Primers labeled with energy transfer coupled dyes for DNA sequencing

• US 5,707,804 A
• Filed: 03/27/1995
• Issued: 01/13/1998
• Est. Priority Date: 02/01/1994
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ju, Jingyue, Mathies, Richard, Glazer, Alexander
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/410,808
• Time in Patent Office: 1,023 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/81 Packaged device or kit

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.

Claims

1. 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.

2. 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.

3. A method according to claim 2, wherein said acceptor-donor pair are 9-phenylxanthenes.

4. 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.

5. A method according to claim 4, wherein said separation is by electrophoresis.

6. 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.

7. A method according to claim 6, wherein said acceptor-donor pair are 9-phenylxanthenes.

8. 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.

9. 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.

10. A method according to claim 8, wherein there are four primers having different acceptor-donor pairs.

11. A method according to claim 8, wherein said acceptor-donor fluorescent pair is separated by not more than 20 nucleotides.

12. A method according to claim 8, wherein at least two acceptor-donor fluorescent pairs are xanthene compounds.

13. A method according to claim 12, wherein said xanthene compounds comprise fluorescein derivatives and rhodamine derivatives.