Method for detecting oligonucleotides using energy transfer dyes with long stoke shift

• US 6,335,440 B1
• Filed: 03/18/1999
• Issued: 01/01/2002
• Est. Priority Date: 05/03/1996
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Spurgeon, Sandra L., Rosenblum, Barnett, Lee, Linda G.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US09/272,097
• Time in Patent Office: 1,020 Days
• Field of Search: 435/6, 435/91.2, 536/25.3, 536/26.6, 549/223, 549/224-227
• US Class Current: 536/26.6
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 311/90 with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/04 derived from triarylmethanes , i.e. central C-atom is substituted by amino, cyano, alkyl
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R₂₁Z₁C(O)R₂₂R₂₆ where R₂₁ is a C_1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z₁ is either NH, sulfur or oxygen, R₂₂ is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R₂₈ includes a functional group which attaches the linker to the acceptor dye.

Claims

1. A compound comprising:

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

5. A compound according to claim 1 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

6. A compound according to claim 1 wherein the functional group comprises a benzene ring.

7. A compound according to claim 1 wherein the donor dye is a member of the fluorescein class of dyes.

8. A compound according to claim 1 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes.

9. A compound according to claim 1 wherein the donor dye is a member of the carboxyfluorescein class of dyes.

10. A compound according to claim 1 wherein the donor dye is selected from the group consisting of carboxyfluoresceins, 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines, N,N,N′

,N′

-tetramethyl carboxyrhodamines, carboxy R110s, and carboxy R6Gs.

11. A compound according to claim 1 wherein the acceptor dye is selected from the group consisting of 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines, N,N,N′

,N′

-tetramethyl carboxyrhodamines, carboxy R110s, carboxy R6Gs, carboxy-X-rhodamines and Cy5.

12. A compound according to claim 1 wherein the acceptor dye is selected from the group consisting DR110-2, DR6G-2, DTMR and DROX.

13. A compound according to claim 1 wherein the compound is attached to a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog.

14. A compound according to claim 1 wherein the compound is attached to a reagent selected from the group consisting of deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside diphosphate and deoxynucleoside triphosphate.

15. A compound according to claim 1 wherein the compound is attached to a reagent selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.

16. A compound according to claim 1 wherein the compound is attached to a reagent selected from the group consisting of dideoxynucleoside, dideoxynucleoside monophosphate, dideoxynucleoside diphosphate and dideoxynucleoside triphosphate.

17. A compound according to claim 1 wherein the compound is attached to a reagent selected from the group consisting of dideoxycytosine, dideoxyadenosine, dideoxyguanosine, and dideoxythymidine.

18. A compound according to claim 1 wherein the compound is attached to an oligonucleotide.

19. A compound according to claim 1 wherein the compound is attached to an oligonucleotide with a 3′

end which is extendable by using a polymerase.

50. A compound according to claim 1 wherein the functional group is an alkene.

51. A compound according to claim 1 wherein the functional group is a diene.

52. A compound according to claim 1 wherein the functional group is an alkyne.

53. A compound according to claim 1 wherein the functional group is a five membered ring having at least one unsaturated bond.

54. A compound according to claim 1 wherein the functional group is a six membered ring having at least one unsaturated bond.

55. A compound according to claim 1 wherein the functional group is a five and six membered ring having a fused ring structure.

2. A compound comprising:

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing substantially all of the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

3. A compound comprising:

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor dye having an emission maximum at least about 100 nm greater than the absorbance maximum of the donor dye.

4. A compound comprising:

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum greater than about 600 nm.

20. A method for sequencing a nucleic acid sequence comprising:

forming extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the separated extended primers;

the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and a compound attached to the oligonucleotide, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

21. A method for sequencing a nucleic acid sequence comprising:

forming extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the separated extended primers;

the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and a compound attached to the oligonucleotide, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing substantially all of the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

22. A method for sequencing a nucleic acid sequence comprising:

forming extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleoside triphosphates at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the separated extended primers;

the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and a compound attached to the oligonucleotide, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum at least about 100 nm greater than the absorbance maximum of the donor dye.

