Granted Patent B2

US 7,157,572 · App. 10/359,826 · Granted Jan 2, 2007

Abstract

Claims (35)

1. A fluorescently labeled reagent comprising:

a reagent selected train the group consisting of a nucleoside, a nucleotide, an oligonucleotide and an oligonucleotide analog; and

an energy transfer fluorescent label attached thereto, the energy transfer fluorescent label comprising:

a donor dye having an absorption maximum at a wavelength between 250 and 400 nm; and

an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maximum of greater than about 500 nm,

whereby excitation of the donor dye with light at said absorption maximum causes said acceptor dye to emit light at said emission maximum.

2. The fluorescently labeled reagent of claim 1 wherein the absorbance maximum of the donor dye is between about 300 and 400 nm.

3. The fluorescently labeled reagent of claim 1 wherein the absorbance maximum of the donor dye is between about 350 and 400nm.

4. The fluorescently labeled reagent of claim 1 wherein the emission maximum of the acceptor dye is greater than about 550 nm.

5. The fluorescently labeled reagent of claim 1 wherein the emission maximum of the acceptor dye is between about 500 and 700 nm.

6. The fluorescently labeled reagent of claim 1 wherein the emission maximum of the acceptor dye is at least about 150 nm greater than the absorption maximum of the donor dye.

7. The fluorescently labeled reagent of claim 1 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.

8. The fluorescently labeled reagent of claim 1 wherein the acceptor dye is selected from the group consisting of 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7dichlororhdamine dyes, carboxyrhodamines, N,N,N′,N′-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G, carboxy-X-rhodamines and Cy5.

9. The fluorescently labeled reagent of claim 1 wherein the acceptor dye is selected from the group consisting of R110, RG6, TAMRA and ROX.

10. The fluorescently labeled reagent of claim 1 that further comprises a linker linking the donor dye to the acceptor dye.

11. The fluorescently labeled reagent of claim 10 wherein the linker absorbs substantially all of the excitation energy emitted by the donor dye and comprises a functional group selected from the group consisting of an alkene, a diene, an alkyne, a five or six membered ring having at least one unsaturated bond and a fused ring structure.

12. The fluorescently labeled reagent of claim 11 wherein the functional group is selected from the group consisting of cyclopenetene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thifuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

13. The fluorescently labeled reagent of claim 1 wherein the reagent is a nucleoside.

14. The fluorescently labeled reagent of claim 1 wherein the reagent is a nucleotide.

15. The fluorescently labeled reagent of claim 14 wherein the nucleotide is a 2′-deoxyribonucleotide.

16. The fluorescently labeled reagent of claim 1 wherein the reagent is an oligonucleotide.

17. The fluorescently labeled reagent of claim 16 wherein the oligonucleotide has a 3′ end which is not extendable using a polymerase.

18. A fluorescently labeled reagent comprising:

a reagent selected from the group consisting of a nucleoside, a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate, an oligonucleotide and an oligonucleotide analog; and

an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye being selected from the group consisting of DYE102, DYE104, DYE106, DYE108, DYE118, and DYE120.

19. The fluorescently labeled reagent of claim 13 wherein the nucleoside is a 2′-deoxyribonucleoside.

20. The fluorescently labeled reagent of claim 19 wherein the 2′-dideoxyribonucleoside is selected from the group consisting of 2′-dideoxyriboadenosine, 2′-dideoxyribocytidine, 2′-dideoxyribognanosine and 2′-dideoxyribothymidine.

21. The fluorescently labeled reagent of claim 13 wherein the nucleotide is a 2′,3′-dideoxyribonucleotide.

22. The fluorescently labeled reagent of claim 21 wherein the 2′,3′-deoxyribonucleotide is selected from the group consisting of a 2′,3′-deoxyriboadenosine, 2′,3′-dideoxyribognanosine and 2′,3′-dideoxyribothymidine.

23. The fluorescently labeled reagent of claim 15 wherein the 2′-deoxyribonucleotide is selected from the group consisting of a 2′-deoxyribonucleotide-5′-monophosphate, a 2′-deoxyribonucleotide-5′-diphosphate and a 2′-deoxyribonucletide-5′-triphosphate.

24. The fluorescently labeled reagent of claim 15 wherein the 2′-dideoxyribonucleotide is selected from the group consisting of 2′-dideoxyribocytidine-5′-triphosphate, 2′-dideoxyriboguanosine-5′-triphosphate, 2′,3′-dideoxyriboguanosine5′-triphosphate and 2′,3′-dideoxyribothymidine-52′deoxyribothymidine-5′-triphosphate.

25. The fluorescently labeled reagent of claim 14 wherein the nucleoside is a 2′,3′-dideoxyribonucleoside.

26. The fluorescently labeled reagent of claim 25 wherein the 2′,3′-dideoxyribonucleotide is selected from the group consisting of a 2′,3′-dideoxyribonucleotide-5′-monophosphate, a 2′,3′-dideoxyribonucleotide-5′-diphosphate and a 2′,3′-dideoxyribonucleotide-5′-triphosphate.

27. The fluorescently labeled reagent of claim 25 wherein the 2′,3′-deoxyribonucleotide is selected from the group consisting of 2′,3′-deoxyriboadenosine-5′,triphosphate, 2′,3′-dideoxyribocytidine-5′-triphosphate, 2′,3+-deoxyriboguanosine-5′-trophosphate and 2′-deoxyribothymidine-5′-triphosphate.

28. The fluorescently labeled reagent of claim 1 wherein the donor dye comprises a coumarin ring, a pyrene ring or a sulfopyrene ring.

Assignments (6)