Energy transfer dyes with enhanced fluorescence

• US 5,945,526 A
• Filed: 03/23/1998
• Issued: 08/31/1999
• Est. Priority Date: 05/03/1996
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Lee, Linda G., Spurgeon, Sandra L., Rosenblum, Barnett
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US09/046,203
• Time in Patent Office: 526 Days
• Field of Search: 536/26.6, 536/24.33, 435/6, 435/91.2, 549/223
• 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 R₂₁ Z₁ C(O)R₂₂ R₂₈ 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. An energy transfer dye having the structure ##STR120## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₂₁ is a C_1-5 alkyl attached to the donor dye;

R₂₂ includes 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 which is attached to the carbonyl carbon; and

R₂₈ includes a functional group which attaches the linker to the acceptor dye.

2. The energy transfer dye according to claim 1 wherein R₂₂ includes a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

3. The energy transfer dye according to claim 1 wherein the dye has the structure ##STR121## wherein Z₂ is selected from the group consisting of NH, sulfur and oxygen;

and

R₂₉ is a C₁₅ alkyl.

4. The energy transfer dye according to claim 1 wherein the donor dye is a member of the xanthene class of dyes.

5. The energy transfer dye according to claim 4 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. The energy transfer dye 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.

7. The energy transfer dye according to claim 6 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

8. The energy transfer dye 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 R110, and carboxy R6G.

9. The energy transfer dye 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 R110, carboxy R6G, carboxy-X-rhodamines and Cy5.

10. The energy transfer dye according to claim 1 wherein the acceptor dye is selected from the group consisting DR110-2, DR6G-2, DTMR and DROX.

11. An energy transfer dye having the structure ##STR122## where Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₁₁ -R₁₃ and R₁₅ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

R₂₁ is a C_1-5 alkyl;

R₂₂ includes 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 which is attached to the carbonyl carbon;

R₂₈ includes a functional group which attaches the linker to the acceptor dye; and

ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes.

12. The energy transfer dye according to claim 11 wherein R₂₂ includes a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

13. The energy transfer dye according to claim 11 wherein the dye has the structure ##STR123## wherein Z₂ is selected from the group consisting of NH, sulfur and oxygen;

and

R₂₉ is a C_1-5 alkyl.

14. The energy transfer dye according to claim 11 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

15. The energy transfer dye according to claim 11 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes.

16. The energy transfer dye according to claim 11 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

17. The energy transfer dye according to claim 11 wherein the donor dye is selected from the group consisting of carboxyfluoresceins, 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, carboxyrhodamines, N,N,N'"'"',N'"'"'-tetramethyl carboxyrhodamines, carboxy R110 and carboxy R6G.

18. The energy transfer dye according to claim 17 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

19. The energy transfer dye according to claim 11 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 R110, carboxy R6G, carboxy-X-rhodamines and Cy5.

20. The energy transfer dye according to claim 11 wherein the acceptor has the general structure ##STR124## wherein:

Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₆ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ -X₅ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof; and

one of X₃ and X₄ is attached to the R₂₈ group.

21. The energy transfer dye according to claim 11 wherein the acceptor dye has the general structure ##STR125## wherein:

R₁ -R₄ are each independently selected from the group consisting of hydrogen, and alkyl or where one or more of the groups of R₁ and R₅, R₂ and R₆, R₃ and R₈, R₄ and R₉ are taken together to form a ring;

R₅ -R₁₀ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl, and substituted phenyl, or where two or more of R₅ -R₁₀ are taken together to form one or more rings;

X₁, X₃ and X₄ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, or alkoxy;

X₂ and X₅ are chlorine; and

one of X₃ and X₄ are attached to R₂₈.

22. The energy transfer dye according to claim 21 wherein the rings formed by substituents R₅ -R₁₀ are 5, 6 or 7 membered rings.

23. The energy transfer dye according to claim 21 wherein one or more of the groups of R₁ and R₅, R₂ and R₆, R₃ and R₈, R₄ and R₉ are taken together to form a 5, 6 or 7 membered ring.

24. The energy transfer dye according to claim 21 wherein R₁ -R₁₀, X₁, X₃ and X₄ are selected to correspond to a dye selected from the group consisting of DR110-2, DR6G-2, DTMR-2, and DROX-2.

