Monitoring amplification of DNA during PCR

• US 6,174,670 B1
• Filed: 06/04/1997
• Issued: 01/16/2001
• 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., Ririe, Kirk M., Rasmussen, Randy P.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/869,276
• Time in Patent Office: 1,322 Days
• Field of Search: 435/6, 435/91.2, 436/94
• US Class Current: 435/6.12
• 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

Methods of monitoring hybridization during polymerase chain reaction are disclosed. These methods are achieved with rapid thermal cycling and use of double stranded DNA dyes or specific hybridization probes. A fluorescence resonance energy transfer pair comprises fluorescein and Cy5 or Cy5.5. Methods for quantitating amplified DNA and determining its purity are carried out by analysis of melting and reannealing curves.

Claims

1. A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps of

amplifying the target sequence by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase and primers for the targeted nucleic acid sequence to the biological sample and thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature;

exciting the biological sample with light at a wavelength absorbed by the donor fluorophore and detecting fluorescent emission from the fluorescence energy transfer pair.

3. The method of claims 1 or 2 wherein the donor and acceptor fluorophores are at a distance of about 0-5 nucleotides.

4. The method of claim 3 wherein the donor and acceptor fluorophores are at a distance of about 0-2 nucleotides.

5. The method of claim 4 wherein the donor and acceptor fluorophores are at a distance of 1 nucleotide.

2. A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps of

amplifying the target sequence by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase and primers for the targeted nucleic acid sequence to the biological sample and thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature;

exciting the sample with light at a wavelength absorbed by the donor fluorophore; and

monitoring temperature dependent fluorescence from the fluorescence energy transfer pair.

6. A method of real time monitoring of a polymerase chain reaction amplification of a target nucleic acid sequence in a biological sample, said method comprising the steps of

(a) adding to the biological sample an effective amount of two nucleic acid primers and a nucleic acid probe, wherein one of said primers and the probe are each labeled with one member of a fluorescence energy transfer pair comprising an acceptor fluorophore and a donor fluorophore, and wherein the labeled probe hybridizes to an amplified copy of the target nucleic acid sequence within 15 nucleotides of the labeled primer;

(b) amplifying the target nucleic acid sequence by polymerase chain reaction;

(c) illuminating the biological sample with light of a selected wavelength that is absorbed by said donor fluorophore; and

(d) detecting the fluorescence emission of the sample.

7. The method of claim 6 further comprising the step of monitoring temperature dependent fluorescence of the sample.

8. The method of claims 6 wherein the donor and acceptor fluorophores are at a distance of about 4-6 nucleotides from one another.

9. An improved method of amplifying a target nucleic acid sequence biological sample, said method comprising the steps of

(a) adding to the biological sample an effective amount of a nucleic-acid-binding fluorescent entity;

(b) amplifying the target nucleic acid sequence using polymerase chain reaction, comprising thermally cycling the biological sample using initial predetermined temperature and time parameters, and then

(i) illuminating the biological sample with a selected wavelength of light that is absorbed by said fluorescent entity during the polymerase chain reaction;

(ii) monitoring fluorescence from said sample to determine the optimal temperature and time parameters for the polymerase chain reaction; and

(iii) adjusting the initial temperature and time parameters in accordance with the fluorescence,

wherein the monitoring fluorescence step consists of monitoring an amplification dependent emission of the fluorescent entity.

10. The method of claim 9 wherein the fluorescent entity comprises a double strand specific nucleic acid binding dye.

11. The method of claim 9 wherein the fluorescent entity comprises a fluorescently labeled oligonucleotide probe that hybridizes to the targeted nucleic acid sequence.

12. The method of claim 9 wherein the fluorescent entity comprises a pair of oligonucleotide probes, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair.

13. The method of claim 12 wherein a donor fluorophore emission spectrum and an acceptor fluorophore absorption spectrum overlap less than 25%, and the acceptor fluorophore has a peak extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup> and upon hybridization of the two probes, the donor and acceptor fluorophores are within 15 nucleotides of one another.

