monitoring emitted radiation from the first oligonucleotide probe; and combining the first probe-oligonucleotide of interest hybrid complex formed in step (b) with the second oligonucleotide probe under conditions that the first oligonucleotide probe will be displaced from the first probe-oligonucleotide of interest complex, while monitoring emitted radiation from the first oligonucleotide probe.
Claim 26
Independent Claim
188 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a corresponding first nucleic acid sequence in the oligonucleotide of interest, and (ii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound which, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that is substantially quenched when the probe hybridizes to a complementary nucleic acid strand; (a′) providing a second oligonucleotide prove comprising a second nucleic acid sequence complementary to the corresponding first nucleic acid sequence in the oligonucleotide of interest; (b) combining the first oligonucleotide probe with the sample suspected of containing the oligonucleotide of interest under hybridizing conditions to form a first probe-oligonucleotide of interest hybrid complex, while monitoring emitted radiation from the first oligonucleotide probe; and combining the first probe-oligonucleotide of interest hybrid complex formed in step (b) with the second oligonucleotide probe under conditions that the first oligonucleotide probe will be displaced from the first probe-oligonucleotide of interest complex, while monitoring emitted radiation from the first oligonucleotide probe.
Claim 27
Dependent Claim
213 words
The method of claim 21 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryloxy, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 28
Dependent Claim
10 words
The method of claim 22 , wherein the dye is
Claim 29
Independent Claim
172 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing an oligonucleotide probe that comprises a single-stranded nucleic acid molecule comprising first and second complementary nucleic acid sequences flanking a third nucleic acid sequence that forms a loop structure when the first and second complementary nucleic acid sequences hybridizes with one another to form a stem structure, wherein the third nucleic acid in the loop structure comprises a nucleic acid sequence complementary to a nucleic acid sequence in the oligonucleotide of interest, and wherein the probe is singly labeled with a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound that provides detectable emission radiation, and further wherein the label is such that when the first and second nucleic sequences are hybridized to form the stem structure, the emitted radiation is substantially quenched, and when the third nucleic acid sequence is hybridized to the oligonucleotide of interest and the first and second nucleic acid sequences are in nonhybridized form, provides detectable emission radiation.
Claim 30
Independent Claim
172 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing an oligonucleotide probe that comprises a single-stranded nucleic acid molecule comprising first and second complementary nucleic acid sequences flanking a third nucleic acid sequence that forms a loop structure when the first and second complementary nucleic acid sequences hybridizes with one another to form a stem structure, wherein the third nucleic acid in the loop structure comprises a nucleic acid sequence complementary to a nucleic acid sequence in the oligonucleotide of interest, and wherein the probe is singly labeled with a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound that provides detectable emission radiation, and further wherein the label is such that when the first and second nucleic sequences are hybridized to form the stem structure, the emitted radiation is substantially quenched, and when the third nucleic acid sequence is hybridized to the oligonucleotide of interest and the first and second nucleic acid sequences are in nonhybridized form, provides detectable emission radiation.
Claim 31
Dependent Claim
213 words
The method of claim 24 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryl, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 32
Dependent Claim
10 words
The method of claim 25 , wherein the dye is
Claim 33
Independent Claim
286 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a corresponding first nucleic acid sequence in the oligonucleotide of interest, (ii) a second nucleic acid sequence complementary to a third nucleic acid sequence in a second probe, and (iii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound which, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that is substantially quenched when the probe hybridizes to a complementary nucleic acid strand; wherein the corresponding first nucleic acid sequence in the oligonucleotide of interest is adjacent to a second nucleic acid sequence in the oligonucleotide of interest, wherein the second nucleic acid sequence in the oligonucleotide of interest is either a wild type nucleic acid sequence or a nucleic acid sequence containing a point mutation, and wherein the method further comprises (a′) providing a second oligonucleotide probe, having third and fourth nucleic acid sequences, wherein the third nucleic acid sequence is complementary to either the wild type or the mutant second nucleic acid sequence in the oligonucleotide of interest and the fourth nucleic acid sequence is complementary to the second nucleic acid sequence in the first oligonucleotide probe; (b) combining the first oligonucleotide probe with the sample suspected of containing the oligonucleotide of interest under hybridizing conditions to form a first probe-oligonucleotide of interest hybridization complex, while monitoring emitted radiation from the first oligonucleotide probe, (b′) incubating the second oligonucleotide probe with the first probe-oligonucleotide complex; and (c) following step (b′), correlating any change in emitted radiation with the presence of the wild type sequence or the mutant sequence.