25. An energy transfer fluorescent dye having the general structure ##STR126## wherein:

Y₁, Y₁ '"'"', Y₂ '"'"', and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,

R₁₁ -R₁₆ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof, and

X₁ -X₅ and X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₆, and X₁ -X₅ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ and X₄ and one of X₃ '"'"' and X₄ '"'"' are taken together to form a linker having the general formula R₂₅ Z₃ C(O)R₃₇ where R₂₅ is a C_1-4 alkyl attached to the donor dye at the X₃ or X₄ substituent, Z₃ is either NH, O or S, C(O) is a carbonyl group and R₃₇ is a substituent attached to the acceptor dye at the X₃ '"'"' or X₁ '"'"' substituent.

26. A fluorescently labeled reagent comprising:

a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and

an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye including a dye having the structure ##STR127## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₂₁ is a C_1-5 alkyl attached to the donor dye;

R₂₂ includes 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 which is attached to the carbonyl carbon; and

R₂₈ includes a functional group which attaches the linker to the acceptor dye.

27. The fluorescently labeled reagent according to claim 26 wherein R₂₂ includes a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

28. The fluorescently labeled reagent according to claim 26 wherein the dye has the structure ##STR128## wherein Z₂ is selected from the group consisting of NH, sulfur and oxygen;

and

R₂₉ is a C_1-5 alkyl.

29. The fluorescently labeled reagent according to claim 26 wherein the donor dye is a member of the xanthene class of dyes.

30. The fluorescently labeled reagent according to claim 29 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

31. The fluorescently labeled reagent according to claim 26 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes.

32. The fluorescently labeled reagent according to claim 26 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 R110, and carboxy R6G.

33. The fluorescently labeled reagent according to claim 26 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 R110, carboxy R6G, carboxy-X-rhodamines and Cy5.

34. The fluorescently labeled reagent according to claim 26 wherein the acceptor dye is selected from the group consisting DR110-2, DR6G-2, DTMR and DROX.

35. The fluorescently labeled reagent according to claim 26 wherein the reagent is selected from the group consisting of deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside diphosphate and deoxynucleoside triphosphate.

36. The fluorescently labeled reagent according to claim 35 wherein the deoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.

37. The fluorescently labeled reagent according to claim 26 wherein the reagent is selected from the group consisting of dideoxynucleoside, dideoxynucleoside monophosphate, dideoxynucleoside diphosphate and dideoxynucleoside triphosphate.

38. The fluorescently labeled reagent according to claim 26 wherein the dideoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.

39. The fluorescently labeled reagent according to claim 35 wherein the reagent is an oligonucleotide.

40. The fluorescently labeled reagent according to claim 39 wherein the oligonucleotide has a 3'"'"' end which is extendable by using a polymerase.

41. A fluorescently labeled reagent comprising:

a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and

an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye including a dye having the structure ##STR129## where Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₁₁ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

R₂₁ is a C_1-5 alkyl;

R₂₂ includes 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 which is attached to the carbonyl carbon;

R₂₈ includes a functional group which attaches the linker to the acceptor dye; and

ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes.

42. The fluorescently labeled reagent according to claim 41 wherein R₂₂ includes a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

43. The fluorescently labeled reagent according to claim 41 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

44. The fluorescently labeled reagent according to claim 41 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes.

45. The fluorescently labeled reagent according to claim 44 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

46. The fluorescently labeled reagent according to claim 41 wherein the donor dye is selected from the group consisting of carboxyfluoresceins, 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, carboxyrhodamines, N,N,N'"'"',N'"'"'-tetramethyl carboxyrhodamines, carboxy R110, and carboxy R6G.

47. The fluorescently labeled reagent according to claim 41 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 R110, carboxy R6G, carboxy-X-rhodamines and Cy5.

48. The fluorescently labeled reagent according to claim 41 wherein the acceptor has the general structure ##STR130## wherein:

Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₆ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ -X₅ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof; and

one of X₃ and X₄ is attached to the R₂₈ group.