14. The method of claim 13 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

104. The method of claim 14 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5.5 as the acceptor.

15. A method for detecting a target nucleic acid sequence of a biological sample, said method comprising the steps of

(a) adding to the biological sample an effective amount of a pair of oligonucleotide probes that hybridize to the target nucleic acid sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair, wherein an emission spectrum of the donor fluorophore and an absorption spectrum of the acceptor fluorophore overlap less than 25%, the acceptor fluorophore has a peak extinction coefficient greater than 100,000 M⁻

1 cm<sup>−

1</sup> and upon hybridization of the two probes, the donor and acceptor fluorophores are within 25 nucleotides of one another;

(b) illuminating the biological sample with a selected wavelength of light that is absorbed by said donor fluorophore; and

(c) detecting fluorescent emission of the biological sample.

16. The method of claim 15 wherein the resonance energy transfer pair comprises fluorescein as the donor.

17. The method of claim 16 wherein the resonance energy transfer pair comprises Cy5 or Cy5.5 as the acceptor.

18. The method of claim 15 wherein the donor and acceptor fluorophores are within about 0-5 nucleotides of one another upon hybridization of the two probes with the target nucleic acid sequence.

19. The method of claim 18 wherein the donor and acceptor fluorophores are within about 0-2 nucleotides of each other.

20. The method of claim 19 wherein the donor and acceptor fluorophores are within 1 nucleotide of each other.

21. A method of real time monitoring of a polymerase chain reaction amplification of a target nuclcic acid sequence in a biological sample, said method comprising the steps of

amplifying the target sequence by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding the two nucleic acid probes, a thermostable polymerase, and primers for the targeted nucleic acid sequence to the biological sample to create an amplification medium and thermally cycling the amplification medium between at least a denaturation temperature and an elongation temperature;

and

illuminating the amplification medium with light having a wavelength absorbed by the donor fluorophore and detecting fluorescent emission from the amplification medium at a plurality of temperatures;

to monitor the temperature dependent fluorescent emission.

22. The method of claim 21 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

23. The method of claim 21 wherein the donor and acceptor fluorophores are within about 0-5 nucleotides of one another.

24. The method of claim 23 wherein the donor and acceptor fluorophores are within about 0-2 nucleotides of one another.

25. The method of claim 24 wherein the donor and acceptor fluorophores are within 1 nucleotide of each other.

26. The method of claim 21 wherein said monitoring step comprises determining melting profiles of the probes melting from said target sequence.

88. The method of claim 21, further comprising monitoring the temperature dependent emission while changing temperature at a rate of ≧

0.1°

C./second.

105. The method of claim 22 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5.5 as the acceptor.

27. A method of real time monitoring of a polymerase chain reaction amplification of a target nucleic acid sequence in a biological sample, said method comprising the steps of

amplifying the target sequence by polymerase chain reaction in the presence of a fluorescent entity, said polymerase chain reaction comprising the steps of adding the fluorescent entity, a thermostable polymerase, and primers for the target nucleic acid sequence to the biological sample to create an amplification medium and thermally cycling the amplification medium between at least a denaturation temperature and an elongation temperature;

exciting the fluorescent entity with light at a wavelength absorbed by the fluorescent entity and detecting fluorescent emission from the fluorescent entity; and

monitoring temperature dependent fluorescence from the fluorescent entity in the amplification medium;

wherein the monitoring step consists of monitoring an amplification dependent signal of the fluorescent entity, and wherein the fluorescent entity is SYBR™

Green I.

28. The method of claim 27 further comprising the step of determining a melting profile of the amplified target sequence.

84. The method of claim 27 wherein the temperature dependent fluorescence is used to identify the amplified target sequence.

85. The method of claim 84 wherein the amplified target sequence is characterized by analysis of melting curves.

86. The method of claim 85 wherein the relative amounts of two or more amplified target sequences are determined by analysis of melting curves.

87. The method of claim 85 wherein areas under the melting curves are found by none ear least squares regression of the sum of multiple gaussians.

89. The method of claim 27, further comprising monitoring the amplification dependent signal of the fluorescent entity while changing temperature at a rate of ≧

0.1°

C./second.

29. A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps of

(a) adding to the biological sample an effective amount of two nucleic acid primers and a nucleic acid probe to form an amplification medium, wherein one of said primers and the probe are each labeled with one member of a fluorescence energy transfer pair comprising an acceptor fluorophore and a donor fluorophore, and wherein the labeled probe Hybridizes to an amplified copy of the target nucleic acid sequence within 15 nucleotides of the labeled primer;

(b) amplifying the target nucleic acid sequence by polymerase chain reaction;

(c) illuminating the amplification medium with light having wavelength that is absorbed by said donor fluorophore and detecting the fluorescence emission from the medium.

