US6472153B1
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Hybridization-triggered fluorescent detection of nucleic acids
Assignee: Epoch Biosciences Inc
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Filing: 1999-10-26
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Granted: 2002-10-29
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Expires: 2019-10-26
Abstract
Abstract Compositions and methods for fluorescent detection of nucleic acids are provided. The compositions can be detected by fluorescence when hybridized to a nucleic acid containing a target sequence, but are non-fluorescent in the non-hybridized state. Alternatively, the fluorescence properties of the compositions change in a detectable manner upon hybridization to a nucleic acid containing a target sequence. Methods for synthesis and methods of use of the compositions are also provided.
Inventors
- 👤 Robert O. Dempcy
- 👤 Irina Aleksandrovna Afonina
- 👤 Nicolaas M. J. Vermeulen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 12:08
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 48
Claim 1
Independent Claim
182 words
A composition comprising: (a) an oligonucleotide (ODN), wherein the oligonucleotide comprises a plurality of nucleotides, a 3′ end and a 5′ end; (b) a minor groove binder (MGB), wherein the minor groove binder is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which molecule binds in a non-intercalating manner into the minor groove of non-single-stranded DNA, RNA or hybrids thereof with an association constant greater than approximately 10 3 M −1 ; and (c) a latent fluorophore (LF); wherein the latent fluorophore is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which binds in an intercalating manner into non-single-stranded DNA, RNA or hybrids thereof, or alternatively lies in the minor groove, or in another manner is oriented to the DNA molecule by the minor groove binder moiety so that it becomes fluorescent or its fluorescence properties are changed in a detectable way; wherein the composition is a covalently bound oligonucleotide, minor groove binder, latent fluorophore combination having a first linking group and a second linking group.
Claim 2
Dependent Claim
182 words
A composition comprising: (a) an oligonucleotide (ODN), wherein the oligonucleotide comprises a plurality of nucleotides, a 3′ end and a 5′ end; (b) a minor groove binder (MGB), wherein the minor groove binder is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which molecule binds in a non-intercalating manner into the minor groove of non-single-stranded DNA, RNA or hybrids thereof with an association constant greater than approximately 10 3 M −1 ; and (c) a latent fluorophore (LF); wherein the latent fluorophore is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which binds in an intercalating manner into non-single-stranded DNA, RNA or hybrids thereof, or alternatively lies in the minor groove, or in another manner is oriented to the DNA molecule by the minor groove binder moiety so that it becomes fluorescent or its fluorescence properties are changed in a detectable way; wherein the composition is a covalently bound oligonucleotide, minor groove binder, latent fluorophore combination having a first linking group and a second linking group.
Claim 3
Dependent Claim
22 words
The composition of claim 1 , wherein the minor groove binder moiety is covalently linked to the 5′ end of the oligonucleotide.
Claim 4
Dependent Claim
22 words
The composition of claim 1 , wherein the minor groove binder moiety is covalently linked to the 3′ end of the oligonucleotide.
Claim 5
Dependent Claim
26 words
The composition of claim 1 , wherein the minor groove binder moiety is covalently linked to the oligonucleotide at one or more of the nucleotide units.
Claim 6
Dependent Claim
24 words
The composition of claim 1 , wherein the first linking group comprises a chain having a backbone of no more than about 100 atoms.
Claim 7
Dependent Claim
24 words
The composition of claim 1 , wherein the second linking group comprises a chain having a backbone of no more than about 50 atoms.
Claim 8
Dependent Claim
14 words
The composition of claim 1 , wherein the composition does not exhibit substantial fluorescence.
Claim 9
Dependent Claim
19 words
The composition of claim 1 , wherein the composition does not exhibit detectable fluorescence emission at a particular wavelength.
Claim 10
Dependent Claim
24 words
The composition of claim 8 , wherein fluorescence emission is increased at the particular wavelength after hybridization of the oligonucleotide to a target sequence.
Claim 11
Dependent Claim
29 words
The composition of claim 1 , wherein the fluorescence emission maximum shifts from a first wavelength to a second wavelength after hybridization of the oligonucleotide to a target sequence.
Claim 12
Dependent Claim
15 words
The composition of claim 1 , wherein the oligonucleotide comprises one or more nucleotide analogues.
