US6492346B1
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US
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Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
Assignee: DRUG ROYALTY TRUST 9
•
Filing: 2000-08-16
•
Granted: 2002-12-10
•
Expires: 2020-08-16
Abstract
Abstract Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI3 group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.
Inventors
- 👤 Joel Hedgpeth
- 👤 Irina A. Afonina
- 👤 Igor V. Kutyavin
- 👤 Eugeny A. Lukhtanov
- 👤 Evgeniy S. Belousov
- 👤 Rich B. Meyer, Jr.
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 09:14
CPC Classifications
C; C07; C07H; C07H21/00; C07H19/00; C07H19/02; C07H19/04; C07H19/23; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813
Claims Analysis
Total Claims: 51
Claim 1
Independent Claim
21 words
An oligonucleotide probe comprising a 5′-end, a 3′-end and one or more detectable labels, wherein the probe is a MGB-oligonucleotide conjugate.
Claim 2
Dependent Claim
21 words
An oligonucleotide probe comprising a 5′-end, a 3′-end and one or more detectable labels, wherein the probe is a MGB-oligonucleotide conjugate.
Claim 3
Dependent Claim
14 words
The probe according to claim 1 wherein the detectable label is a fluorescent label.
Claim 4
Dependent Claim
12 words
The probe according to claim 2 wherein the label is a fluorescein.
Claim 5
Dependent Claim
12 words
The probe according to claim 2 wherein the label is a cyanine.
Claim 6
Dependent Claim
12 words
The probe according to claim 2 wherein the label is a rhodamine.
Claim 7
Dependent Claim
16 words
The probe according to claim 1 wherein the MGB is located at the oligonucleotide 5′ end.
Claim 8
Dependent Claim
16 words
The probe according to claim 1 wherein the MGB is located at the oligonucleotide 3′ end.
Claim 9
Dependent Claim
16 words
The probe according to claim 1 wherein the label is located at the oligonucleotide 5′ end.
Claim 10
Dependent Claim
16 words
The probe according to claim 1 wherein the label is located at the oligonucleotide 3′ end.
Claim 11
Dependent Claim
31 words
The probe according to claim 1 wherein the MGB is selected from the group consisting of a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC 5 ).
Claim 12
Dependent Claim
10 words
The probe according to claim 2 comprising multiple fluorescent labels.
Claim 13
Dependent Claim
26 words
The probe according to claim 11 wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.
Claim 14
Dependent Claim
20 words
The probe according to claim 2 further comprising a quenching agent which quenches the fluorescence emission of the fluorescent label.
Claim 15
Dependent Claim
13 words
The probe according to claim 13 wherein the fluorescent label is a fluorescein.
Claim 16
Dependent Claim
12 words
The probe according to claim 14 wherein the quenching agent is tetramethylrhodamine.
Claim 17
Dependent Claim
26 words
A MGB-oligonucleotide conjugate for use as a primer comprising a 5′ end and a 3′ end, wherein the 3′ end is extendible by a polymerizing enzyme.
Claim 18
Dependent Claim
26 words
A MGB-oligonucleotide conjugate for use as a primer comprising a 5′ end and a 3′ end, wherein the 3′ end is extendible by a polymerizing enzyme.
Claim 19
Dependent Claim
17 words
The primer according to claim 16 , wherein the 3′ end comprises a free 3′ hydroxyl group.
Claim 20
Dependent Claim
17 words
The primer according to claim 16 , wherein the primer has been extended by a polymerizing enzyme.
Claim 21
Dependent Claim
20 words
The primer according to claim 18 , wherein the primer has been extended during the synthesis of a cDNA molecule.
Claim 22
Dependent Claim
17 words
The primer according to claim 18 , wherein the primer has been extended during an amplification reaction.
Claim 23
Dependent Claim
13 words
The probe according to claim 1 , wherein inosine is substituted for guanosine.
Claim 24
Dependent Claim
13 words
The probe according to claim 1 , wherein 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted for guanine.
Claim 25
Dependent Claim
13 words
The probe according to claim 1 , wherein 4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.
Claim 26
Dependent Claim
13 words
The probe according to claim 1 , wherein 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted for adenine.
Claim 27
Dependent Claim
13 words
The primer according to claim 16 , wherein inosine is substituted for guanosine.
Claim 28
Dependent Claim
13 words
The primer according to claim 16 , wherein 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted for guanine.
