US6312894B1
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Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
Assignee: DRUG ROYALTY TRUST 9
•
Filing: 1998-04-03
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Granted: 2001-11-06
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Expires: 2018-04-03
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
2026-10-29 09:55
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: 92
Claim 1
Independent Claim
97 words
A method for hybridizing nucleic acids, comprising the steps of: (a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a minor groove binder (MGB)-oligonucleotide conjugate; 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 2
Independent Claim
97 words
A method for hybridizing nucleic acids, comprising the steps of: (a) providing a first nucleic acid and a second nucleic acid, (b) incubating the nucleic acids under hybridization conditions, and (c) identifying hybridized nucleic acids; wherein at least one of the nucleic acids comprises a minor groove binder (MGB)-oligonucleotide conjugate; 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 3
Dependent Claim
18 words
The method according to claim 1 , wherein the MGB-oligonucleotide conjugate is a probe comprising a detectable label.
Claim 4
Dependent Claim
15 words
The method according to claim 2 , wherein the detectable label is a fluorescent label.
Claim 5
Dependent Claim
23 words
The method according to claim 3 , wherein the MGB-oligonucleotide conjugate further comprises an agent that quenches the emission of the fluorescent label.
Claim 6
Dependent Claim
31 words
The method according to claim 4 , further comprising the step of altering the spatial relationship between the fluorescent label and the agent which quenches the emission of the fluorescent label.
Claim 7
Dependent Claim
23 words
The method according to claim 2 , wherein the method further comprises the step of releasing label from the probe subsequent to hybridization.
Claim 8
Dependent Claim
23 words
The method according to claim 5 , wherein the method further comprises the step of releasing label from the probe subsequent to hybridization.
Claim 9
Dependent Claim
18 words
The method according to claim 6 , wherein release of label occurs as a result of exonuclease hydrolysis.
Claim 10
Dependent Claim
18 words
The method according to claim 7 , wherein release of label occurs as a result of exonuclease hydrolysis.
Claim 11
Dependent Claim
14 words
The method according to claim 3 , wherein more than one probe is used.
Claim 12
Dependent Claim
15 words
The method according to claim 10 , wherein a first and second probe is used.
Claim 13
Dependent Claim
41 words
The method according to claim 11 , wherein the first probe comprises a fluorescence donor and the second probe comprises a fluorescence acceptor, and further wherein the emission wavelengths of the fluorescence donor overlap the absorption wavelengths of the fluorescence acceptor.
Claim 14
Dependent Claim
18 words
The method according to claim 1 wherein the MGB-oligonucleotide conjugate is a primer comprising a free 3′-hydroxyl group.
Claim 15
Dependent Claim
19 words
The method according to claim 13 , further comprising the step of extending the primer with a polymerizing enzyme.
Claim 16
Dependent Claim
15 words
The method according to claim 14 , wherein the polymerizing enzyme is a thermostable enzyme.
Claim 17
Dependent Claim
18 words
The method according to claim 13 , wherein the MGB-oligonucleotide conjugate is a primer in an amplification reaction.
Claim 18
Dependent Claim
16 words
The method according to claim 16 , wherein the amplification reaction is a polymerase chain reaction.
Claim 19
Independent Claim
68 words
A method for primer extension, comprising the steps of: (a) providing a sample containing a target sequence, (b) providing one or more oligonucleotide primers complementary to regions of the target sequence, (c) providing a polymerizing enzyme and nucleotide substrates, and (d) incubating the sample, the oligonucleotide primers, the enzyme and the substrates under conditions favorable for polymerization; wherein at least one of the primers comprises a MGB-oligonucleotide conjugate.
Claim 20
Independent Claim
68 words
A method for primer extension, comprising the steps of: (a) providing a sample containing a target sequence, (b) providing one or more oligonucleotide primers complementary to regions of the target sequence, (c) providing a polymerizing enzyme and nucleotide substrates, and (d) incubating the sample, the oligonucleotide primers, the enzyme and the substrates under conditions favorable for polymerization; wherein at least one of the primers comprises a MGB-oligonucleotide conjugate.
Claim 21
Dependent Claim
14 words
The method according to claim 18 , wherein the method is an amplification reaction.
Claim 22
Dependent Claim
16 words
The method according to claim 19 , wherein the amplification reaction is a polymerase chain reaction.
