Fluorescent nucleobase conjugates having anionic linkers
First Claim
1. A nucleic acid sequencing kit comprising at least one labeled nucleoside triphosphate comprising a conjugate comprising a dye labeled nucleobase of the form:
- (1) B-L-D, wherein B is a nucleobase, L is an anionic linker, and D comprises at least one fluorescent dye that comprises a xanthene, a rhodamine or a fluorescein, or(2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkers such that at least one of L1 and L2 is an anionic linker, and D1 and D2 are members of a fluorescent donor/acceptor pair, such that one of D1 and D2 is a donor dye capable of absorbing light at a first wavelength and emitting energy in response thereto, and the other of D1 and D2 is an acceptor dye capable of absorbing energy emitted by the donor dye and fluorescing at a second wavelength in response thereto, and at least one of D1 and D2 comprises a xanthene, a rhodamine or a fluorescein,wherein L or at least one of L1 and L2 comprises at least one anionic phosphate or anionic phosphate and one or more of the following components;
a 3′
-extendable primer,a polymerase enzyme,one or more 3′
extendable nucleotides which are not labeled with conjugate, and/or a buffering agent.
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Accused Products
Abstract
Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequencing using labeled terminators.
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Citations
24 Claims
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1. A nucleic acid sequencing kit comprising at least one labeled nucleoside triphosphate comprising a conjugate comprising a dye labeled nucleobase of the form:
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(1) B-L-D, wherein B is a nucleobase, L is an anionic linker, and D comprises at least one fluorescent dye that comprises a xanthene, a rhodamine or a fluorescein, or (2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkers such that at least one of L1 and L2 is an anionic linker, and D1 and D2 are members of a fluorescent donor/acceptor pair, such that one of D1 and D2 is a donor dye capable of absorbing light at a first wavelength and emitting energy in response thereto, and the other of D1 and D2 is an acceptor dye capable of absorbing energy emitted by the donor dye and fluorescing at a second wavelength in response thereto, and at least one of D1 and D2 comprises a xanthene, a rhodamine or a fluorescein, wherein L or at least one of L1 and L2 comprises at least one anionic phosphate or anionic phosphate and one or more of the following components; a 3′
-extendable primer,a polymerase enzyme, one or more 3′
extendable nucleotides which are not labeled with conjugate, and/or a buffering agent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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Specification