Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system
First Claim
1. A method for the amplification of a nucleic acid sequence, comprising the steps of:
- providing a target nucleic acid sequence;
providing a first polynucleotide sequence having at least one donor chromophore, the first polynucleotide sequence being complementary to at least a portion of the target nucleic acid sequence;
providing a second polynucleotide sequence having at least one acceptor chromophore, the second polynucleotide sequence being complementary to a least a portion of the target sequence;
performing polymerase chain reaction to amplify the target nucleic acid sequence;
hybridizing the first and second polynucleotide sequences to the target nucleic acid sequence, such that when the first polynucleotide sequence and the second polynucleotide sequence are hybridized to the target nucleic acid sequence, the donor chromophore and acceptor chromophore are in an energy transfer relationship that substantially eliminates secondary quenching; and
irradiating the mixture to detect hybridizations of the first and second polynucleotide sequences by fluorescence energy transfer from the one or more donor chromophores of the first polynucleotide sequence to the one or more acceptor chromophores of the second polynucleotide sequence.
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Abstract
The present invention contemplates monitoring the amplification of nucleic acid using chromophore-containing polynucleotides having at least two donor chromophores operatively linked to the polynucleotide by linker arms, such that the chromophores are positioned by linkage along the length of the polynucleotide at a donor-donor transfer distance, and at least one fluorescing acceptor chromophore operatively linked to the polynucleotide by a linker arm, such that the fluorescing acceptor chromophore is positioned by linkage at a donor-acceptor transfer distance from at least one of the donor chromophores, to form a photonic structure for collecting photonic energy and transferring the energy to an acceptor chromophore, and methods using the photonic structures.
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Citations
24 Claims
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1. A method for the amplification of a nucleic acid sequence, comprising the steps of:
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providing a target nucleic acid sequence;
providing a first polynucleotide sequence having at least one donor chromophore, the first polynucleotide sequence being complementary to at least a portion of the target nucleic acid sequence;
providing a second polynucleotide sequence having at least one acceptor chromophore, the second polynucleotide sequence being complementary to a least a portion of the target sequence;
performing polymerase chain reaction to amplify the target nucleic acid sequence;
hybridizing the first and second polynucleotide sequences to the target nucleic acid sequence, such that when the first polynucleotide sequence and the second polynucleotide sequence are hybridized to the target nucleic acid sequence, the donor chromophore and acceptor chromophore are in an energy transfer relationship that substantially eliminates secondary quenching; and
irradiating the mixture to detect hybridizations of the first and second polynucleotide sequences by fluorescence energy transfer from the one or more donor chromophores of the first polynucleotide sequence to the one or more acceptor chromophores of the second polynucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18)
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16. A method for the amplification of a nucleic acid sequence, comprising the steps of:
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providing a target nucleic acid sequence;
providing a first polynucleotide sequence having at least one donor chromophore, the first polynucleotide sequence being complementary to at least a portion of the target nucleic acid sequence;
providing a second polynucleotide sequence having at least one acceptor chromophore, the second polynucleotide sequence being complementary to a least a portion of the target sequence;
providing four different nucleoside triphosphates, a thermostable amplification enzyme, and two primers, wherein the primers are substantially complementary to the target polynucleotide sequence;
denaturing the target nucleic acid sequence to form single stranded nucleic acids at an appropriate temperature;
hybridizing the first and second polynucleotide sequences to the target nucleic acid sequence, such that when the first polynucleotide sequence and the second polynucleotide sequence are hybridized to the target nucleic acid sequence, the donor chromophore and acceptor chromophore are in an energy transfer relationship that substantially eliminates secondary quenching;
irradiating the mixture to detect hybridizations of the first and second polynucleotide sequences by fluorescence energy transfer from the one or more donor chroniophores of the first polynucleotide sequence to the one or more acceptor chromophores of the second polynucleotide sequence; and
elongating the target polynucleotide sequence by adding the nucleotide triphosphates. - View Dependent Claims (19, 20, 21, 22, 23, 24)
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Specification