Patent US7205105B2 successfully refreshed from Google Patents.
US7205105B2
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US
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Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis
Assignee: ELITechGroup Inc
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Filing: 2002-06-06
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Granted: 2007-04-17
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Expires: 2022-06-06
Abstract
Abstract Oligonucleotide probes/conjugates are provided along with method for their use in assays to monitor amplification wherein the signal produced does not rely on 5′ nuclease digestion.
Inventors
- 👤 Irina A. Afonina
- 👤 Yevgeniy S. Belousov
- 👤 Robert O. Dempcy
- 👤 Igor V. Kutyavin
- 👤 Sergey G. Lokhov
- 👤 Eugeny A. Lukhtanov
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 14:48
CPC Classifications
C; C07; C07C; C07C245/00; C07C245/02; C07C245/06; C07C245/08; C07D; C07D519/00; C07F; C07F9/00; C07F9/02; C07F9/06; C07F9/22; C07F9/24; C07F9/2404; C07F9/2408; C07F9/547; C07F9/553; C07F9/572; C07F9/655; C07F9/6552; C07F9/65522; C07F9/6561; C07H; C07H21/00; C09; C09B; C09B29/00; C09B29/06; C09B29/08; C09B29/0805; C09B29/0807; C09B29/0809; C09B29/0811; C09B29/0813; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/6844; C12Q1/686; C07B; C07B2200/00; C07B2200/11; C40; C40B; C40B40/00
Claims Analysis
Total Claims: 9
Claim 1
Independent Claim
175 words
A method for distinguishing between wild-type, mutant and heterozygous target polynucleotides, said method comprising: (a) contacting a sample containing a target polynucleotide with two probes wherein a first probe is specific for said wild-type target polynucleotide and a second probe is specific for said mutant target polynucleotide, each of said probes having a formula: wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore; wherein said first and second probes have different fluorophores and each of said probes forms a stable hybrid only with the target sequence that is perfectly complementary to the ODN portion of said probes; and (b) measuring the fluorescence produced on hybrid formation for each fluorophore, wherein said measuring is carried out at two wavelengths and is measured as a function of temperature, and using melting curve analysis to indicate the presence or absence of each of said wild-type, mutant and heterozygous target polynucleotides.
Claim 2
Independent Claim
175 words
A method for distinguishing between wild-type, mutant and heterozygous target polynucleotides, said method comprising: (a) contacting a sample containing a target polynucleotide with two probes wherein a first probe is specific for said wild-type target polynucleotide and a second probe is specific for said mutant target polynucleotide, each of said probes having a formula: wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore; wherein said first and second probes have different fluorophores and each of said probes forms a stable hybrid only with the target sequence that is perfectly complementary to the ODN portion of said probes; and (b) measuring the fluorescence produced on hybrid formation for each fluorophore, wherein said measuring is carried out at two wavelengths and is measured as a function of temperature, and using melting curve analysis to indicate the presence or absence of each of said wild-type, mutant and heterozygous target polynucleotides.
Claim 3
Dependent Claim
37 words
A method in accordance with claim 1 , wherein the melting temperatures (T m ) for each hybrid produced between said first and second probes and their respective targets are within about 5° C. of each other.
Claim 4
Dependent Claim
32 words
A method in accordance with claim 1 , wherein the ODN portion of each of said probes is an oligonucleotide or modified oligonucleotide having from 8 to 18 bases or modified bases.
Claim 5
Dependent Claim
32 words
A method in accordance with claim 1 , wherein the ODN portion of each of said probes is an oligonucleotide or modified oligonucleotide having from 10 to 15 bases or modified bases.
Claim 6
Dependent Claim
34 words
A method in accordance with claim 1 , wherein the fluorophore portions of each of said probes are selected from the group consisting of 5-FAM™, 6-FAM™, TET™, JOE™, HEX™, VIC™, NED™, TAMRA™, ROX™and YY™.
Claim 7
Dependent Claim
23 words
A method in accordance with claim 1 , wherein the ODN portion of each of said probes contains at least one modified base.
Claim 8
Dependent Claim
51 words
A method in accordance with claim 6 , wherein each modified base is independently selected from the group consisting of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 4-amino-1H-pyrazolo[3,4-d]pyrimidine, 1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione, 6-amino-3-prop-1-ynyl-5-hydropyrazol[3,4-d]pyrimidine-4-one, 6-amino-3-(3-hydroxyprop-1-ynyl)1-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 3-prop-1-ynyl-4,6-diaminopyrazolo[3,4-d]pyrimidine, 2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, 3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, 5-prop-1-ynyl-1,3-dihydropyrimidine 2,4-dione, 5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, 6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, 6-amino-5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2-one, 6-amino-5-(3-aminoprop-1-ynyl)-1,3 -dihydropyrimidine-2-one, 5-[4-amino-3-(3-methoxyprop-1ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol, 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, 4-(4,6-Diamino-1H-pyrazololl[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, 6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one, 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-chioro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-lodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine, 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine and 3-chioro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.
Claim 9
Dependent Claim
72 words
A method in accordance with claim 1 , wherein said sample is further contacted with a set of primers under amplification conditions and each of said primers contains from one to ten modified bases selected from the group consisting of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 4-amino-1H-pyrazolo[3,4-d]pyrimidine, 1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione, 6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-hydroxyprop-1-yny)1-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 3-prop-1-ynyl-4,6-diaminopyrazolo[3,4-d]pyrimidine, 2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, 3-(2-aminoethynyl)pyrazolo [3,4-d]pyrimidine-4,6-diamine, 5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione, 5-(3 -hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, 6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, 6-amino-5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2-one, 6-amino-5-(3-aminoprop-1-ynyl)-1,3-dihydropyrimidine-2-one, 5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol, 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, 6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one, 5-(4-hydroxy-but-1-ynyl) -1H-pyrimidine-2,4-dione, 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-lodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine, 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine and 3-chioro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.
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