Claim 23
Independent Claim
99 words
A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps: (a) providing a polynucleotide comprising a target sequence, (b) providing at least two MGB-oligonucleotide conjugates, wherein one of the at least two MGB-oligonucleotide conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the MGB-oligonucleotide conjugates has a single-nucleotide mismatch with the target sequence; (c) separately incubating each of the MGB-oligonucleotide conjugates with the polynucleotide under hybridization conditions; and (d) determining the hybridization strength between each of the MGB-oligonucleotide conjugates and the polynucleotide.
Claim 24
Independent Claim
99 words
A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps: (a) providing a polynucleotide comprising a target sequence, (b) providing at least two MGB-oligonucleotide conjugates, wherein one of the at least two MGB-oligonucleotide conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the MGB-oligonucleotide conjugates has a single-nucleotide mismatch with the target sequence; (c) separately incubating each of the MGB-oligonucleotide conjugates with the polynucleotide under hybridization conditions; and (d) determining the hybridization strength between each of the MGB-oligonucleotide conjugates and the polynucleotide.
Claim 25
Independent Claim
105 words
A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps: (a) providing a MGB-oligonucleotide conjugate of defined sequence, (b) providing at least two polynucleotides, each of which comprises a target sequence, wherein one of the polynucleotides has a target sequence that is perfectly complementary to the MGB-oligonucleotide conjugate and at least one other of the polynucleotides has a target sequence having a single-nucleotide mismatch with the MGB-oligonucleotide conjugate; (c) separately incubating each of the polynucleotides with the MGB-oligonucleotide conjugate under hybridization conditions; and (d) determining the hybridization strength between each of the polynucleotides and the MGB-oligonucleotide conjugate.
Claim 26
Independent Claim
105 words
A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps: (a) providing a MGB-oligonucleotide conjugate of defined sequence, (b) providing at least two polynucleotides, each of which comprises a target sequence, wherein one of the polynucleotides has a target sequence that is perfectly complementary to the MGB-oligonucleotide conjugate and at least one other of the polynucleotides has a target sequence having a single-nucleotide mismatch with the MGB-oligonucleotide conjugate; (c) separately incubating each of the polynucleotides with the MGB-oligonucleotide conjugate under hybridization conditions; and (d) determining the hybridization strength between each of the polynucleotides and the MGB-oligonucleotide conjugate.
Claim 27
Independent Claim
78 words
A method of ligating two or more oligonucleotides, each of which is hybridized to adjacent sites on a target nucleic acid, comprising the steps of: (a) providing a sample containing a target sequence, (b) providing at least two oligonucleotides which are complementary to adjacent sites on the target sequence, (c) incubating the sample and the oligonucleotides under conditions favorable for ligation, and (d) identifying ligated nucleic acids; wherein at least one of the oligonucleotides comprises a MGB-oligonucleotide conjugate.
Claim 28
Independent Claim
78 words
A method of ligating two or more oligonucleotides, each of which is hybridized to adjacent sites on a target nucleic acid, comprising the steps of: (a) providing a sample containing a target sequence, (b) providing at least two oligonucleotides which are complementary to adjacent sites on the target sequence, (c) incubating the sample and the oligonucleotides under conditions favorable for ligation, and (d) identifying ligated nucleic acids; wherein at least one of the oligonucleotides comprises a MGB-oligonucleotide conjugate.
Claim 29
Independent Claim
16 words
A method for primer-dependent nucleotide sequence analysis wherein a MGB-oligonucleotide conjugate is used as a primer.
Claim 30
Independent Claim
16 words
A method for primer-dependent nucleotide sequence analysis wherein a MGB-oligonucleotide conjugate is used as a primer.
Claim 31
Independent Claim
60 words
A method for determining the sequence of a polynucleotide comprising the steps of: (a) providing an array of oligonucleotide probes of different sequences, (b) incubating the polynucleotide and the array under hybridization conditions, and (c) determining to which of the oligonucleotide probes in the array the polynucleotide hybridizes; wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.
Claim 32
Independent Claim
60 words
A method for determining the sequence of a polynucleotide comprising the steps of: (a) providing an array of oligonucleotide probes of different sequences, (b) incubating the polynucleotide and the array under hybridization conditions, and (c) determining to which of the oligonucleotide probes in the array the polynucleotide hybridizes; wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.
