METHODS OF ANALYZING POLYNUCLEOTIDES EMPLOYING ENERGY TRANSFER DYES
First Claim
1. A method of analyzing a polynucleotide, comprising the steps of:
- (a) forming a plurality of differently sized fluorescently labeled extended oligonucleotide primers that are complementary to the polynucleotide by enzymatically extending an oligonucletoide primer that is complementary to a region of the polynucleotide and that is labeled with an energy transfer dye in the presence of the polynucleotide, nucleotides suitable for effecting template-dependent primer extension and at least one terminating nucleotide, such that the labeled primer is extended until the terminating nucleotide is incorporated into the labeled extended primer;
(b) separating the fluorescently labeled extended oligonucleotide primers based upon their sizes; and
(c) detecting the fluorescently labeled extended primers based upon their fluorescence, wherein the energy transfer dye comprises;
(i) a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;
(ii) a 4,7-dichlororhodamine acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto; and
(iii) a non-nucleosidic linker linking the xanthene donor dye to the 4,7-dichlororhodamine acceptor dye.
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Accused Products
Abstract
Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate 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 R21Z1C(O)R22R28 where R21 is a C1-5 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.
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Citations
31 Claims
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1. A method of analyzing a polynucleotide, comprising the steps of:
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(a) forming a plurality of differently sized fluorescently labeled extended oligonucleotide primers that are complementary to the polynucleotide by enzymatically extending an oligonucletoide primer that is complementary to a region of the polynucleotide and that is labeled with an energy transfer dye in the presence of the polynucleotide, nucleotides suitable for effecting template-dependent primer extension and at least one terminating nucleotide, such that the labeled primer is extended until the terminating nucleotide is incorporated into the labeled extended primer;
(b) separating the fluorescently labeled extended oligonucleotide primers based upon their sizes; and
(c) detecting the fluorescently labeled extended primers based upon their fluorescence, wherein the energy transfer dye comprises;
(i) a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;
(ii) a 4,7-dichlororhodamine acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto; and
(iii) a non-nucleosidic linker linking the xanthene donor dye to the 4,7-dichlororhodamine acceptor dye. - 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, 25, 26, 27, 28, 29, 30)
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31. In a method of analyzing a sequence of a polynucleotide employing fluorescently labeled primers and terminating nucleotides, the improvement comprising utilizing at least one primer that is labeled with an energy transfer dye comprising:
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(i) a xanthene donor dye;
(ii) a 4,7-dichlororhodamine acceptor dye; and
(iii) a non-nucleosidic linker covalently linking the xanthene donor dye to the 4,7-dichlororhodamine acceptor dye.
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Specification