US5707804A
Expired / Lapsed
US
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Primers labeled with energy transfer coupled dyes for DNA sequencing
Assignee: University of California
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Filing: 1995-03-27
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Granted: 1998-01-13
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Expires: 2015-03-27
Abstract
Abstract Compositions are provided for making oligonucleotides carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donor 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 under separation conditions, by varying the distance between the donor and acceptor in a given pair, and to have enhanced emission intensities from the acceptor. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids.
Inventors
- 👤 Richard Mathies
- 👤 Alexander Glazer
- 👤 Jingyue Ju
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 00:18
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/582; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; G01N33/53; G01N33/536; G01N33/542; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
105 words
A method of identification and detection of components in a multicomponent mixture employing different fluorescent labels to detect at least two components of interest, wherein said labels are characterized by: (1) having a donor-acceptor fluorescent pair bonded to an oligonucleotide 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; said method comprising: binding different labels to different components of said multi-component mixture; detecting each of said labeled components by irradiating at the absorption wavelength of said donor and detecting the fluorescence of each of said labels.
Claim 2
Independent Claim
105 words
A method of identification and detection of components in a multicomponent mixture employing different fluorescent labels to detect at least two components of interest, wherein said labels are characterized by: (1) having a donor-acceptor fluorescent pair bonded to an oligonucleotide 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; said method comprising: binding different labels to different components of said multi-component mixture; detecting each of said labeled components by irradiating at the absorption wavelength of said donor and detecting the fluorescence of each of said labels.
Claim 3
Dependent Claim
30 words
A method according to claim 1, wherein said donor absorbs light in the wavelength range of 350-800 nm and said acceptor emits light in the wavelength range of 450-1000 nm.
Claim 4
Dependent Claim
12 words
A method according to claim 2, wherein said acceptor-donor pair are 9-phenylxanthenes.
Claim 5
Independent Claim
141 words
A method of separating components of a multicomponent mixture, wherein each of the different components of interest are labeled with different labels, wherein said labels are characterized by: (1) having a donor-acceptor fluorescent pair bonded to an oligonucleotide chain with efficient energy transfer from said donor to said acceptor; (2) each of the labels absorbs at substantially the same wavelength and emits at a different wavelength; (3) and each of the different labels has substantially the same mobility in said separation as a result of varying the spacing of said donor-acceptor pair along said oligonucleotide chain; said method comprising: binding different labels to different components of said multi-component mixture; separating said multi-component mixture into individual fractions; and detecting each of said labeled components by irradiating at an absorption wavelength of said donor and detecting the fluorescence of each of said labels.
Claim 6
Independent Claim
141 words
A method of separating components of a multicomponent mixture, wherein each of the different components of interest are labeled with different labels, wherein said labels are characterized by: (1) having a donor-acceptor fluorescent pair bonded to an oligonucleotide chain with efficient energy transfer from said donor to said acceptor; (2) each of the labels absorbs at substantially the same wavelength and emits at a different wavelength; (3) and each of the different labels has substantially the same mobility in said separation as a result of varying the spacing of said donor-acceptor pair along said oligonucleotide chain; said method comprising: binding different labels to different components of said multi-component mixture; separating said multi-component mixture into individual fractions; and detecting each of said labeled components by irradiating at an absorption wavelength of said donor and detecting the fluorescence of each of said labels.
Claim 7
Dependent Claim
12 words
A method according to claim 4, wherein said separation is by electrophoresis.
Claim 8
Dependent Claim
30 words
A method according to claim 4, wherein said donor absorbs light in the wavelength range of 350-800 nm and said acceptor emits light in the wavelength range of 450-1000 nm.
Claim 9
Dependent Claim
12 words
A method according to claim 6, wherein said acceptor-donor pair are 9-phenylxanthenes.
Claim 10
Dependent Claim
229 words
In a method for sequencing a nucleic acid which employs primers for copying a single stranded nucleic acid and dideoxynucleotides for terminating the chain at a particular nucleotide resulting from said copying, said method comprising: cloning a nucleic acid fragment to be sequenced into a vector comprising a primer binding sequence 5' to said fragment complementary to a primer, or ligating an oligonucleotide primer binding sequence to a DNA fragment to be sequenced, where the sequence of said oligonucleotide is complementary to a primer; copying said fragment with a DNA polymerase in the presence of said primer, dNTPs and each of a plurality of dideoxynucleotides in separate reaction vessels, to generate single stranded DNA sequencing fragments; and separating the resulting mixture of single stranded DNA sequencing fragments and determining the sequence by means of the bands present on the gel; the improvement which comprises: employing primers which are characterized by: (1) having an acceptor-donor fluorescent pair bonded to a nucleic acid chain complementary to said primer binding sequence, where the donor transfers energy to said acceptor for enhanced fluorescence of said acceptor; (2) each of the primers absorbs at substantially the same wavelength and emits at a different wavelength; and (3) each of the primers has substantially the same mobility in said separation, resulting from varying the spacing and fluorophores of said donor-acceptor pair along said nucleic acid chain.
Claim 11
Dependent Claim
229 words
In a method for sequencing a nucleic acid which employs primers for copying a single stranded nucleic acid and dideoxynucleotides for terminating the chain at a particular nucleotide resulting from said copying, said method comprising: cloning a nucleic acid fragment to be sequenced into a vector comprising a primer binding sequence 5' to said fragment complementary to a primer, or ligating an oligonucleotide primer binding sequence to a DNA fragment to be sequenced, where the sequence of said oligonucleotide is complementary to a primer; copying said fragment with a DNA polymerase in the presence of said primer, dNTPs and each of a plurality of dideoxynucleotides in separate reaction vessels, to generate single stranded DNA sequencing fragments; and separating the resulting mixture of single stranded DNA sequencing fragments and determining the sequence by means of the bands present on the gel; the improvement which comprises: employing primers which are characterized by: (1) having an acceptor-donor fluorescent pair bonded to a nucleic acid chain complementary to said primer binding sequence, where the donor transfers energy to said acceptor for enhanced fluorescence of said acceptor; (2) each of the primers absorbs at substantially the same wavelength and emits at a different wavelength; and (3) each of the primers has substantially the same mobility in said separation, resulting from varying the spacing and fluorophores of said donor-acceptor pair along said nucleic acid chain.
Claim 12
Dependent Claim
25 words
A method according to claim 8, wherein one of the members of said acceptor-donor fluorescent pair is bonded to the 5' terminus of said primer.
Claim 13
Dependent Claim
15 words
A method according to claim 8, wherein there are four primers having different acceptor-donor pairs.
Claim 14
Dependent Claim
19 words
A method according to claim 8, wherein said acceptor-donor fluorescent pair is separated by not more than 20 nucleotides.
Claim 15
Dependent Claim
16 words
A method according to claim 8, wherein at least two acceptor-donor fluorescent pairs are xanthene compounds.
Claim 16
Dependent Claim
16 words
A method according to claim 12, wherein said xanthene compounds comprise fluorescein derivatives and rhodamine derivatives.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-01-12 | 2001-07-14 | 2002-01-13 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-01-12 | 2005-07-14 | 2006-01-13 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-01-12 | 2009-07-14 | 2010-01-13 | $7700.00 | Window Closed / Lapsed |