Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
First Claim
1. 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 and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.
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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.
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Citations
91 Claims
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1. A method for hybridizing nucleic acids, comprising the steps of:
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(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 and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 21, 50, 51, 68)
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10. A method for primer extension, comprising the steps of:
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(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 and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (11, 12, 69)
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13. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:
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(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 further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels, 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.
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14. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:
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(a) providing a MGB-oligonucleotide conjugate of defined sequence and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels, (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. - View Dependent Claims (52, 53, 70)
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15. 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:
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(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 and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (16, 54, 55, 71)
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- 17. A method for primer-dependent nucleotide sequence analysis wherein a MGB-oligonucleotide conjugate further comprising multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels is used as a primer.
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18. A method for determining the sequence of a polynucleotide comprising the steps of:
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(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 and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (58, 59, 73)
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19. A method for examining gene expression comprising the steps of:
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(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 and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (60, 61, 74)
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20. A method for identifying one or more mutations in a gene of interest comprising the steps of:
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(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 and further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (22, 62, 63, 75)
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23. A method for cDNA synthesis, wherein a MGB-oligonucleotide conjugate further comprising multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels is used as a primer.
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24. 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:
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(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; (c) wherein said MGB-oligonucleotide conjugate comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 64, 65, 76)
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35. 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:
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(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; (c) wherein the MGB-oligonucleotide conjugate further comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 66, 67, 77)
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78. A method for identifying one or more nucleotide polymorphisms in a polynucleotide sample, the method comprising:
- providing an array of support-bound oligonucleotide probes of different sequences;
incubating a polynucleotide sample with said array under hybridization conditions;
incubating a plurality of different minor groove binder-oligonucleotide conjugate probes with said polynucleotide sample with said array under hybridization conditions to form one or more minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids, wherein said minor groove binder comprises multiple fluorescent labels wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels; and
detecting the presence of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids on said array. - View Dependent Claims (79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91)
- providing an array of support-bound oligonucleotide probes of different sequences;
Specification