Method for nucleic acid analysis using fluorescence resonance energy transfer

• US 6,177,249 B1
• Filed: 04/20/1999
• Issued: 01/23/2001
• Est. Priority Date: 12/18/1995
• Status: Expired due to Term

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Kwok, Pui-Yan, Chen, Xiangning
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/295,026
• Time in Patent Office: 644 Days
• Field of Search: 435/6, 436/501
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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.

Claims

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.

2. The kit according to claim 1 wherein one fluorophore is selected from the group consisting of fluorescein, cascade blue, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid and the other fluorophore is selected from the group consisting of 6-carboxy-X-rhodamine, N,N,N′

,N′

-tetramethyl-6-carboxyrhodamine, Texas Red, Eosin, fluorescein, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid.

3. The kit according to claim 2 wherein one fluorophore is fluorescein and the other fluorophore is 6-carboxy-X-rhodamine or N,N,N′

,N′

-tetramethyl-6-carboxyrhodamine.

5. The kit according to claim 3 wherein the first fluorophore is selected from the group consisting of fluorescein, cascade blue, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid and the other fluorophore is selected from the group consisting of 6-carboxy-X-rhodamine, N,N,N′

,N′

-tetramethyl-6-carboxyrhodamine, Texas Red, Eosin, fluorescein, 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, 4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid and 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid.

6. The kit according to claim 5 wherein one fluorophore is fluorescein and the other fluorophore is Texas Red.

7. The kit of claim 1, wherein the fluorophore that is linked to the polynucleotide is separated by at least about 20 nucleotides from the fluorophore that is linked to the dideoxynucleotide after the dideoxynucleotide has been added by 3′

extension to the polynucleotide.

8. The kit of claim 7, wherein the fluorophore that is linked to the polynucleotide is separated by at least about 40 nucleotides from the fluorophore that is linked to the dideoxynucleotide after the dideoxynucleotide has been added by 3′

extension to the polynucleotide.

4. A kit for detecting the presence of a target site of at least one nucleotide in a nucleic acid, the kit comprising:

(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.

9. The kit of claim 4 wherein the first fluorophore that is linked to the first polynucleotide is separated by at least about 20 nucleotides from the second fluorophore that is linked to the second polynucleotide after the second polynucleotide has been added by 3′

extension to the first polynucleotide.

10. The kit of claim 9 wherein the first fluorophore that is linked to the first polynucleotide is separated by at least about 40 nucleotides from the second fluorophore that is linked to the second polynucleotide after the second polynucleotide has been added by 3′

extension to the first polynucleotide.