Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer
First Claim
1. A method for detecting presence of a target site of at least one nucleotide in a sequence of contiguous nucleotides in a sample of nucleic acid comprising:
- (a) synthesizing, in the sample, an oligonucleotide on and directed by a template of the contiguous nucleotides including the target site wherein the oligonucleotide comprises two fluorophores each of which is covalently linked to a separate nucleotide; and
(b) detecting a fluorescence energy transfer from one fluorophore to the other upon denaturation and release of the oligonucleotide from the contiguous nucleotides including the target site wherein the detecting of fluorescence energy transfer indicates the presence of the target site.
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Abstract
A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.
286 Citations
14 Claims
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1. A method for detecting presence of a target site of at least one nucleotide in a sequence of contiguous nucleotides in a sample of nucleic acid comprising:
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(a) synthesizing, in the sample, an oligonucleotide on and directed by a template of the contiguous nucleotides including the target site wherein the oligonucleotide comprises two fluorophores each of which is covalently linked to a separate nucleotide; and (b) detecting a fluorescence energy transfer from one fluorophore to the other upon denaturation and release of the oligonucleotide from the contiguous nucleotides including the target site wherein the detecting of fluorescence energy transfer indicates the presence of the target site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for detecting presence of a target site of at least one nucleotide in a sequence of contiguous nucleotides in a sample of nucleic acid comprising:
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(a) performing, in the sample, a reaction which synthesizes an oligonucleotide on and directed by a template of the contiguous nucleotides including the target site if the target site is present in said sample, wherein the oligonucleotide comprises two fluorophores each of which is covalently linked to a separate nucleotide; and (b) detecting a fluorescence energy transfer from one fluorophore to the other upon denaturation and release of oligonucleotide if it is synthesized in step (a), wherein the detecting of fluorescence energy transfer indicates the presence of the target site.
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Specification