Claim 29
Dependent Claim
13 words
The primer according to claim 16 , wherein 4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.
Claim 30
Dependent Claim
13 words
The primer according to claim 16 , wherein 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted for adenine.
Claim 31
Dependent Claim
10 words
A composition comprising the probe according to claim 1 .
Claim 32
Dependent Claim
10 words
A composition comprising the primer according to claim 16 .
Claim 33
Dependent Claim
17 words
The probe according to claim 1 , wherein the probe is less than 20 nucleotides in length.
Claim 34
Dependent Claim
17 words
The primer according to claim 16 , wherein the primer is less than 20 nucleotides in length.
Claim 35
Dependent Claim
14 words
A kit for amplification comprising one or more primers according to claim 16 .
Claim 36
Dependent Claim
15 words
A kit for hybridization analysis comprising one or more probes according to claim 1 .
Claim 37
Dependent Claim
19 words
A kit for use in a hydrolyzable probe assay comprising one or more probes according to claim 1 .
Claim 38
Dependent Claim
19 words
A kit for use in single nucleotide mismatch detection comprising one or more probes according to claim 1 .
Claim 39
Dependent Claim
19 words
A kit for use in single nucleotide mismatch detection comprising one or more primers according to claim 16 .
Claim 40
Dependent Claim
19 words
A kit for use in nucleotide sequence analysis comprising one or more of the probes of claim 1 .
Claim 41
Dependent Claim
19 words
A kit for use in nucleotide sequence analysis comprising one or more of the primers of claim 16 .
Claim 42
Dependent Claim
26 words
A MGB-oligonucleotide conjugate that hybridizes to a target nucleic acid to form a hybrid, wherein the melting temperature of the hybrid is independent of base composition.
Claim 43
Dependent Claim
26 words
A MGB-oligonucleotide conjugate that hybridizes to a target nucleic acid to form a hybrid, wherein the melting temperature of the hybrid is independent of base composition.
Claim 44
Dependent Claim
54 words
The probe according to claim 1 , wherein the minor groove binder is a molecule having a molecular weight of approximately 150 to approximately 2,000 Daltons that binds in a non-intercalating manner into the minor groove of a double-stranded nucleic acid with an association constant of greater than approximately 10 3 M −1 .
Claim 45
Dependent Claim
44 words
The probe according to claim 1 , wherein said MGB is a radical of a compound selected from the group consisting of netropsin, distamycin and lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, diarylamidines, pentamidine, stilbamidine, berenil, CC-1065, pyrroloindole, indole polypeptides, Hoechst 33258, and 4′-6-diamidino-2-phenylindole.
Claim 46
Dependent Claim
13 words
The probe according to claim 1 , wherein said MGB is crescent shaped.
Claim 47
Dependent Claim
54 words
The MGB-oligonucleotide conjugate of claim 40 , wherein the minor groove binder is a molecule having a molecular weight of approximately 150 to approximately 2,000 Daltons that binds in a non-intercalating manner into the minor groove of a double-stranded nucleic acid with an association constant of greater than approximately 10 3 M −1 .
Claim 48
Dependent Claim
45 words
The MGB-oligonucleotide conjugate of claim 40 , wherein said MGB is a radical of a compound selected from the group consisting of netropsin, distamycin and lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, a diarylamidine, pentamidine, stilbamidine berenil, CC-1065, pyrroloindole, indole polypeptides, Hoechst 33258, and 4′-6-diamidino-2-phenylindole.
Claim 49
Dependent Claim
13 words
The MGB-oligonucleotide conjugate of claim 40 , wherein said MGB is crescent shaped.
Claim 50
Dependent Claim
45 words
The MGB-oligonucleotide conjugate of claim 40 , wherein said MGB is selected from the group consisting of a dimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 2 ), a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ), tetramer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 4 ) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC 5 ).
Claim 51
Dependent Claim
45 words
The MGB-oligonucleotide conjugate of claim 40 , wherein said MGB is an analog of a compound selected from the group consisting of netropsin, distamycin and lexitropsin, mithramycin, chromomycin A3, olivomycin, anthramycin, sibiromycin, a diarylamidine, pentamidine, stilbamidine berenil, CC-1065, pyrroloindole, indole polypeptides, Hoechst 33258, and 4′-6-diamidino-2-phenylindole.
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