Method for nucleic acid analysis using fluorescence resonance energy transfer
First Claim
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1. A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising:
- (a) a polynucleotide covalently linked to one fluorophore and capable of binding to the target site or immediately 3′
to the target site; and
(b) a dideoxynucleotide covalently linked to a second fluorophore, which is capable of binding to the nucleic acid immediately 5′
to the polynucleotide and capable of adding by 3′
extension to the polynucleotide, wherein the emission spectrum of one of the fluorophores overlaps the excitation spectrum of the other, packaged in a container.
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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.
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Citations
10 Claims
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1. A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising:
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(a) a polynucleotide covalently linked to one fluorophore and capable of binding to the target site or immediately 3′
to the target site; and
(b) a dideoxynucleotide covalently linked to a second fluorophore, which is capable of binding to the nucleic acid immediately 5′
to the polynucleotide and capable of adding by 3′
extension to the polynucleotide, wherein the emission spectrum of one of the fluorophores overlaps the excitation spectrum of the other, packaged in a container.- View Dependent Claims (2, 3, 5, 6, 7, 8)
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4. A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising:
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(a) a first polynucleotide covalently linked to a first fluorophore; and
(b) a second polynucleotide covalently linked to a second fluorophore wherein the second polynucleotide is capable of being ligated to the first polynucleotide to form an oligonucleotide which binds to the target site, wherein the emission spectrum of one of the fluorophores overlaps the excitation spectrum of the other, packaged in a container. - View Dependent Claims (9, 10)
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Specification