23. A method for sequencing a nucleic acid sequence comprising:

forming extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the separated extended primers;

the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and a compound attached to the oligonucleotide, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum greater than about 600 nm.

24. A method for sequencing a nucleic acid sequence comprising:

forming extended primers by hybridizing a nucleic acid sequence with a primer in the presence of deoxynucleoside triphosphates, at least one fluorescently labeled dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxyucteoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the fluorescently labeled dideoxynucleotides attached to the separated extended primers;

the fluorescently labeled dideoxynucleotide triphosphate including a dideoxynucleotide triphosphate and a compound attached to the dideoxynucleotide triphosphate, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

25. A method for sequencing a nucleic acid sequence comprising:

forming extended primers by hybridizing a nucleic acid sequence with a primer in the presence of deoxynucleoside triphosphates, at least one fluorescently labeled dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxyucteoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the fluorescently labeled dideoxynucleotides attached to the separated extended primers;

the fluorescently labeled dideoxynucleotide triphosphate including a dideoxynucleotide triphosphate and a compound attached to the dideoxynucleotide triphosphate, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing substantially all of the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

26. A method for sequencing a nucleic acid sequence comprising:

forming extended primers by hybridizing a nucleic acid sequence with a primer in the presence of deoxynucleoside triphosphates, at least one fluorescently labeled dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxyucteoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the fluorescently labeled dideoxynucleotides attached to the separated extended primers;

the fluorescently labeled dideoxynucleotide triphosphate including a dideoxynucleotide triphosphate and a compound attached to the dideoxynucleotide triphosphate, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum at least about 100 nm greater than the absorbance maximum of the donor dye.

27. A method for sequencing a nucleic acid sequence comprising:

forming extended primers by hybridizing a nucleic acid sequence with a primer in the presence of deoxynucleoside triphosphates, at least one fluorescently labeled dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxyucteoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the fluorescently labeled dideoxynucleotides attached to the separated extended primers;

the fluorescently labeled dideoxynucleotide triphosphate including a dideoxynucleotide triphosphate and a compound attached to the dideoxynucleotide triphosphate, the compound including a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum greater than about 600 nm.

28. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, at least one of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

29. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, at least one of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing substantially all of the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

30. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, at least one of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum at least about 100 nm greater than the absorbance maximum of the donor dye.

31. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, at least one of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum greater than about 600 nm.

32. An energy transfer dye comprising:

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a fluorecein or rhodamine donor dye capable of absorbing light at a first wavelength, the functional group having a second substituent comprising a fluorecein or rhodamine acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

wherein the first substituent of the functional group is a substituent of the donor dye at the five or six ring position of the fluorecein or rhodamine donor dye and the second substituent of the functional group is a substituent of the acceptor dye at the five or six ring position of the fluorecein or rhodamine acceptor dye.

33. An energy transfer dye according to claim 32 wherein the acceptor has an emission maximum at least about 100 nm greater than the absorbance maximum of the donor dye.

34. An energy transfer dye according to claim 32 wherein the acceptor has an emission maximum greater than about 600 nm.

35. An energy transfer dye according to claim 32 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes.

36. An energy transfer dye according to claim 32 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes and the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

37. An energy transfer dye according to claim 32 wherein the donor dye is selected from the group consisting of carboxyfluoresceins, 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines, N,N,N′

,N′

-tetramethyl carboxyrhodamines, carboxy R110s, and carboxy R6Gs.

38. An energy transfer dye according to claim 32 wherein the acceptor dye is selected from the group consisting of 4,7-dichlorofluoresceln dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines, N,N,N′

,N′

-tetramethyl carboxyrhodamines, carboxy R110s, carboxy R6Gs, carboxy-X-rhodamines and Cy5.

