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US 9,315,859 B2Granted
DDAO compounds as fluorescent reference standards
AssigneeLife Technologies Corporation
InventorsScott C. Benson, Cinna Monighetti, Sandy M. Koepf
Life of this patent
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Abstract
According to the present teachings, methods and compositions are provided that utilize at least one reference dye of formula (I): In some embodiments, a method comprises measuring a detection signal of a reporter dye and at least one reference dye of formula (I). In some embodiments, a composition comprises a reference dye of formula (1), a buffer, a selection of nucleotides and a protein.
Claims16
A method comprising:
- (a) contacting a sample which may comprise at least one target polynucleotide with at least one probe and a reference dye to form a mixture, wherein each of the at least one probes comprises a target-specific moiety, a reporter dye and a quencher dye, wherein each target-specific moiety is an oligonucleotide and is specific for a different target and each reporter dye is different from the other reporter dyes and is different from the reference dye, and wherein the reference dye has formula (I):
- wherein:
- each of R1 to R3 and R6 to R8 is independently —H, halogen, —CO2H, —CO2R, —SO3H, —SO3R, —CH2CO2H, —CH2CO2R, —CH2SO3H, —CH2SO3R, —CH2NH2, —CH2NHR, —NO2, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, and substituted C1-C6 alkoxy, wherein R is C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, and substituted C1-C6 alkoxy;
- R4 and R5 taken separately are selected from C1-C6 alkyl, and C1-C6 substituted alkyl, or R4 and R5 taken together are selected from C3-C7 cycloalkyl, C4-C7 unsaturated cycloalkyl, C3-C7 substituted cycloalkyl, or C4-C7 substituted unsaturated cycloalkyl;
- (b) amplifying a target polynucleotide in the mixture to form an amplified mixture;
- (c) measuring fluorescence emitted by the reference dye;
- and
- (d) measuring fluorescence emitted by at least one reporter dye.
5 more elements
A method comprising:
- (a) contacting a sample which may comprise at least one target polynucleotide with at least one probe and a reference dye to form a mixture, wherein each of the at least one probes comprises a target-specific moiety, a reporter dye and a quencher dye, wherein each target-specific moiety is an oligonucleotide and is specific for a different target and each reporter dye is different from the other reporter dyes and is different from the reference dye, and wherein the reference dye has formula:
- (b) amplifying a target polynucleotide in the mixture;
- (c) measuring fluorescence emitted by the reference dye;
- and
- (d) measuring fluorescence emitted by at least one reporter dye.
2 more elements
A method comprising:
- (a) contacting a sample which may comprise at least one target polynucleotide with two to six probes and a reference dye to form a mixture, wherein each of the two to six probes comprises a target-specific moiety, a reporter dye and a quencher dye, wherein each target-specific moiety is an oligonucleotide and is specific for a different target and each reporter dye is different from the other reporter dyes and is different from the reference dye, and wherein the reference dye has formula (I):
- wherein:
- each of R1 to R3 and R6 to R8 is independently —H, halogen, —CO2H, —CO2R, —SO3H, —SO3R, —CH2CO2H, —CH2CO2R, —CH2SO3H, —CH2SO3R, —CH2NH2, —CH2NHR, —NO2, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, and substituted C1-C6 alkoxy, wherein R is C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, and substituted C1-C6 alkoxy;
- R4 and R5 taken separately are selected from C1-C6 alkyl, and C1-C6 substituted alkyl, or R4 and R5 taken together are selected from C3-C7 cycloalkyl, C4-C7 unsaturated cycloalkyl, C3-C7 substituted cycloalkyl, or C4-C7 substituted unsaturated cycloalkyl;
- (b) amplifying a target polynucleotide in a PCR, wherein at least one reporter dye separates from the respective quencher dye during the PCR, thereby releasing fluorescence of the at least one reporter dye;
- (c) measuring fluorescence emitted by the reference dye;
- and
- (d) measuring fluorescence emitted by the at least one reporter dye.
5 more elements
Dependencies are inferred from the claim text.