Probes labeled with energy transfer couples dyes exemplified with DNA fragment analysis
First Claim
1. A method of identification and detection of single or double stranded nucleic acids in a mixture of nucleic acids with different fluorescent labels covalently bonded to said nucleic acids to detect at least two different nucleic acids of interest having different mobilities in a separation means, wherein said labels are characterized by:
- (1) having a molecular weight not exceeding about 10,000 daltons and comprising a donor-acceptor fluorescent pair bonded to a backbone chain with energy transfer from said donor to said acceptor; and
(2) each of the labels absorbs at substantially the same wavelength and emits at a different wavelength, with the proviso that one of the labels may have the same fluorescer as the donor and acceptor;
said method comprising;
preparing a mixture of said nucleic acids wherein a portion of said nucleic acids are sample nucleic acids to which are bonded labels which emit light at one or more first wavelengths and a portion of said nucleic acids are standard molecular weight nucleic acids which have labels which emit light at one or more second wavelengths, said first and second wavelengths being different;
separating or analyzing said mixture by said separation means as a result of different mobilities of said sample nucleic acids and said standard nucleic acids; and
detecting each of said labeled nucleic acids by irradiating with a common light source at the absorption wavelength of said donor and detecting the fluorescence of each of said labels.
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Abstract
Compositions are provided comprising sets of fluorescent labeled oligonucleotides carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility and enhanced emission intensities under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in analyzing double stranded and single stranded nucleic acid fragments using capillary electrophoresis.
164 Citations
17 Claims
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1. A method of identification and detection of single or double stranded nucleic acids in a mixture of nucleic acids with different fluorescent labels covalently bonded to said nucleic acids to detect at least two different nucleic acids of interest having different mobilities in a separation means, wherein said labels are characterized by:
- (1) having a molecular weight not exceeding about 10,000 daltons and comprising a donor-acceptor fluorescent pair bonded to a backbone chain with energy transfer from said donor to said acceptor; and
(2) each of the labels absorbs at substantially the same wavelength and emits at a different wavelength, with the proviso that one of the labels may have the same fluorescer as the donor and acceptor;said method comprising; preparing a mixture of said nucleic acids wherein a portion of said nucleic acids are sample nucleic acids to which are bonded labels which emit light at one or more first wavelengths and a portion of said nucleic acids are standard molecular weight nucleic acids which have labels which emit light at one or more second wavelengths, said first and second wavelengths being different; separating or analyzing said mixture by said separation means as a result of different mobilities of said sample nucleic acids and said standard nucleic acids; and detecting each of said labeled nucleic acids by irradiating with a common light source at the absorption wavelength of said donor and detecting the fluorescence of each of said labels. - View Dependent Claims (2, 3, 4, 5, 6)
- (1) having a molecular weight not exceeding about 10,000 daltons and comprising a donor-acceptor fluorescent pair bonded to a backbone chain with energy transfer from said donor to said acceptor; and
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7. A method of identification and detection of single or double stranded nucleic acids in a mixture of nucleic acids with different fluorescent labels covalently bonded thereto to detect at least two different nucleic acids of interest, wherein said labels are characterized by:
- (1) having a molecular weight not exceeding about 10,000 daltons and comprising a donor-acceptor fluorescent pair bonded to a nucleic acid backbone chain with energy transfer from said donor to said acceptor; and
(2) each of the labels absorbs at substantially the same wavelength and emits at a different wavelength, with the proviso that one of the labels may have the same fluorescer as the donor and acceptor;said method comprising; preparing a mixture of said nucleic acids wherein a portion of said nucleic acids are sample nucleic acids which have labels which emit light at one or more first wavelengths and a portion of said nucleic acids are standard nucleic acids which have labels which emit light at one or more second wavelengths, said first and second wavelengths being different; separating said mixture by means of capillary electrophoresis in the presence of a resolution enhancing agent; and detecting each of said labeled components by irradiating with a common light source at the absorption wavelength of said donor and detecting the fluorescence of each of said labels. - View Dependent Claims (8, 9, 10, 11, 12, 13)
- (1) having a molecular weight not exceeding about 10,000 daltons and comprising a donor-acceptor fluorescent pair bonded to a nucleic acid backbone chain with energy transfer from said donor to said acceptor; and
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14. A kit comprising at least two fluorescent compounds, each of said fluorescent compounds characterized by:
(1) having an acceptor-donor fluorescent pair bonded to a nucleic acid backbone chain and separated by not more than 20 nucleotides, where said donor transfers energy to said acceptor for fluorescence of said acceptor;
(2) each of the fluorescent compounds absorbs at substantially the same wavelength and emits at a different wavelength; and
(3) each of said fluorescent compounds has substantially the same mobility in electrophoretic or chromatographic separation, resulting from varying the spacing of said donor-acceptor pair along said backbone.
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15. A kit for use in simultaneously detecting a plurality of components comprising at least two labels,
each of said labels characterized by: - (1) having a donor-acceptor fluorescent pair wherein said donor and said acceptor are covalently bonded to a chain at specific locations on said chain such that there is efficient energy transfer to said acceptor; and
(2) each of said labels absorbs light at substantially the same wavelength and emits light at a different wavelength. - View Dependent Claims (16, 17)
- (1) having a donor-acceptor fluorescent pair wherein said donor and said acceptor are covalently bonded to a chain at specific locations on said chain such that there is efficient energy transfer to said acceptor; and
Specification