39. An energy transfer dye according to claim 32 wherein the acceptor dye is selected from the group consisting DR110-2, DR6G-2, DTMR and DROX.

40. An energy transfer dye according to claim 32 wherein the energy transfer dye is coupled to a member of the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog.

41. An energy transfer dye according to claim 32 wherein the energy transfer dye is coupled to a member of the group consisting of a deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside diphosphate and deoxynucleoside triphosphate.

42. An energy transfer dye according to claim 32 wherein the energy transfer dye is coupled to a member of the group consisting of a deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.

43. An energy transfer dye according to claim 32 wherein the energy transfer dye is coupled to a member of the group consisting of a dideoxynucleoside, dideoxynucleoside monophosphate, dideoxynucleoside diphosphate and dideoxynucleoside triphosphate.

44. An energy transfer dye according to claim 32 wherein the energy transfer dye is coupled to a member of the group consisting of a dideoxycytosine, dideoxyadenosine, dideoxyguanosine, and dideoxythymidine.

45. An energy transfer dye according to claim 32 wherein the energy transfer dye is coupled to an oligonucleotide.

46. An energy transfer dye according to claim 32 wherein the energy transfer dye is coupled to an oligonucleotide with a 3′

end which is extendable by using a polymerase.

47. A method for sequencing a nucleic acid sequence comprising:

forming extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the extended primers;

the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced, and an energy transfer dye comprising

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a fluorecein or rhodamine donor dye capable of absorbing light at a first wavelength, the functional group having a second substituent comprising a fluorecein or rhodamine acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

wherein the first substituent of the functional group is a substituent of the donor dye at the five or six ring position of the fluorecein or rhodamine donor dye and the second substituent of the functional group is a substituent of the acceptor dye at the five or six ring position of the fluorecein or rhodamine acceptor dye.

48. A method for sequencing a nucleic acid sequence comprising:

forming extended primers by hybridizing a nucleic acid sequence with a primer in the presence of deoxynucleoside triphosphates, at least one fluorescently labeled dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxyucteoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the extended primers; and

fluorescently detecting the fluorescently labeled dideoxynucleotides attached to the separated extended primers;

the fluorescently labeled dideoxynucleotide triphosphate including a dideoxynucleotide triphosphate and an energy transfer dye comprising

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a fluorecein or rhodamine donor dye capable of absorbing light at a first wavelength, the functional group having a second substituent comprising a fluorecein or rhodamine acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

wherein the first substituent of the functional group is a substituent of the donor dye at the five or six ring position of the fluorecein or rhodamine donor dye and the second substituent of the functional group is a substituent of the acceptor dye at the five or six ring position of the fluorecein or rhodamine acceptor dye.

49. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, at least one fluorescent dye comprising

a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a fluorecein or rhodamine donor dye capable of absorbing light at a first wavelength, the functional group having a second substituent comprising a fluorecein or rhodamine acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

wherein the first substituent of the functional group is a substituent of the donor dye at the five or six ring position of the fluorecein or rhodamine donor dye and the second substituent of the functional group is a substituent of the acceptor dye at the five or six ring position of the fluorecein or rhodamine acceptor dye.

56. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, multiple of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

57. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, multiple of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing substantially all of the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response.

58. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, multiple of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum at least about 100 nm greater than the absorbance maximum of the donor dye.

59. A kit for sequencing a nucleic acid sequence comprising:

at least four fluorescently labeled dideoxynucleotide triphosphates, each having a different fluorescent dye attached to a different dideoxynucleotide triphosphate, multiple of the fluorescently labeled dideoxynucleotide triphosphates comprising as the fluorescent dye a compound comprising a functional group selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure, the functional group having a first substituent comprising a donor dye having a xanthene ring structure, the donor dye being capable of absorbing light at a first wavelength and emitting excitation energy in response and a second substituent comprising an acceptor dye capable of absorbing excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum greater than about 600 nm.