UV excitable fluorescent energy transfer dyes
First Claim
Patent Images
1. 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 having an absorption maxima at a wavelength between 250 nm and 400 nm; and
an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than 500 nm.
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Abstract
Novel energy transfer dyes which can be used with shorter wavelength light sources are provided. These dyes include a donor dye with an absorption maxima at a wavelength between about 250 to 450 nm and an acceptor dye which is capable of absorbing energy emitted from the donor dye. One of the energy transfer dyes has a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure and an acceptor dye which is capable of absorbing energy emitted from the donor dye, wherein the donor dye has an absorption maxima between about 250 and 450 nm and the acceptor dye has an emission maxima at a wavelength greater than about 500 nm.
119 Citations
26 Claims
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1. A method for sequencing a nucleic acid sequence comprising:
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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, andan energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including a donor dye having an absorption maxima at a wavelength between 250 nm and 400 nm; and
an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than 500 nm. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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2. A method for sequencing a nucleic acid sequence comprising:
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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, andan energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye including a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure; and
an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than 500 nm.
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3. A method for sequencing a nucleic acid sequence comprising:
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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 fluoresently 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 having an absorption maxima at a wavelength between 250 nm and 400 nm; and
an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than 500 nm. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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4. A method for sequencing a nucleic acid sequence comprising:
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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 which is a member of a class of dyes having a coumarin or pyrene ring structure; and
an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than 500 nm.
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25. A method for sequencing a nucleic acid sequence comprising:
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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, andan energy transfer fluorescent dye selected from the group consisting of DYE102, DYE104, DYE106, DYE108, DYE118 and DYE120.
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26. A method for sequencing a nucleic acid sequence comprising:
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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 selected from the group consisting of DYE102, DYE104, DYE106, DYE108, DYE118 and DYE120.
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Specification
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Current AssigneePerkinelmer Incorporated, Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
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Original AssigneePE Corp.
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InventorsLee, Linda G.
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Primary Examiner(s)Riley, Jezia
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Application NumberUS09/385,352Time in Patent Office935 DaysField of Search435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 549/381, 436/94US Class Current435/6.12CPC Class CodesC07H 19/10 with the saccharide radical...C07H 19/20 with the saccharide radical...C07H 21/00 Compounds containing two or...C07H 21/04 with deoxyribosyl as saccha...C09B 11/24 Phthaleins containing amino...C12Q 1/6818 involving interaction of tw...C12Q 1/6869 Methods for sequencingC12Q 2563/107 fluorescenceY10T 436/143333 Saccharide [e.g., DNA, etc.]