Claim 13
Dependent Claim
21 words
The composition of claim 11 , wherein the oligonucleotide is a PNA/DNA chimera, wherein PNA is a polyamide (peptide) nucleic acid.
Claim 14
Dependent Claim
12 words
The composition of claim 11 , wherein inosine is substituted for guanosine.
Claim 15
Dependent Claim
16 words
The composition of claim 11 , wherein the oligonucleotide comprises one or more pyrazolopyrimidine nucleotide residues.
Claim 16
Dependent Claim
12 words
The composition of claim 14 , wherein 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one is substituted for guanine.
Claim 17
Dependent Claim
12 words
The composition of claim 14 , wherein 4-amino-1H-pyrazolo[3,4-d]pyrimidine is substituted for adenine.
Claim 18
Dependent Claim
12 words
The composition of claim 14 , wherein 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-6(7H)-dione is substituted for adenine.
Claim 19
Dependent Claim
80 words
A method for detecting a target sequence in a polynucleotide, wherein the method comprises: (a) combining a composition according to claim 1 with a sample containing a polynucleotide, wherein the oligonucleotide portion of the composition comprises a sequence which hybridizes to the target sequence, to form a hybridization mixture; (b) incubating the hybridization mixture under conditions which yield specific hybridization; and (c) thereafter measuring fluorescence of the hybridization mixture, wherein fluorescence is indicative of the presence of the target sequence.
Claim 20
Dependent Claim
23 words
The method according to claim 18 , wherein the polynucleotide is in a sample comprising a plurality of other polynucleotides having different sequences.
Claim 21
Dependent Claim
16 words
The method according to claim 18 , wherein the sample comprises a signal amplification hybridization reaction.
Claim 22
Dependent Claim
56 words
The method according to claim 18 , wherein a plurality of target sequences are detected; wherein, for each target sequence, there is a unique ODN-MGB-LF comprising an ODN portion that is complementary to the target sequence and a LF whose fluorescence is distinguishable from that of other LFs conjugated to ODNs complementary to different target sequences.
Claim 23
Dependent Claim
20 words
The method according to claim 18 , wherein the composition of step (a) is the ODN-MGB-LF of claim 36 .
Claim 24
Dependent Claim
119 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 the steps of: (a) contacting the mixture of polynucleotides with a composition according to claim 1 , wherein the composition forms a stable hybrid only with said target sequence that is perfectly complementary to the oligonucleotide portion of the composition and wherein the composition does not form a stable hybrid with any of the related sequences; and (b) measuring fluorescence, wherein fluorescence is indicative of the presence of the target sequence.
Claim 25
Dependent Claim
28 words
The method according to claim 23 , wherein one or more of the related sequences has a single-nucleotide mismatch with respect to the ODN portion of the composition.
Claim 26
Dependent Claim
20 words
The method according to claim 23 , wherein the composition of step (a) is the ODN-MGB-LF of claim 36 .
Claim 27
Dependent Claim
88 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 composition according to claim 1 , wherein the oligonucleotide portion of the composition has a sequence that is complementary to the target sequence, and wherein the composition forms stable hybrids with the related sequences; and (b) measuring fluorescence, wherein fluorescence is indicative of the presence of the one or more related sequences.
Claim 28
Dependent Claim
20 words
The method according to claim 26 , wherein the composition of step (a) is the ODN-MGB-LF of claim 36 .
Claim 29
Dependent Claim
81 words
A method for primer extension, wherein the method comprises the steps of: (a) providing a sample containing a target sequence; (b) providing one or more oligonucleotide primers, wherein each primer is complementary to a region 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 ODN-MGB-LF conjugate according to claim 1 .
Claim 30
Dependent Claim
17 words
The method according to claim 28 , wherein the products of polymerization are detected in real time.
Claim 31
Dependent Claim
20 words
The method according to claim 28 , wherein the composition of step (d) is the ODN-MGB-LF of claim 36 .
Claim 32
Dependent Claim
13 words
A ODN-MGB-LF according to claim 1 that is immobilized on a solid support.