Claim 34
Independent Claim
286 words
A method for detecting an oligonucleotide of interest in a sample, comprising: (a) providing a first oligonucleotide probe comprising (i) a first nucleic acid sequence complementary to a corresponding first nucleic acid sequence in the oligonucleotide of interest, (ii) a second nucleic acid sequence complementary to a third nucleic acid sequence in a second probe, and (iii) a label, wherein the label is a fluorescent dye that comprises a dipyrrometheneboron difluoride compound which, when the probe is in single-stranded, nonhybridized form, provides detectable emission radiation that is substantially quenched when the probe hybridizes to a complementary nucleic acid strand; wherein the corresponding first nucleic acid sequence in the oligonucleotide of interest is adjacent to a second nucleic acid sequence in the oligonucleotide of interest, wherein the second nucleic acid sequence in the oligonucleotide of interest is either a wild type nucleic acid sequence or a nucleic acid sequence containing a point mutation, and wherein the method further comprises (a′) providing a second oligonucleotide probe, having third and fourth nucleic acid sequences, wherein the third nucleic acid sequence is complementary to either the wild type or the mutant second nucleic acid sequence in the oligonucleotide of interest and the fourth nucleic acid sequence is complementary to the second nucleic acid sequence in the first oligonucleotide probe; (b) combining the first oligonucleotide probe with the sample suspected of containing the oligonucleotide of interest under hybridizing conditions to form a first probe-oligonucleotide of interest hybridization complex, while monitoring emitted radiation from the first oligonucleotide probe, (b′) incubating the second oligonucleotide probe with the first probe-oligonucleotide complex; and (c) following step (b′), correlating any change in emitted radiation with the presence of the wild type sequence or the mutant sequence.
Claim 35
Dependent Claim
213 words
The method of claim 27 , wherein the dye has the chemical structure wherein R 1 through R 7 are independently selected from the group consisting of: hydrogen; halogen; alkyl; carboxyalkyl; acyl; aryl; arylalkyl; sulfonyl; formyl; substituted ethenyl having the formula —CX═CY—Z wherein X, Y and Z are independently halogen, C 1 -C 10 alkyl, cyano, ester, amide, ethenyl, polyethenyl, aryl or heteroaryl; and —L—G, wherein L is a linking moiety and G is a reactive group enabling binding of the dye to an amino or carboxylic acid moiety; or wherein (a) either R 1 and R 2 , or R 2 and R 3 , and/or (b) either R 4 and R 5 , or R 5 and R 6 , together form a benzene ring substituted with 0 to 4 substituents selected from the group consisting of hydrogen, halogen, cyano, sulfonyl, sulfonate, carboxyl, carboxylate, alkyl, perfluoroalkyl, alkoxy, alkylthio, nitro, amino, monoalkylamino, dialkylamino, alkylamido, aryl, aryloxy, heteroaryl, heteroaryloxy, arylamino, heteroarylamino, or arylamido, or wherein two ortho substituents are linked to form an additional such aromatic ring, with the proviso that at least one substituent within the group of R 1 through R 7 is heteroaryl, and at least a second substituent within the group of R 1 through R 7 is —L—G.
Claim 36
Dependent Claim
10 words
The method of claim 28 , wherein the dye is
References Cited (Backward Citations)
US4820630A
Cited
US4774339A
Cited
US5124246A
Cited
US5248782A
Cited
US5187288A
Cited
US5274113A
Cited
WO1993009185A1
Cited
US5451663A
Cited
US5433896A
Cited
WO1996015270A1
Cited
EP0713921A2
Cited
US5616464A
Cited
WO1997000967A1
Cited
EP0808829A1
Cited
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