Monitoring amplification with fret probes

• US 7,160,998 B2
• Filed: 03/26/2003
• Issued: 01/09/2007
• Est. Priority Date: 06/04/1996
• Status: Expired due to Fees

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Wittwer, Carl T., Rasmussen, Randy P., Lay, Marla
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US10/397,759
• Publication Number:
  US 20040002098A1
• Time in Patent Office: 1,385 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3
• US Class Current: 536/24.3
• 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 is directed to kits for monitoring a nucleic acid during amplification. More particularly, the present invention relates to kits comprising a first oligonucleotide labeled with a first fluorophore, a second oligonucleotide labeled with a second fluorophore, and components for amplifying the locus, wherein the first and second fluorophores comprise a fluorescence resonance energy transfer pair. Hybridization of at least the first oligonucleotide to the amplified locus places the first and second fluorophores in a resonance energy transfer relationship.

Claims

1. A kit for conducting real time monitoring of a PCR amplification of a locus of a nucleic acid sample, said kit comprising:

a first oligonucleotide labeled with a first fluorophore at the 3′

end of the first oligonucleotide; and

a second oligonucleotide labeled with a second fluorophore, wherein said second oligonucleotide is modified to comprise a terminal 3′

phosphate;

wherein the first and second fluorophores comprise a fluorescence resonance energy transfer pair, and the first and second oligonucleotides are configured to hybridize to the locus such that hybridization of the first and second oligonucleotides to the locus, during said PCR, places the first and second fluorophores in a resonance energy transfer relationship.

2. The kit of claim 1 wherein the first and second oligonucleotides are configured such that hybridization of the first and second oligonucleotides to the locus places the first and second fluorophores within 0 to 25 nucleotides of each other.

3. The kit of claim 2 wherein the first and second oligonucleotides are configured such that hybridization of the first and second oligonucleotides to the locus places the first and second fluorophores within 0 to 5 nucleotides of each other.

4. The kit of claim 2 wherein the first fluorophore is a donor fluorophore and the second fluoropbore is an acceptor fluorophore.

5. The kit of claim 4 wherein the acceptor fluorophore has an extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup>.

6. The kit of claim 4 wherein the acceptor fluorophore has a maximum excitation wavelength of about 650 nm to about 675 nm.

7. The kit of claim 1 further comprising a pair of primers configured for amplifying the locus.

8. The kit of claim 7 further comprising deoxynucleotides, buffer, and a thermostable polymerase.

9. The kit of claim 1 wherein the locus is the factor V Leiden mutation locus.

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

a first oligonucleotide labeled with a first fluorophore at the 3′

end of the first oligonucleotide;

a second oligonucleotide labeled with a second fluorophore; and

primers configured for amplifying the locus;

wherein the first and second fluorophores comprise a fluorescence resonance energy transfer pair, and the first and second oligonucleotides are configured to hybridize to the locus such that hybridization of the first and second oligonucleotides to the locus places the first and second fluorophores in a resonance energy transfer relationship, and wherein said primers and the first and second oligonucleotides are provided mixed together in a single reaction mixture.

11. The kit of claim 10 further comprising deoxynucleotides, a buffer, and a thermostable polymnerase.

20. The kit of claim 10 wherein said second oligonucleotide is modified to comprise a terminal 3′

phosphate.

21. The kit of claim 10 wherein the acceptor fluorophore has an extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup>.

22. The kit of claim 10 wherein the locus is the factor V Leiden mutation locus.

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

an oligonucleotide labeled with a first fluorophore;

a pair of primers configured for amplifying the locus, one of the primers labeled with a second fluorophore;

wherein the first and second fluorophores comprise a fluorescence resonance energy transfer pair, and the oligonucleotide is configured to hybridize to the locus such that hybridization of the oligonucleotide and the labeled primer to an amplified copy of the locus places the first and second fluorophores in a resonance energy transfer relationship.

13. The kit of claim 12 wherein the first fluorophore is a donor fluorophore and the second fluorophore is an acceptor fluorophore.

14. The kit of claim 12 wherein the oligonucleotide hybridizes to an amplified copy of the locus within 15 nucleotides of the labeled primer.

15. The kit of claim 12 wherein the locus is the factor V Leiden mutation locus.

16. The kit of claim 12 wherein the locus is the MTHFR locus.

17. The kit of claim 12 wherein the acceptor fluorophore of said fluorescence resonance energy transfer pair has an extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup>.

18. The kit of claim 12 wherein the acceptor fluorophore of said fluorescence resonance energy transfer pair has a maximum excitation wavelength of about 650 nm to about 675 nm.

19. The kit of claim 12 further comprising deoxynucleotides, a buffer, and a thermostable polymerase.