49. The fluorescently labeled reagent according to claim 41 wherein the acceptor dye has the general structure ##STR131## wherein:

R₁ -R₄ are each independently selected from the group consisting of hydrogen, and alkyl or where one or more of the groups of R₁ and R₅, R₂ and R₆, R₃ and R₇, R₄ and R₈ are taken together to form a ring;

R₅ -R₁₀ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl, and substituted phenyl, or where two or more of R₅ -R₁₀ are taken together to form one or more rings;

X₁, X₃ and X₄ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, or alkoxy;

X₂ and X₅ are chlorine; and

one Of X₃ and X₄ are attached to R₂₈.

50. The fluorescently labeled reagent according to claim 49 wherein R₁ -R₁₀, X₁, X₃ and X₄ are selected to correspond to a dye selected from the group consisting of DR110-2, DR6G-2, DTMR-2, and DROX-2.

51. The fluorescently labeled reagent according to claim 41 wherein the reagent is selected from the group consisting of deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside diphosphate and deoxynucleoside triphosphate.

52. The fluorescently labeled reagent according to claim 51 wherein the deoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.

53. The fluorescently labeled reagent according to claim 41 wherein the reagent is selected from the group consisting of dideoxynucleoside, dideoxynucleoside monophosphate, dideoxynucleoside diphosphate and dideoxynucleoside triphosphate.

54. The fluorescently labeled reagent according to claim 41 wherein the dideoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.

55. The fluorescently labeled reagent according to claim 41 wherein the reagent is an oligonucleotide.

56. The fluorescently labeled reagent according to claim 55 wherein the oligonucleotide has a 3'"'"' end which is extendable by using a polymerase.

57. A fluorescently labeled reagent comprising:

a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and

an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye including a dye having the structure ##STR132## wherein;

Y₁, Y₁ '"'"', Y₂ '"'"' and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,

R₁₁ -R₁₆ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido nitrile alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof, and

X₁ -X₅ and X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₆, and X₁ -X₅ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ and X₄ and one of X₃ '"'"' and X₄ '"'"' are taken together to form a linker one of X₃ and X₄ and one of X₃ '"'"' and X₄ '"'"' are taken together to form a linker having the general formula R₂₅ Z₃ C(O)R₃₇ where R₂₅ is a C_1-4 alkyl attached to the donor dye at the X₃ or X₄ substituent, Z₃ is either NH, O or S, C(O) is a carbonyl group and R₃₇ is a substituent attached to the acceptor dye at the X₃ '"'"' or X₁ '"'"' substituent.

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

forming a mixture of 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 mixture of extended primers; and

determining the sequence of the nucleic acid sequence by fluorescently measuring the mixture of extended primers formed;

the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and having a 3'"'"' end extendable by a polymerase, and

an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye having the structure ##STR133## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₂₁ is a C_1-5 alkyl attached to the donor dye;

R₂₂ includes 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 which is attached to the carbonyl carbon; and

R₂₈ includes a functional group which attaches the linker to the acceptor dye.

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

forming a mixture of 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 mixture of extended primers; and

determining the sequence of the nucleic acid sequence by fluorescently measuring the mixture of extended primers formed;

the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and having a 3'"'"' end extendable by a polymerase, and

an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye having the structure ##STR134## where Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₁₁ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

R₂ 1 is a C_1-5 alkyl;

R₂₂ includes 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 which is attached to the carbonyl carbon;

R₂₈ includes a functional group which attaches the linker to the acceptor dye; and

ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes.

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

forming a mixture of 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 deoxynucleoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the mixture of extended primers; and

determining the sequence of the nucleic acid sequence by detecting the fluorescently labeled dideoxynucleotide attached to the separated mixture of extended primers;

the fluorescently labeled dideoxynucleoside triphosphate including

a dideoxynucleoside triphosphate, and

an energy transfer fluorescent dye attached to the dideoxynucleoside triphosphate, the energy transfer dye having the structure ##STR135## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₂₁ is a C_1-5 alkyl attached to the donor dye;

R₂₂ includes 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 which is attached to the carbonyl carbon; and

R₂₈ includes a functional group which attaches the linker to the acceptor dye.