Claim 33
Independent Claim
59 words
A method for examining gene expression comprising the steps of: (a) providing an array of oligonucleotide probes of different sequences, (b) incubating a population of polynucleotides with the array under hybridization conditions, and (c) determining to which of the oligonucleotide probes in the array the population hybridizes; wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.
Claim 34
Independent Claim
59 words
A method for examining gene expression comprising the steps of: (a) providing an array of oligonucleotide probes of different sequences, (b) incubating a population of polynucleotides with the array under hybridization conditions, and (c) determining to which of the oligonucleotide probes in the array the population hybridizes; wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.
Claim 35
Independent Claim
65 words
A method for identifying one or more mutations in a gene of interest comprising the steps of: (a) providing an array of oligonucleotide probes of different sequences, (b) incubating a polynucleotide sample with the array under hybridization conditions, and (c) determining to which of the oligonucleotide probes in the array the polynucleotide hybridizes; wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.
Claim 36
Independent Claim
65 words
A method for identifying one or more mutations in a gene of interest comprising the steps of: (a) providing an array of oligonucleotide probes of different sequences, (b) incubating a polynucleotide sample with the array under hybridization conditions, and (c) determining to which of the oligonucleotide probes in the array the polynucleotide hybridizes; wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.
Claim 37
Dependent Claim
23 words
The method of hybridization according to claim 1 , wherein the melting temperature of the hybridized nucleic acid is independent of base composition.
Claim 38
Dependent Claim
28 words
The method of hybridization according to claim 27 wherein the melting temperature of the hybridized nucleic acid is dependent primarily on the length of the hybridized nucleic acid.
Claim 39
Independent Claim
14 words
A method for cDNA synthesis, wherein a MGB-oligonucleotide conjugate is used as a primer.
Claim 40
Independent Claim
14 words
A method for cDNA synthesis, wherein a MGB-oligonucleotide conjugate is used as a primer.
Claim 41
Independent Claim
115 words
A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence, the method comprising: (a) contacting the mixture of polynucleotides with a Minor Groove Binder (MGB)-oligonucleotide conjugate, wherein the MGB-oligonucleotide conjugate forms a stable hybrid only with said target sequence that is perfectly complementary to the oligonucleotide and wherein the MGB-oligonucleotide conjugate does not form a stable hybrid with any of the related sequences; and (b) measuring hybrid formation, whereby hybrid formation is indicative of the presence of said target sequence.
Claim 42
Independent Claim
115 words
A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence, the method comprising: (a) contacting the mixture of polynucleotides with a Minor Groove Binder (MGB)-oligonucleotide conjugate, wherein the MGB-oligonucleotide conjugate forms a stable hybrid only with said target sequence that is perfectly complementary to the oligonucleotide and wherein the MGB-oligonucleotide conjugate does not form a stable hybrid with any of the related sequences; and (b) measuring hybrid formation, whereby hybrid formation is indicative of the presence of said target sequence.
Claim 43
Dependent Claim
27 words
The method according to claim 31 , wherein an oligonucleotide that is not conjugated to a MGB does not form a stable hybrid with the target sequence.
Claim 44
Dependent Claim
16 words
The method according to claim 32 , wherein the related sequence is a single nucleotide mismatch.
Claim 45
Dependent Claim
11 words
The method according to claim 33 wherein the polynucleotide is DNA.
Claim 46
Dependent Claim
13 words
The method according to claim 33 wherein the polynucleotide comprises a coding region.
Claim 47
Dependent Claim
13 words
The method according to claim 33 wherein the polynucleotide comprises a non-coding region.
Claim 48
Dependent Claim
13 words
The method according to claim 33 wherein the polynucleotide comprises a mutant sequence.
Claim 49
Dependent Claim
17 words
The method according to claim 33 wherein the target sequence comprises an allele of a polymorphic sequence.
Claim 50
Dependent Claim
20 words
The method according to claim 38 wherein the other polynucleotides in the mixture comprise different alleles of the polymorpbic sequence.
Claim 51
Dependent Claim
16 words
The method according to claim 31 wherein the MGB-oligonucleotide conjugate is attached to a solid support.
Claim 52
Dependent Claim
18 words
The method according to claim 31 wherein the polynucleotides in said mixture are attached to a solid support.
