Probes labeled with energy transfer coupled dyes
First Claim
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1. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
- (a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label consists of a first specific chemical structure comprising a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first backbone chain, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label consists of a second specific chemical structure comprising a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second backbone chain, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label consists of a third specific chemical structure comprising a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third backbone chain, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label consists of a fourth specific chemical structure comprising a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth backbone chain, with energy transfer from said fourth donor to said fourth acceptor, further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids produced in said step (a); and
(c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.
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Abstract
Fluorescent labels having at least one donor and at least one acceptor fluorophore bonded to a polymeric backbone in energy transfer relationship, as well as methods for their use, are provided. Of particular interest are the subject labels wherein the polymeric backbone is a nucleic acid and the donor fluorophore is bonded to the 5′ terminus of said nucleic acid. Such labels find use as primers in applications involving nucleic acid chain extension, such as sequencing, PCR and the like.
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Citations
30 Claims
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1. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label consists of a first specific chemical structure comprising a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first backbone chain, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label consists of a second specific chemical structure comprising a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second backbone chain, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label consists of a third specific chemical structure comprising a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third backbone chain, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label consists of a fourth specific chemical structure comprising a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth backbone chain, with energy transfer from said fourth donor to said fourth acceptor, further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids produced in said step (a); and
(c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.- View Dependent Claims (9, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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2. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first backbone chain, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second backbone chain, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third backbone chain, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth backbone chain, provided further that said first donor and said first acceptor are covalently bonded to said first backbone chain to produce a first fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second backbone chain to produce a second fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third backbone chain to produce a third fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth backbone chain to produce a fourth fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said fourth donor to said fourth acceptor, and further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids produced in said step (a); and
(c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.
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3. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label is substantially pure and comprises a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first backbone chain, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label is substantially pure and comprises a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second backbone chain, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label is substantially pure and comprises a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third backbone chain, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label is substantially pure and comprises a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth backbone chain, with energy transfer from said fourth donor to said fourth acceptor, further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template produced in said step (a); and
(c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.
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4. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first backbone chain, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second backbone chain, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third backbone chain, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth backbone chain, provided further that said first donor and said first acceptor are covalently bonded to said first backbone chain to produce a substantially pure first fluorescent energy-transfer label with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second backbone chain to produce a substantially pure second fluorescent energy-transfer label with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third backbone chain to produce a substantially pure third fluorescent energy-transfer label with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth backbone chain to produce a substantially pure fourth fluorescent energy-transfer label with energy transfer from said fourth donor to said fourth acceptor, and further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids produced in said step (a); and
(c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.
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5. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label consists of a first specific chemical structure comprising a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first covalently bonded chain of atoms, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label consists of a second specific chemical structure comprising a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second covalently bonded chain of atoms, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label consists of a third specific chemical structure comprising a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third covalently bonded chain of atoms, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label consists of a fourth specific chemical structure comprising a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth covalently bonded chain of atoms, with energy transfer from said fourth donor to said fourth acceptor, further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids produced in said step (a); and
(c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.- View Dependent Claims (11, 13, 15, 17, 19)
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6. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first covalently bonded chain of atoms, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second covalently bonded chain of atoms, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third covalently bonded chain of atoms, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth covalently bonded chain of atoms, provided further that said first donor and said first acceptor are covalently bonded to said first covalently bonded chain of atoms to produce a first fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second covalently bonded chain of atoms to produce a second fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third covalently bonded chain of atoms to produce a third fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth covalently bonded chain of atoms to produce a fourth fluorescent energy-transfer label having essentially only one specific chemical structure with energy transfer from said fourth donor to said fourth acceptor, and further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids produced in said step (a); and
(c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.
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7. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label is substantially pure and comprises a first fluorescent donor and a first fluorescent acceptor covalently bonded to a first covalently bonded chain of atoms, with energy transfer from said first donor to said first acceptor, said second fluorescent energy-transfer label is substantially pure and comprises a second fluorescent donor and a second fluorescent acceptor covalently bonded to a second covalently bonded chain of atoms, with energy transfer from said second donor to said second acceptor, said third fluorescent energy-transfer label is substantially pure and comprises a third fluorescent donor and a third fluorescent acceptor covalently bonded to a third covalently bonded chain of atoms, with energy transfer from said third donor to said third acceptor, and said fourth fluorescent energy-transfer label is substantially pure and comprises a fourth fluorescent donor and a fourth fluorescent acceptor covalently bonded to a fourth covalently bonded chain of atoms, with energy transfer from said fourth donor to said fourth acceptor, further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template produced in said step (a); and
(c) identifying and distinguishing said different size deoxyribonucleic acids complementary to said deoxyribonucleic acid template separated in said step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.
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8. A method of identifying and distinguishing different deoxyribonucleic acids produced in chain termination sequencing reactions, said method comprising:
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(a) producing a first, a second, a third, and a fourth set of different size deoxyribonucleic acids complementary to a deoxyribonucleic acid template using a nucleic acid polymerase, wherein;
said first set consists essentially of different size deoxyribonucleic acids covalently bonded to a first fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to adenine, said second set consists essentially of different size deoxyribonucleic acids covalently bonded to a second fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to guanine, said third set consists essentially of different size deoxyribonucleic acids covalently bonded to a third fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to thymine, and said fourth set consists essentially of different size deoxyribonucleic acids covalently bonded to a fourth fluorescent energy-transfer label and terminates with a chain terminator comprising a nitrogenous base complementary to cytosine, provided that said first fluorescent energy-transfer label comprises a first fluorescent donor, a first fluorescent acceptor, and a first covalently bonded chain of atoms, said second fluorescent energy-transfer label comprises a second fluorescent donor, a second fluorescent acceptor, and a second covalently bonded chain of atoms, said third fluorescent energy-transfer label comprises a third fluorescent donor, a third fluorescent acceptor, and a third covalently bonded chain of atoms, and said fourth fluorescent energy-transfer label comprises a fourth fluorescent donor, a fourth fluorescent acceptor, and a fourth covalently bonded chain of atoms, provided further that said first donor and said first acceptor are covalently bonded to said first covalently bonded chain of atoms to produce a substantially pure first fluorescent energy-transfer label with energy transfer from said first donor to said first acceptor, said second donor and said second acceptor are covalently bonded to said second covalently bonded chain of atoms to produce a substantially pure second fluorescent energy-transfer label with energy transfer from said second donor to said second acceptor, said third donor and said third acceptor are covalently bonded to said third covalently bonded chain of atoms to produce a substantially pure third fluorescent energy-transfer label with energy transfer from said third donor to said third acceptor, and said fourth donor and said fourth acceptor are covalently bonded to said fourth covalently bonded chain of atoms to produce a substantially pure fourth fluorescent energy-transfer label with energy transfer from said fourth donor to said fourth acceptor, and further provided that the absorption wavelengths of said first donor, said second donor, said third donor, and said fourth donor are substantially the same and said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor, each have different emission wavelengths;
(b) separating said different size deoxyribonucleic acids produced in said step (a); and
(c) identifying and distinguishing the deoxyribonucleic acids separated in step (b) by irradiating with light absorbed by said first donor, said second donor, said third donor, and said fourth donor; and
detecting light emission from said first acceptor, said second acceptor, said third acceptor, and said fourth acceptor.
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Specification