US6884584B2
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Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
Assignee: Epoch Biosciences Inc
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Filing: 2002-04-22
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Granted: 2005-04-26
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Expires: 2022-04-22
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 (CDPI 3 ), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI 3 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 00:03
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: 15
Claim 1
Independent Claim
133 words
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 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 − ; and detecting the presence of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids on said array.
Claim 2
Independent Claim
133 words
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 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 − ; and detecting the presence of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids on said array.
Claim 3
Dependent Claim
16 words
The method of claim 1 , wherein the nucleotide polymorphisms are one or more single-nucleotide polymorphisms.
Claim 4
Dependent Claim
22 words
The method of claim 1 , wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 12.5% to 100%.
Claim 5
Dependent Claim
28 words
The method of claim 3 , wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 18° C.
Claim 6
Dependent Claim
22 words
The method of claim 1 , wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 25% to 87.5%.
Claim 7
Dependent Claim
28 words
The method of claim 5 , wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 8° C.
Claim 8
Dependent Claim
22 words
The method of claim 1 , wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 37.5% to 75%.
Claim 9
Dependent Claim
28 words
The method of claim 7 , wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 4° C.
Claim 10
Dependent Claim
22 words
The method of claim 1 , wherein the A/T content of said minor groove binder-oligonucleotide conjugate probe ranges from 37.5% to 87.5%.
Claim 11
Dependent Claim
28 words
The method of claim 9 , wherein the range of the melting temperatures of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is less than 4° C.
Claim 12
Dependent Claim
65 words
The method of claim 1 , wherein said minor groove binder is a member selected from the group consisting of: netropsin; distamycin; lexitropsin; mithramycin; chromomycin A 3 ; olivomycin; anthramycin; sibiromycin; pentamidine; stilbamidine; berenil; CC-1065; Hoechst 33258; 4′-6-diamidino-2-phenylindole (DAPI); a dimer, a trimer, a tetramer and a pentamer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI); N-methylpyrrole-4-carbox-2-amide (MPC), and a dimer, a trimer, a tetramer and a pentamer of MPC.
Claim 13
Dependent Claim
17 words
The method of claim 1 , wherein said minor groove binder-oligonucleotide conjugate probe(s) contain a detectable label.
Claim 14
Dependent Claim
29 words
The method of hybridization according to claim 1 , wherein the melting temperature of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is relatively independent of base composition.
Claim 15
Dependent Claim
33 words
The method of hybridization according to claim 1 wherein the melting temperature of said minor groove binder-oligonucleotide conjugate probe-target nucleic acid hybrids is dependent primarily on the length of the hybridized nucleic acid.
References Cited (Backward Citations)
US4458066A
Cited
US4358535B1
Cited
US4358535A
Cited
US5449767A
Cited
EP0070685A2
Cited
US4835263A
Cited
US4883750A
Cited
US4683202A
Cited
US4683202B1
Cited
US4868105A
Cited
US4683195A
Cited
US4683195B1
Cited
US4800159A
Cited
US4868103A
Cited
US5525464A
Cited
US5492806A
Cited
US5124246A
Cited
EP0320308B1
Cited
EP0336731B1
Cited
WO1990003370A1
Cited
WO1990014353A1
Cited
US5237101A
Cited
US5210015A
Cited
US5512667A
Cited
WO1992010588A1
Cited
WO1992020698A1
Cited
US5419966A
Cited
WO1993003736A1
Cited
US5574142A
Cited
WO1994017092A1
Cited
US5786138A
Cited
US5446137A
Cited
US5446137B1
Cited
US5646126A
Cited
WO1995029184A1
Cited
US5556752A
Cited
WO1996017957A1
Cited
WO1996032496A2
Cited
US5801155A
Cited
US6312894B1
Cited
WO1996040711A1
Cited
WO1997012896A1
Cited
US5659022A
Cited
US5776907A
Cited
WO1998002448A1
Cited
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