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US 7,456,281 B2Granted
Nucleic acid melting analysis with saturation dyes
AssigneeIdaho Technology, Inc.
InventorVirginie E. Dujols
Life of this patent
Estimating the term…
Abstract
Methods are provided for nucleic acid analysis wherein a target nucleic acid is mixed with a dsDNA binding dye to form a mixture. Optionally, an unlabeled probe is included in the mixture. A melting curve is generated for the target nucleic acid by measuring fluorescence from the dsDNA binding dye as the mixture is heated. Dyes for use in nucleic acid analysis and methods for making dyes are also provided.
Claims22
A compound having the formula:
- wherein
- the moiety represents an optionally-substituted fused monocyclic or polycyclic aromatic ring or an optionally-substituted fused monocyclic or polycyclic nitrogen-containing heteroaromatic ring;
- X is oxygen, sulfur, selenium, tellurium or a moiety selected from C(CH3)2 and NR1, where R1 is hydrogen or C1-6 alkyl;
- R2 is selected from the group consisting of C1-6 alkyl, C3-8 cycloalkyl, aryl, aryl(C1-3 alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, optionally substituted cyclic heteroatom-containing moieties, and optionally substituted acyclic heteroatom-containing moieties;
- t=0 or 1;
- Z is a charge selected from 0 or 1;
- R3 is selected from the group consisting of hydrogen, C1-6 alkyl, and arylcarbonyl, or R2 and R3 are taken together to form —CH2)w—, wherein w is 1 to 5;
- R9 and R10 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, and arylcarbonyl;
- n=0, 1, or 2;
- and
- v=0 or 1;
- with the proviso that v=0 when R2 and R3 are not taken together to form —(CH2)w—;
- wherein when v=0, Q is an heterocycle selected from the group of structures consisting of:
- and
- wherein when v=1, Q is an heterocycle selected from the group of structures consisting of:
- wherein R4, R5, R6, R7, R8, R12, and R13 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, alkylnitrilethio, aryl, heteroaryl, alkoxy, arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, trialkylammoniumalkylcarbonylthio, and nucleosidylthio, each of which may be optionally substituted;
- an acyclic heteroatom-containing moiety, a cyclic heteroatom-containing moiety, a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety.
14 more elements
A compound having the formula:
- wherein
- the moiety represents an optionally-substituted fused monocyclic or polycyclic aromatic ring or an optionally-substituted fused monocyclic or polycyclic nitrogen-containing heteroaromatic ring;
- X is oxygen, sulfur, selenium, tellurium or a moiety selected from C(CH3)2 and NR1, where R1 is hydrogen or C1-6 alkyl;
- R2 is selected from the group consisting of C1-6 alkyl, C3-8 cycloalkyl, aryl, aryl(C1-3 alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, optionally substituted cyclic heteroatom-containing moietics, and optionally substituted acyclic heteroatom-containing moieties;
- t=0 or 1;
- Z is a charge selected from 0 or 1;
- R 3 is selected from the group consisting of hydrogen, C1-6 alkyl, and arylcarbonyl, or R2 and R3 are taken together to form —(CH2)w—, wherein w is 1 to 5;
- R9 and R10 are each independently selected from the group consisting of hydrogen, C1-5 alkyl, and arylcarbonyl;
- n=0, 1, or 2;
- and
- v=0 or 1;
- with the proviso that v=0 when R2 and R3 are not taken together to form —(CH2)w—;
- wherein when v=0, Q is an heterocycle selected from the group of structures consisting of:
- and
- wherein when v=1, Q is an heterocycle selected from the group of structures consisting of:
- wherein R4, R5, R6, R7, R8, R12, and R13 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, alkylnitrilethio, aryl, heteroaryl, alkoxy, alkylthio, arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, trialkylammoniumalkylcarbonylthio, and nucleosidylthio, each of which may be optionally substituted;
- an acyclic heteroatom-containing moiety, a cyclic heteroatom-containing moiety, a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety, and
- wherein at least one of R4, R5,R6, R7, and R8 is selected from the group consisting of arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, trialkylammoniumalkylcarbonylthio, alkylnitrilethio, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, and nucleosidylthio, each of which may be optionally substituted.
15 more elements
A compound having the formula:
- wherein
- the moiety represents an optionally-substituted fused monocyclic or polycyclic aromatic ring or an optionally-substituted fused monocyclic or polycyclic nitrogen-containing heteroaromatic ring;
- X is oxygen, sulfur, selenium, tellurium or a moiety selected from C(CH3)2 and NR1, where R2 is hydrogen or C1-6 alkyl;
- R2 is selected from the group consisting of C1-6 alkyl, C3-8 cycloalkyl, aryl, aryl(C1-3alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, optionally substituted cyclic heteroatom-containing moieties, and optionally substituted acyclic heteroatom-containing moieties;
- t=0 or 1;
- Z is a charge selected from 0 or 1;
- R3 is selected from the group consisting of hydrogen, C1-6 alkyl, and arylcarbonyl, or R2 and R3 are taken together to form —(CH2)w—, wherein w is 1 to 5;
- R9 and R10 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, and arylcarbonyl;
- n=0, 1, or 2;
- and
- v=0 or 1;
- with the proviso that v=0 when R2 and R3 are not taken together to form —(CH2)w—;
- wherein when v=0, Q is an heterocycle selected from the group of structures consisting of:
- and
- wherein when v=1, Q is an heterocycle selected from the group of structures consisting of:
- wherein R4, R5, R6, R7, R8, R12, and R13 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynl, alkenylalkynyl, alkylnitrilethio, aryl, heteroaryl, alkoxy, alkylthio, arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, trialkylammoniumalkylcarbonylthio, and nucleosidylthio, each of which may be optionally substituted;
- an acyclic heteroatom-containing moiety, a cyclic heteroatom-containing moiety, a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety, with the proviso that R4 is not selected from the group consisting hydrogen, N,N-dimethylpiperazinyl, pyrimidinylthio, methylthio, thiol, amino, and hydroxyl.
14 more elements
Dependencies are inferred from the claim text.