Energy transfer dyes with enhanced fluorescence

An energy transfer fluorescent dye comprising:

• a donor dye with a xanthene ring structure the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• an acceptor dye which is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine and 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 or 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 his a sufficiently short length that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye.

An energy transfer fluorescent dye comprising:

• a donor dye which absorbs light at a first wavelength and emitting excitation energy in response, the donor dye having the general structure ##STR61## wherein:
• Y.sub.1 and Y.sub.2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,
• R.sub.11 -R.sub.16 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
• X.sub.1, X.sub.2, X.sub.4 and 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;
• an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor dye having the general structure ##STR62## wherein:
• Y.sub.1 and Y.sub.2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,
• R.sub.11 -R.sub.16 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
• X.sub.1, X.sub.2, X.sub.4 and X.sub.5 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
• a linker forming the X.sub.3 substituents of the donor and acceptor dyes.

A fluorescently labeled reagent comprising:

• a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate and nuleoside triphosphate, modified to be attached 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 with a xanthene ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• 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
• a linker attaching the donor dye to the acceptor dye, the linker having a 4' end which includes a R.sub.1 XC(O)R.sub.2 group where R.sub.1 is a C.sub.1-5 alkyl attached to a 4' ring position on the xanthene ring structure of the donor dye, X is selected from the group consisting of NH, sulfur and oxygen, C(O) is a carbonyl group, and R.sub.2 attaches the linker to the acceptor dye and includes a functional group which is attached to the carbonyl carbon selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond and a fused ring structure.

A fluorescently labeled reagent comprising:

• a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate and nucleoside triphosphate, modified to be attached 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 with a xanthene ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• an acceptor dye which is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine and 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 or 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.

A fluorescently labeled oligonucleotide primer comprising:

• an oligonucleotide;
• and
• an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including
• a donor dye with a xanthene ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• 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
• a linker attaching the donor dye to the acceptor dye, the linker having a 4' end which includes a R.sub.1 XC(O)R.sub.2 group where R.sub.1 is a C.sub.1-5 alkyl attached to 4' ring position on the xanthene ring structure of the donor dye, X is selected from the group consisting of NH, sulfur and oxygen, C(O) is a carbonyl group, and R.sub.2 attaches the linker to the acceptor dye a includes a functional group which is attached to the carbonyl carbon selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond and a fused ring structure.

A fluorescently labeled oligonucleotide primer comprising:

• an oligonucleotide;
• and
• an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including
• a donor dye with a xanthene ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response,
• an acceptor dye which is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine and 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 or 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.

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
• an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including
• a donor dye with a xanthene ring structure the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• 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
• a linker attaching the donor dye to the acceptor dye, the linker having a 4' end which includes a R.sub.1 XC(O)R.sub.2 group where R.sub.1 is a C.sub.1-5 alkyl attached to a 4' ring position on the xanthene ring structure of the donor dye, X is selected from the group consisting of NH, sulfur and oxygen, C(O) is a carbonyl group, and R.sub.2 atttaches the linker to the acceptor dye and includes a functional group which is attached to the carbonyl carbon selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond and a fused ring structure.

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 fluorescantly 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
• an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including
• a donor dye with a xanthene ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response,
• an acceptor dye which is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine and 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 or 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.

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 deoxyucteoside 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 dideoxynucleotide triphosphate including
• a dideoxynucleotide triphosphate, and
• an energy transfer fluorescent dye attached to the dideoxynucleotide triphosphate, the dye including
• a donor dye with a xanthene ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• 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
• a linker attaching the donor dye to the acceptor dye, the linker having a 4' end which includes a R.sub.1 XC(O)R.sub.2 group where R.sub.1 is a C.sub.1-5 alkyl attached to a 4' ring position on the xanthene ring structure of the donor dye, X is selected from the group consisting of NH, sulfur and oxygen, C(O) is a carbonyl group, and R.sub.2 attaches the linker to the acceptor dye and includes a functional group which is attached to the carbonyl carbon selected from the group consisting of an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond and a fused ring structure.

A method for sequencing 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 dideoxynucleotide triphosphate including
• a dideoxynucleotide triphosphate, and
• an energy transfer fluorescent dye attached to the dideoxynucleotide triphosphate, the dye including
• a donor dye with a xanthene ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response,
• an acceptor dye which is a member of a class of dyes selected from the group consisting of xanthene, cyanine, phthalocyanine and squaraine and 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 or 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.

An energy transfer fluorescent dye comprising:

• a donor dye which has a carboxy fluorescein ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• an acceptor dye which has a 5-carboxy-X-rhodamine ring structure and is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;
• 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.

A fluorescently labeled reagent comprising;

• a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate and nucleoside triphosphate, modified to be attached 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 which has a carboxy fluorescein ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• an acceptor dye which has a 5-carboxy-X-rhodamine ring structure and is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;
• and
• a linker attaching the donor dye to the acceptor dye which has a sufficiently short length that the acceptor dye absorbs substantially al of the excitation energy emitted by the donor dye.

A fluorescently labeled oligonucleotide primer comprising:

• oliganucleotide;
• and
• an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including
• a donor dye which has a carboxy fluorescein ring structure, the donor dye absorbing light at a first wavelength and emitting excitation energy in response;
• an acceptor dye which has a 5-carboxy-X-rhodamine ring structure and is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;
• 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.