Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer

• US 5,945,283 A
• Filed: 12/17/1996
• Issued: 08/31/1999
• 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): Marschel, Ardin H.
• Application Number:
  US08/767,979
• Time in Patent Office: 987 Days
• Field of Search: 435/6, 435/810, 436/501, 935/77, 935/78
• 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 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.

2. The method according to claim 1 wherein the synthesizing step comprises template-directed 3'"'"' extension of a polynucleotide, which is covalently linked to one fluorophore and bound either to the target site or immediately 3'"'"' to the target site on the template, by addition of a dideoxynucleoside triphosphate covalently linked to the other fluorophore.

3. The method according to claim 2 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,4-adiaza-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.

4. The method according to claim 3 wherein one fluorophore is fluorescein and the other fluorophore is 6-carboxy-X-rhodamine or N,N,N'"'"',N'"'"'-tetramethyl-6-carboxyrhodamine.

5. The method according to claim 3 wherein detecting an increase in emission of one fluorophore upon excitation of the other indicates the presence of the target site in the nucleic acid.

6. The method according to claim 3 wherein detecting a quenching of one fluorophore indicates the presence of the target site in the nucleic acid.

7. The method according to claim 3 wherein the method detects single nucleotide polymorphism.

8. The method according to claim 1 wherein the synthesizing step comprises a template-directed ligation of a first polynucleotide, which is covalently linked to one fluorophore and bound to the target site on the template, to a second polynucleotide covalently linked to the other fluorophore.

9. The method according to claim 8 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,4-adiaza-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,4-adiaza-S-indacene-propionic acid.

10. The method according to claim 9 wherein one fluorophore is fluorescein and the other fluorophore is 6-carboxy-X-rhodamine or N,N,N'"'"',N'"'"'-tetramethyl-6-carboxyrhodamine.

11. The method according to claim 9 wherein detecting an increase in emission of one fluorophore upon excitation of the other indicates the presence of the target site in the nucleic acid.

12. The method according to claim 9 wherein detecting a quenching of one fluorophore indicates the presence of the target site in the nucleic acid.

13. The method according to claim 9 wherein the method detects single nucleotide polymorphism.

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:

(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.