Claim 53
Independent Claim
86 words
A method for detecting one or more sequences related to a target sequence, wherein the one or more related sequences are present in a sample of polynucleotides, the method comprising: (a) contacting the sample with a Minor Groove Binder (MGB)-oligonucleotide conjugate, wherein the oligonucleotide has a sequence that is complementary to the target sequence, and wherein the MGB-oligonucleotide conjugate forms stable hybrids with the related sequences; and (b) measuring hybrid formation, wherein hybrid formation is indicative of the presence of the one or more related sequences.
Claim 54
Independent Claim
86 words
A method for detecting one or more sequences related to a target sequence, wherein the one or more related sequences are present in a sample of polynucleotides, the method comprising: (a) contacting the sample with a Minor Groove Binder (MGB)-oligonucleotide conjugate, wherein the oligonucleotide has a sequence that is complementary to the target sequence, and wherein the MGB-oligonucleotide conjugate forms stable hybrids with the related sequences; and (b) measuring hybrid formation, wherein hybrid formation is indicative of the presence of the one or more related sequences.
Claim 55
Dependent Claim
26 words
The method according to claim 42 , wherein an oligonucleotide that is not conjugated to a MGB does not form stable hybrids with the related sequence.
Claim 56
Dependent Claim
22 words
The method according to claim 43 , wherein the related sequence has a single nucleotide mismatch with respect to the target sequence.
Claim 57
Dependent Claim
11 words
The method according to claim 42 wherein the polynucleotides are DNA.
Claim 58
Dependent Claim
15 words
The method according to claim 42 wherein the polynucleotides comprise one or more coding regions.
Claim 59
Dependent Claim
15 words
The method according to claim 42 wherein the polynucleotides comprise one or more non-coding regions.
Claim 60
Dependent Claim
15 words
The method according to claim 42 wherein the polynucleotides comprise one or more mutant sequences.
Claim 61
Dependent Claim
19 words
The method according to claim 48 wherein the polynucleotides comprise a plurality of different mutations in the same gene.
Claim 62
Dependent Claim
13 words
The method according to claim 48 wherein the gene is a viral gene.
Claim 63
Dependent Claim
13 words
The method according to claim 50 wherein the virus has an RNA genome.
Claim 64
Dependent Claim
14 words
The method according to claim 51 wherein the virus is a human immunodeficiency virus.
Claim 65
Dependent Claim
17 words
The method according to claim 42 wherein the target sequence comprises an allele of a polymorphic sequence.
Claim 66
Dependent Claim
19 words
The method according to claim 53 wherein the polynucleotides in the mixture comprise different alleles of the polymorphic sequence.
Claim 67
Dependent Claim
16 words
The method according to claim 42 wherein the MGB-oligonucleotide conjugate is attached to a solid support.
Claim 68
Dependent Claim
18 words
The method according to claim 42 wherein the polynucleotides in said mixture are attached to a solid support.
Claim 69
Dependent Claim
20 words
The method according to claim 1 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 70
Dependent Claim
20 words
The method according to claim 18 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 71
Dependent Claim
20 words
The method according to claim 22 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 72
Dependent Claim
20 words
The method according to claim 23 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 73
Dependent Claim
20 words
The probe according to claim 23 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 74
Dependent Claim
20 words
The primer according to claim 23 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 75
Dependent Claim
20 words
The method according to claim 24 , therein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 76
Dependent Claim
20 words
The method according to claim 25 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 77
Dependent Claim
20 words
The method according to claim 26 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 78
Dependent Claim
20 words
The method according to claim 27 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 79
Dependent Claim
20 words
The method according to claim 31 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 80
Dependent Claim
20 words
The method according to claim 42 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate is a peptide nucleic acid.
Claim 81
Dependent Claim
19 words
The method according to claim 1 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 82
Dependent Claim
19 words
The method according to claim 18 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 83
Dependent Claim
19 words
The method according to claim 22 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 84
Dependent Claim
19 words
The method according to claim 23 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 85
Dependent Claim
19 words
The probe according to claim 23 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 86
Dependent Claim
19 words
The primer according to claim 23 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 87
Dependent Claim
19 words
The method according to claim 24 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 88
Dependent Claim
19 words
The method according to claim 25 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 89
Dependent Claim
19 words
The method according to claim 26 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 90
Dependent Claim
19 words
The method according to claim 27 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 91
Dependent Claim
19 words
The method according to claim 31 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
Claim 92
Dependent Claim
19 words
The method according to claim 42 , wherein the oligonucleotide portion of the MGB-oligonucleotide conjugate comprises a peptide linkage.
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