Methods of sequencing and detection using energy transfer labels with cyanine dyes as donor chromophores
First Claim
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1. In a method for sequencing a nucleic acid fragment, comprising:
- (i) hybridizing an oligonucleotide primer to said fragment,(ii) copying said fragment with a DNA polymerase in separate reaction vessels, each reaction vessel containing dNTPs, one of a set of distinct labeled oligonucleotide primers, all of which bind to a complementary sequence in said fragment, and one of a plurality of dideoxynucleotides, to generate single stranded DNA sequencing fragments, each labeled oligonucleotide primer comprising an oligonucleotide backbone having bonded thereto a donor fluorophore and an acceptor fluorophore in energy transfer relationship, the donor fluorophore of each labeled oligonucleotide primer absorbing light at a wavelength that is substantially common among all labeled oligonucleotide primers and the acceptor fluorophores emitting light energy at wavelengths that differ among labeled oligonucleotide primers in said set, and(iii) separating the resulting mixture of single-stranded DNA sequencing fragments into bands and determining the sequence by means of the order of the bands, the improvement in which the donor fluorophore of each labeled oligonucleotide primer is a cyanine dye characterized by a quantum yield of between about 0.01 and about 0.25, and each acceptor fluorophore has a quantum yield of at least 0.3.
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Abstract
Cyanine dyes are used as the donor fluorophore in energy transfer labels in which light energy is absorbed by a donor fluorophore and transferred to an acceptor fluorophore which responds to the transfer by emitting fluorescent light for detection. The cyanine dyes impart an unusually high sensitivity to the labels thereby improving their usefulness in a wide variety of biochemical procedures, particularly nucleic acid sequencing, nucleic acid fragment sizing, and related procedures.
30 Citations
28 Claims
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1. In a method for sequencing a nucleic acid fragment, comprising:
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(i) hybridizing an oligonucleotide primer to said fragment, (ii) copying said fragment with a DNA polymerase in separate reaction vessels, each reaction vessel containing dNTPs, one of a set of distinct labeled oligonucleotide primers, all of which bind to a complementary sequence in said fragment, and one of a plurality of dideoxynucleotides, to generate single stranded DNA sequencing fragments, each labeled oligonucleotide primer comprising an oligonucleotide backbone having bonded thereto a donor fluorophore and an acceptor fluorophore in energy transfer relationship, the donor fluorophore of each labeled oligonucleotide primer absorbing light at a wavelength that is substantially common among all labeled oligonucleotide primers and the acceptor fluorophores emitting light energy at wavelengths that differ among labeled oligonucleotide primers in said set, and (iii) separating the resulting mixture of single-stranded DNA sequencing fragments into bands and determining the sequence by means of the order of the bands, the improvement in which the donor fluorophore of each labeled oligonucleotide primer is a cyanine dye characterized by a quantum yield of between about 0.01 and about 0.25, and each acceptor fluorophore has a quantum yield of at least 0.3. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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2. In a method for the identification and detection of single stranded nucleic acid fragments in a mixture of nucleic acid fragments, said method comprising:
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(i) hybridizing different nucleic acid fragments in said mixture to different probes, each probe comprising an oligonucleotide backbone having bonded thereto a donor fluorophore and an acceptor fluorophore in energy transfer relationship, the donor fluorophore of each labeled oligonucleotide primer absorbing light at a wavelength that is substantially common among all labeled oligonucleotide primers and the acceptor fluorophores emitting light energy at wavelengths that differ among the different probes, (ii) separating said nucleic acid fragments thus hybridized, according to mobilities of said nucleic acid fragments, and (iii) detecting said hybridized nucleic acid fragments thus separated, by irradiation at the absorption wavelength of said donor fluorophores and detection of fluorescence emitted by each of said acceptor fluorophores, the improvement in which the donor fluorophore of each label is a cyanine dye having a quantum yield of between about 0.01 and about 0.25, and each acceptor fluorophore has a quantum yield of at least 0.3.
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3. In a method for the identification and detection of single or double stranded nucleic acid fragments in a mixture of nucleic acid fragments, said method comprising:
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(i) labeling each nucleic acid fragment of said mixture by means of apolymerase chain reaction employing a primer comprising an oligonucleotide backbone having bonded thereto a donor fluorophore and an acceptor fluorophore in energy transfer relationship, (ii) separating said nucleic acid fragments thus labeled, according to mobilities of said nucleic acid fragments, and (iii) detecting said hybridized nucleic acid fragments thus separated, by irradiation at the absorption wavelength of said donor fluorophore and detection of fluorescence emitted by said acceptor fluorophore, the improvement in which said donor fluorophore is a cyanine dye having a quantum yield of between about 0.01 and about 0.25, and said acceptor fluorophore is a dye has a quantum yield of at least 0.3.
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4. In a method for the identification and detection of single stranded nucleic acid fragments in a mixture of nucleic acid fragments, said method comprising:
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(i) ligating different nucleic acid fragments in said mixture to different labels, each probe comprising an oligonucleotide backbone having bonded thereto a donor fluorophore and an acceptor fluorophore in energy transfer relationship, the donor fluorophore of each label light at a wavelength that is substantially common among all labels and the acceptor fluorophores emitting light energy at wavelengths that differ among the different labels, (ii) separating said nucleic acid fragments thus ligated, according to mobilities of said nucleic acid fragments, and (iii) detecting said hybridized nucleic acid fragments thus separated, by irradiation at the absorption wavelength of said donor fluorophores and detection of fluorescence emitted by each of said acceptor fluorophores, the improvement in which the donor fluorophore of each label is a cyanine dye having a quantum yield of between about 0.01 and about 0.25, and each acceptor fluorophore has a quantum yield of at least 0.3.
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Specification