Fluorescent donor-acceptor pair with low spectral overlap

• US 6,245,514 B1
• Filed: 09/17/1999
• Issued: 06/12/2001
• Est. Priority Date: 06/04/1996
• Status: Expired due to Term

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Wittwer, Carl T.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/398,629
• Time in Patent Office: 634 Days
• Field of Search: 435/6, 435/810, 435/91.2, 436/94, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 3/5082 Test tubes per se
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 21/0303 Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment G01N21/031 - G01N21/15 take precedence
• CPC Class Codes: G01N 21/07 Centrifugal type cuvettes G01N21/09 takes precedence
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 35/025 : having a carousel or turntable for reaction cells or cuvettes
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to fluorescence resonance energy transfer pairs for detecting the presence of a target analyte wherein the donor fluorophore'"'"'s emission spectrum and the acceptor fluorophore'"'"'s absorption spectrum overlap by less than 25%. In a preferred embodiment, the present invention relates to the use of fluorescein and Cy5 or Cy5.5 as fluorescence resonance energy transfer pairs for use as labels on oligonucleotides for analysis of a nucleic acid locus during amplification.

Claims

1. A fluorescence energy transfer pair for detecting a target analyte, said pair comprising a first probe labeled with fluorescein and a second probe labeled with Cy5 or Cy5.5, wherein in the presence of the target analyte said first probe and said second probe are positioned so that the fluorescein and Cy5 or Cy5.5 are in a fluorescence resonance energy transfer relationship.

2. A fluorescence resonance energy transfer pair for producing a fluorescent signal under predetermined conditions, said pair comprising a donor fluorophore having an emission spectrum and an acceptor fluorophore having an absorption spectrum, wherein the donor fluorophore'"'"'s emission spectrum and the acceptor fluorophore'"'"'s absorption spectrum overlap less than 25%, and wherein under the predetermined conditions the donor fluorophore is in a fluorescence resonance energy transfer relationship with the acceptor fluorophore.

3. The fluorescence resonance energy transfer pair of claim 2 wherein the donor fluorophore is fluorescein and the acceptor fluorophore is Cy5 or Cy5.5.

4. The fluorescence resonance energy transfer pair of claim 2 wherein the donor fluorophore is provided as a label on a first oligonucleotide, the acceptor fluorophore is provided as a label on a second oligonucleotide, and the predetermined conditions include hybridization of the first and second oligonucleotides to a target sequence.

5. The fluorescence resonance energy transfer pair of claim 2 wherein the donor fluorophore and acceptor fluorophore are provided on a single oligonucleotide probe and held in the fluorescent resonance energy transfer relationship, wherein the oligonucleotide probe has specificity for a target sequence and the donor fluorophore and acceptor fluorophore are released from the fluorescent resonance energy transfer relationship during amplification of the target sequence in the presence of the oligonucleotide probe.

6. The fluorescence resonance energy transfer pair of claim 2 wherein the acceptor fluorophore has an extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup>.

7. A fluorescence resonance energy transfer pair for producing a fluorescent signal under predetermined conditions, said pair comprising a donor fluorophore having an emission spectrum and an acceptor fluorophore having an absorption spectrum, wherein the donor fluorophore'"'"'s emission spectrum and the acceptor fluorophore'"'"'s absorption spectrum overlap by about 15%, and wherein under the predetermined conditions the donor fluorophore is in a fluorescence resonance energy transfer relationship with the acceptor fluorophore.

8. The fluorescence resonance energy transfer pair of claim 7 wherein the donor fluorophore is fluorescein and the acceptor fluorophore is Cy5.5.

9. A kit for amplification and analysis of a locus of a nucleic acid sample, comprising:

a probe system comprising

a first oligonucleotide;

a donor fluorophore having an emission spectrum; and

an acceptor fluorophore having an absorption spectrum, wherein the donor fluorophore and the acceptor fluorophore comprise a fluorescence resonance energy transfer pair such that the donor fluorophore'"'"'s emission spectrum and the acceptor fluorophore'"'"'s absorption spectrum overlap by less than 25%, and wherein one of the donor and acceptor fluorophores are provided as a label on the first oligonucleotide and wherein upon hybridization of the first oligonucleotide to the locus the first and second fluorophores are in a resonance energy transfer relationship; and

components for amplifying the locus.

10. The kit of claim 9 wherein the components are for conducting nucleic acid amplification by polymerase chain reaction.

11. The kit of claim 10 wherein the probe system comprises a second oligonucleotide, and wherein the first oligonucleotide is labeled with the donor fluorophore, and the second oligonucleotide is labeled with the acceptor fluorophore.

12. The kit of claim 9 wherein analysis of the locus includes quantification, detection, or sequence analysis.

13. The kit of claim 9 wherein the donor fluorophore is fluorescein.

14. The kit of claim 13 wherein the acceptor fluorophore has an extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup>.

15. The kit of claim 14 wherein the acceptor fluorophore is Cy5.

16. The kit of claim 11 wherein the donor fluorophore'"'"'s emission spectrum and the acceptor fluorophore'"'"'s absorption spectrum overlap by about 15%.

17. The kit of claim 16 wherein the donor fluorophore is fluorescein and the acceptor fluorophore is Cy5.5.

18. The kit of claim 9 wherein the components for amplifying the locus include a pair of primers configured for amplifying the locus, wherein one of said primers and the first oligonucleotide are each labeled with one member of the fluorescence energy transfer pair.

19. A kit for the amplification and analysis of a locus of a nucleic acid sample, comprising:

an oligonucleotide labeled with a donor fluorophore having an emission spectrum and an acceptor fluorophore having an absorption spectrum wherein the donor fluorophore and the acceptor fluorophore comprise a fluorescence resonance energy transfer pair such that the donor fluorophore'"'"'s emission spectrum and the acceptor fluorophore'"'"'s absorption spectrum overlap by less than 25%, and the donor fluorophore and acceptor fluorophore are held in a fluorescent energy transfer relationship; and

components for amplifying the locus including a polymerase and a pair of primers;

wherein the donor and acceptor fluorophores are released from the resonance energy transfer relationship upon amplification of the locus.

20. The kit of claim 19 wherein analysis of the locus includes quantification, detection, or sequence analysis.

21. The kit of claim 19 wherein the donor fluorophore is fluorescein.

22. The kit of claim 21 wherein the acceptor fluorophore has an extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup>.

23. The kit of claim 22 wherein the acceptor fluorophore is Cy5.

24. The kit of claim 19 wherein the donor fluorophore is fluorescein and the acceptor fluorophore is Cy5.5.