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

forming a mixture of 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 deoxynucleoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the mixture of extended primers; and

determining the sequence of the nucleic acid sequence by detecting the fluorescently labeled dideoxynucleotide attached to the separated mixture of extended primers;

the fluorescently labeled dideoxynucleoside triphosphate including

a dideoxynucleoside triphosphate, and

an energy transfer fluorescent dye attached to the dideoxynucleoside triphosphate, the dye having the structure ##STR136## where Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

Z₁ is selected from the group consisting of NH, sulfur and oxygen;

R₁₁ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

R₂₁ is a C_1-5 alkyl;

R₂₂ includes 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 which is attached to the carbonyl carbon;

R₂₈ includes a functional group which attaches the linker to the acceptor dye; and

ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes.

63. The energy transfer dye according to claim 61 wherein the acceptor dye has a rhodamine ring structure.

62. An energy transfer fluorescent dye comprising:

a donor dye which absorbs light at a first wavelength and emits excitation energy in response;

an acceptor dye selected from the group consisting of xanthene, cyanine and phthalocyanine dyes which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum that is greater than about 600 nm; and

a linker attaching the donor dye to the acceptor dye which has a sufficiently short length that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye.

64. An energy transfer fluorescent dye comprising:

a donor dye which absorbs light at a first wavelength and emits excitation energy in response;

an acceptor dye selected from the group consisting of xanthene, cyanine and phthalocyanine dyes which is capable of absorbing the 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; and

a linker attaching the donor dye to the acceptor dye which has a sufficiently short length that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye.

65. The energy transfer dye according to claim 64 wherein the acceptor dye has a rhodamine ring structure.

71. The energy transfer dye according to claim 65 wherein the linker functional group is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

66. An energy transfer dye comprising:

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

a linker linking the donor dye to the acceptor dye such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker 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.

67. The energy transfer dye according to claim 66 wherein the energy transfer dye has the structure:

##STR137## wherein Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; and

R₁₁ -R₁₃ and R₁₅ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof.

68. The energy transfer dye according to claim 66 wherein the energy transfer dye has the structure:

##STR138## wherein Y₁ '"'"' and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.

69. The energy transfer dye according to claim 66 wherein the energy transfer dye has the structure:

##STR139## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₃, R_15-R17 and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₃, and R₁₅ -R₁₇ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.

70. The energy transfer dye according to claim 66 wherein the energy transfer dye has the structure:

##STR140## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₆ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ -X₅ and X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₆ and X₁ -X₅ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ and X₄ and one of X₃ '"'"' and X₄ '"'"' are taken together to form the linker.

72. The energy transfer dye according to claim 66 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

73. The energy transfer dye according to claim 66 wherein the donor dye is a member of the xanthene class of dyes.

74. The energy transfer dye according to claim 73 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

75. The energy transfer dye according to claim 66 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes.

76. The energy transfer dye according to claim 75 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

77. The energy transfer dye according to claim 66 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.

78. The energy transfer dye according to claim 66 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.

79. The energy transfer dye according to claim 66 wherein the acceptor dye is selected from the group consisting DR110-2, DR6G-2, DTMR and DROX.

82. The fluorescently labeled reagent according to claim 70 wherein the energy transfer fluorescent dye has the structure:

##STR142## wherein Y₁ '"'"' and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.

80. A fluorescently labeled reagent comprising:

a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and

an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye

including

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

a linker linking the donor dye to the acceptor dye such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker 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.

81. The fluorescently labeled reagent according to claim 80 wherein the energy transfer fluorescent dye has the structure:

##STR141## wherein Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; and

R_11-R13 and R₁₅ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof.

83. The fluorescently labeled reagent according to claim 80 wherein the energy transfer fluorescent dye has the structure:

##STR143## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₃, R₁₅ -R₁₇ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₃ and R₁₅ -R₁₇ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.

84. The fluorescently labeled reagent according to claim 80 wherein the energy transfer fluorescent dye has the structure:

##STR144## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₆ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ -X₅ and X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₆ and X₁ -X₅ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ -X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ and X₄ and one of X₃ '"'"' and X₄ '"'"' are taken together to form the linker.

85. The fluorescently labeled reagent according to claim 80 wherein the linker functional group is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

86. The fluorescently labeled reagent according to claim 80 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

87. The fluorescently labeled reagent according to claim 80 wherein the donor dye is a member of the xanthene class of dyes.

88. The fluorescently labeled reagent according to claim 87 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

89. The fluorescently labeled reagent according to claim 80 wherein the donor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine and asymmetric benzoxanthene dyes.

