FRET-LABELED COMPOUNDS AND USES THEREFOR
First Claim
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1. A compound comprising a label portion and a reactant portion in an analytical reaction,a) wherein the label portion comprises a FRET label;
- b) wherein the compound is configured such that the FRET label has an emission spectrum comprising at least two peaks that distinctly identify the reactant portion in the analytical reaction.
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Abstract
FRET-labeled compounds are provided for use in analytical reactions. In certain embodiments, FRET-labeled nucleotide analogs are used in place of naturally occurring nucleoside triphosphates or other analogs in analytical reactions comprising nucleic acids, for example, template-directed nucleic acid synthesis, DNA sequencing, RNA sequencing, single-base identification, hybridization, binding assays, and other analytical reactions.
123 Citations
67 Claims
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1. A compound comprising a label portion and a reactant portion in an analytical reaction,
a) wherein the label portion comprises a FRET label; b) wherein the compound is configured such that the FRET label has an emission spectrum comprising at least two peaks that distinctly identify the reactant portion in the analytical reaction. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 25)
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2. (canceled)
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18. (canceled)
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22-24. -24. (canceled)
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26-35. -35. (canceled)
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36. A method of distinguishing between two labeled compounds in an analytical reaction, comprising:
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a) labeling a first compound with a FRET pair in a first orientation to generate a first labeled compound, wherein the first orientation ensures a first FRET efficiency; b) labeling a second compound with the FRET pair in a second orientation to generate a second labeled compound, wherein the second orientation ensures a second FRET efficiency; c) combining the first labeled compound and the second labeled compound in an analytical reaction; d) subjecting the analytical reaction to excitation radiation; e) detecting a signal emitted from the analytical reaction; and f) analyzing the signal to determine a signal FRET efficiency, wherein if the signal FRET efficiency is equal to the first FRET efficiency, the signal originated from the first labeled compound, and if the signal FRET efficiency is equal to the second FRET efficiency, the signal originated from the second labeled compound, thereby distinguishing between the two labeled compounds in the analytical reaction. - View Dependent Claims (38, 42, 43, 44)
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37. (canceled)
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39-41. -41. (canceled)
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45-56. -56. (canceled)
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57. A method of determining an identity and relative position of a nucleotide in a template nucleic acid, comprising:
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a) providing the template nucleic acid complexed with a polymerase enzyme capable of template-dependent synthesis of a complementary nascent nucleic acid strand, in a first complex; b) contacting the first complex with a plurality of differentially labeled compounds, wherein each of the plurality of differentially labeled compounds comprises; i) an individually detectable label, wherein a first subset of the differentially labeled compounds comprises an individually detectable label that undergoes resonance energy transfer at a submaximal efficiency that is less than 90%, and a second subset of the differentially labeled compounds comprises an individually detectable label that does not undergo resonance energy transfer; and ii) a different base selected from A, T, G and C, wherein each of the plurality of differentially labeled compounds that comprises a given base comprise an identical individually detectable label; and c) detecting whether any of the differentially labeled compounds are incorporated into the nascent nucleic acid strand, incorporation of one of the differentially labeled compounds being indicative of complementarity between a base in the differentially labeled compound and a position in the template nucleic acid that is being processed by the polymerase enzyme. - View Dependent Claims (58, 59, 60)
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61-65. -65. (canceled)
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66. A method for distinguishing between binding of multiple reactants, comprising:
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a) providing a first reactant comprising a donor FRET chromophore; b) providing a second reactant comprising a first acceptor FRET chromophore, wherein the first acceptor FRET chromophore generates a first emission spectrum during binding to the first reactant; c) providing a third reactant comprising a second acceptor FRET chromophore, wherein the second acceptor FRET chromophore is identical to the first acceptor FRET chromophore, and further wherein the second acceptor FRET chromophore generates a second emission spectrum during binding to the first reactant, wherein the second emission spectrum is detectably distinct from the first emission spectrum; d) exposing the first reactant to both the second reactant and the third reactant under conditions that promote binding to the first reactant; and e) monitoring spectral emissions from the first reactant, wherein detection of the first emission spectrum indicates binding of the second reactant to the first reactant and detection of the second emission spectrum indicates binding of the third reactant to the first reactant.
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67-68. -68. (canceled)
Specification