Energy transfer dyes with enchanced fluorescence

Claim 1

An energy transfer dye having the structure ##STR121## 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; C(O) is a carbonyl group; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 21 is a C 1-5 alkyl attached to the donor dye; R 22 is a substituent 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 R 28 includes a functional group which attaches the linker to the acceptor dye.

Claim 84

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 ##STR139## 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; C(O) is a carbonyl group; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 21 is a C 1-5 alkyl attached to the donor dye; R 22 is a substituent 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 R 28 includes a functional group which attaches the linker to the acceptor dye.

Claim 85

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 ##STR139## 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; C(O) is a carbonyl group; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 21 is a C 1-5 alkyl attached to the donor dye; R 22 is a substituent 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 R 28 includes a functional group which attaches the linker to the acceptor dye.

Claim 86

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 ##STR140## where C(O) is a carbonyl group; Y 1 and Y 2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 11 -R 17 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; R 21 is a C 1-5 alkyl; R 22 is a substituent 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; R 28 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.

Claim 87

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 ##STR140## where C(O) is a carbonyl group; Y 1 and Y 2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 11 -R 17 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; R 21 is a C 1-5 alkyl; R 22 is a substituent 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; R 28 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.

Claim 88

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 ##STR141## 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; C(O) is a carbonyl group; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 21 is a C 1-5 alkyl attached to the donor dye; R 22 is a substituent 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 R 28 includes a functional group which attaches the linker to the acceptor dye.

Claim 89

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 ##STR141## 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; C(O) is a carbonyl group; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 21 is a C 1-5 alkyl attached to the donor dye; R 22 is a substituent 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 R 28 includes a functional group which attaches the linker to the acceptor dye.

Claim 90

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 ##STR142## where C(O) is a carbonyl group; Y 1 and Y 2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 11 -R 17 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; R 21 is a C 1-5 alkyl; R 22 is a substituent 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; R 28 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.

Claim 91

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 ##STR142## where C(O) is a carbonyl group; Y 1 and Y 2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z 1 is selected from the group consisting of NH, sulfur and oxygen; R 11 -R 17 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; R 21 is a C 1-5 alkyl; R 22 is a substituent 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; R 28 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.