90. The fluorescently labeled reagent according to claim 89 wherein the acceptor dye is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine dyes.

91. The fluorescently labeled reagent according to claim 80 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.

92. The fluorescently labeled reagent according to claim 80 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.

93. The fluorescently labeled reagent according to claim 80 wherein the acceptor dye is selected from the group consisting DR110-2, DR6G-2, DTMR and DROX.

94. The fluorescently labeled reagent according to claim 80 wherein the reagent is selected from the group consisting of deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside diphosphate and deoxynucleoside triphosphate.

95. The fluorescently labeled reagent according to claim 94 wherein the deoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.

96. The fluorescently labeled reagent according to claim 80 wherein the reagent is selected from the group consisting of dideoxynucleoside, dideoxynucleoside monophosphate, dideoxynucleoside diphosphate and dideoxynucleoside triphosphate.

97. The fluorescently labeled reagent according to claim 96 wherein the dideoxynucleotides are selected from the group consisting of dideoxycytosine, dideoxyadenosine, dideoxyguanosine, and dideoxythymidine.

98. The fluorescently labeled reagent according to claim 80 wherein the reagent is an oligonucleotide.

99. The fluorescently labeled reagent according to claim 80 wherein the oligonucleotide has a 3'"'"' end which is extendable by using a polymerase.

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

forming a mixture of 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 mixture of extended primers; and

determining the sequence of the nucleic acid sequence by fluorescently measuring the mixture of extended primers formed;

the fluorescently labeled oligonucleotide primer including

an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and having a 3'"'"' end extendable by a polymerase, and

an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response,

an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, and

a linker linking the donor dye to the acceptor dye such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker 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.

101. The method according to claim 100 wherein the energy transfer fluorescent dye has the structure:

##STR145## wherein Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; and

R₁₁ -R₁₃ and R₁₅ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof.

102. The method according to claim 100 wherein the energy transfer fluorescent dye has the structure:

##STR146## wherein Y₁ '"'"' and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.

103. The method according to claim 100 wherein the energy transfer fluorescent dye has the structure:

##STR147## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₃, R₁₅ -R₁₇ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₃ and R₁₅ -R₁₇ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.

104. The method according to claim 100 wherein the energy transfer fluorescent dye has the structure:

##STR148## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₆ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ -X₅ and X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₆ and X₁ -X₅ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ and X₄ and one of X₃ '"'"' and X₄ '"'"' are taken together to form the linker.

109. The method according to claim 100 wherein the energy transfer fluorescent dye has the structure:

##STR152## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₆ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ -X₅ and X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₆, and X₁ -X₅ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ and X₄ and one of X₃ '"'"' and X₄ '"'"' are taken together to form the linker.

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

forming a mixture of 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 deoxynucleoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer;

separating the mixture of extended primers; and

determining the sequence of the nucleic acid sequence by detecting the fluorescently labeled dideoxynucleotide attached to the separated mixture of extended primers;

the fluorescently labeled dideoxynucleoside triphosphate including

a dideoxynucleoside triphosphate, and

an energy transfer fluorescent dye attached to the dideoxynucleoside triphosphate, the energy transfer dye including

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response,

an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, and

a linker linking the donor dye to the acceptor dye such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker 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.

106. The method according to claim 105 wherein the energy transfer fluorescent dye has the structure:

##STR149## wherein Y₁ and Y₂ are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; and

R₁₁ -R₁₃ and R₁₅ -R₁₇ are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof.

107. The method according to claim 105 wherein the energy transfer fluorescent dye has the structure:

##STR150## wherein Y₁ '"'"' and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.

108. The method according to claim 105 wherein the energy transfer fluorescent dye has the structure:

##STR151## wherein;

Y₁, Y₁ '"'"', Y₂ and Y₂ '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;

R₁₁ -R₁₃, R₁₅ -R₁₇ and R₁₁ '"'"'-R₁₆ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof;

X₁ '"'"'-X₅ '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof;

Y₁, Y₂, R₁₁ -R₁₃ and R₁₅ -R₁₇ are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

Y₁ '"'"', Y₂ '"'"', R₁₁ '"'"'-R₁₆ '"'"', and X₁ '"'"'-X₅ '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

one of X₃ '"'"' and X₄ '"'"' includes the linker.