Claim 33
Dependent Claim
77 words
A method for detecting a target sequence, wherein the method comprises: (a) immobilizing a ODN-MGB-LF according to claim 1 at an address on a solid support, wherein the ODN is complementary to the target sequence; (b) exposing the support to a solution potentially containing the target sequence under conditions favorable to hybridization; and (c) thereafter measuring fluorescence of the support, wherein fluorescence at the address is indicative of the presence of the target sequence in the solution.
Claim 34
Dependent Claim
31 words
The method according to claim 32 , wherein a plurality of ODN-MGB-LFs, having different sequences in their ODN portions, are immobilized at distinct addresses on the support in an ordered array.
Claim 35
Dependent Claim
16 words
A kit for hybridization analysis, wherein the kit comprises a composition according to claim 1 .
Claim 36
Dependent Claim
33 words
The composition of claim 1 , wherein the LF is covalently attached to the ODN through said first linking group and the MGB is attached to the LF through said second linking group.
Claim 37
Dependent Claim
24 words
The composition of claim 35 , wherein the third linking group comprises a chain having a backbone of no more than about 50 atoms.
Claim 38
Dependent Claim
24 words
The composition of claim 35 , wherein the fourth linking group comprises a chain having a backbone of no more than about 50 atoms.
Claim 39
Dependent Claim
33 words
The composition of claim 1 , wherein the MGB is covalently attached to the ODN through said first linking group and the MGB is attached to the LF through said second linking group.
Claim 40
Dependent Claim
60 words
A composition comprising: wherein X has a backbone chain length between 0 and 48 atoms long, wherein the backbone contains atoms selected from C, N, O and S, and wherein the backbone optionally contains one or more of —NH—, —O—, —NH—C(═O)—, —C═(O)—NH—, —NH—C(═O)—NH—, —NH—C(═S)—NH—, —SO 2 —, —SO 2 —NH—, —S—or —S—S— groups; and wherein Y is a leaving group.
Claim 41
Dependent Claim
60 words
A composition comprising: wherein X has a backbone chain length between 0 and 48 atoms long, wherein the backbone contains atoms selected from C, N, O and S, and wherein the backbone optionally contains one or more of —NH—, —O—, —NH—C(═O)—, —C═(O)—NH—, —NH—C(═O)—NH—, —NH—C(═S)—NH—, —SO 2 —, —SO 2 —NH—, —S—or —S—S— groups; and wherein Y is a leaving group.
Claim 42
Dependent Claim
16 words
The compound of claim 39 , wherein the leaving group forms part of an activated ester.
Claim 43
Dependent Claim
25 words
The compound of claim 40 , wherein the activated ester is selected from the group consisting of hydroxysulfosuccinimide esters, hydroxysucciniminde esters, tetrafluorophenyl esters and anhydrides.
Claim 44
Dependent Claim
52 words
A method for the synthesis of a ODN-MGB-LF conjugate, wherein the method comprises: (a) treating the composition of claim 39 with an acid; and (b) reacting the product of step (a) with an amino-tailed ODN and with a LF, wherein the LF comprises a linking group which terminates in a reactive group.
Claim 45
Dependent Claim
22 words
A composition comprising: wherein K is a cleavable linker; m is between 1 and 99; and n is between 1 and 47.
Claim 46
Dependent Claim
22 words
A composition comprising: wherein K is a cleavable linker; m is between 1 and 99; and n is between 1 and 47.
Claim 47
Dependent Claim
9 words
The composition of claim 43 , wherein K is
Claim 48
Dependent Claim
84 words
A method for the synthesis of a ODN-MGB-LF conjugate, wherein the method comprises: (a) removing the DMTr group from the composition of claim 43 ; (b) using the product of step (a) for oligonucleotide synthesis to generate a CPG-MGB-ODN; (c) removing the MGB-ODN of step (b) from the CPG by cleavage at the cleavable linker K; and (d) reacting the product of step (c) with a LF, wherein the LF comprises a linking group which terminates in a reactive group, to produce a ODN-MGB-LF.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2005-10-28 | 2006-04-29 | 2006-10-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2009-10-28 | 2010-04-29 | 2010-10-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2013-10-28 | 2014-04-29 | 2014-10-29 | $7700.00 | Window Closed / Lapsed |