30. The method of claim 29 further comprising the step of monitoring the temperature dependent fluorescence of the sample.

31. The method of claim 30 wherein said monitoring step comprises determining a melting profile of the amplified target sequence.

32. The method of claim 29 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

33. The method of claim 29 wherein the donor and acceptor fluorophores are at a distance of about 4-6 nucleotides from one another when the labeled probe is hybridized to an amplified copy of the target nucleic acid sequence.

90. The method of claim 30, further comprising monitoring the fluorescence while changing temperature at a rate of ≧

0.1°

C./second.

34. A method of detecting a difference at a selected locus in a first nucleic acid as compared to a second nucleic acid, comprising the steps of:

(a) providing a pair of oligonucleotide primers configured for amplifying, by polymerase chain reaction, a selected segment of the first nucleic acid and a corresponding segment of the second nucleic acid, wherein the selected segment and corresponding segment each comprises the selected locus, to result in amplified products containing a copy of the selected locus;

(b) providing a pair of oligonucleotide probes, one of said probes being labeled with an acceptor fluorophore and the other probe being labeled with a donor fluorophore of a fluorogenic resonance energy transfer pair such that upon hybridization of the two probes with the amplified products the donor and acceptor are in resonance energy transfer relationship, wherein one of the probes is configured for hybridizing to the amplified products such that said one of the probes spans the selected locus and exhibits a first melting profile when the difference is present in the first nucleic acid that is distinguishable from a second melting profile of the second nucleic acid;

(c) amplifying the selected segment of first nucleic acid and the corresponding segment of the second nucleic acid by polymerase chain reaction in the presence of effective amounts of probes to result in an amplified selected segment and an amplified corresponding segment, at least a portion thereof having both the probes hybridized thereto with the fluorogenic resonance energy transfer pair in resonance energy transfer relationship;

(d) illuminating the amplified selected segment and the amplified corresponding segment with the probes hybridized thereto with light to elicit fluorescence by the fluorogenic resonance energy transfer pair;

(e) measuring fluorescence emission as a function of temperature to determine the first melting profile of said one of the probes melting from the amplified selected segment of first nucleic acid and the second melting profile of said one of the probes melting from the amplified corresponding segment of second nucleic acid; and

(f) comparing the first melting profile to the second melting profile, wherein a difference therein indicates the presence of the difference in the sample nucleic acid.

35. The method of claim 34 wherein the resonance energy transfer pair comprises fluorescein as the donor and a member selected from the group consisting of Cy5 and Cy5.5 as the acceptor.

36. The method of claim 34 wherein the donor and the acceptor are coupled to the probes such that when both probes are hybridized to the amplified selected segment or the amplified corresponding segment, the donor and acceptor flurophores are separated by no more than about 25 nucleotide residues.

37. The method of claim 36 wherein the donor and the acceptor are coupled to probes such that when the probes are hybridized to the amplified selected segment or the amplified corresponding segment, the donor and acceptor are flurophores separated by about 0-5 nucleotide residues.

38. The method of claim 37 wherein the resonance energy transfer donor and the resonance energy transfer acceptor flurophores are coupled to the probes such that when the probes are hybridized to the amplified selected segment or the amplified corresponding segment the donor and acceptor flurophores are separated by about 0-2 nucleotide residues.

39. The method of claim 38 wherein the resonance energy transfer donor and the resonance energy transfer acceptor are coupled to the probes such that when the probes are hybridized to the amplified selected segment or the amplified corresponding segment the donor and acceptor flurophores are separated by 1 nucleotide residue.

40. A method of detecting a difference at a selected locus in a first nucleic acid as compared to a second nucleic acid, comprising the steps of:

(a) providing a pair of oligonucleotide primers configured for amplifying, by polymerase chain reaction, a selected segment of the first nucleic acid and a corresponding segment of the second nucleic acid, wherein the selected segment and corresponding segment each comprises the selected locus, to result in amplified products containing a copy of the selected locus;

(b) providing an oligonucleotide probe, wherein one of said primers and the probe are each labeled with one member of a fluorescence energy transfer pair comprising an donor fluorophore and an acceptor fluorophore, and wherein the labeled probe and labeled primer hybridize to the amplified products such that the donor and acceptor are in resonance energy transfer relationship, and wherein the probe is configured for hybridizing to the amplified products such that said probe spans the selected locus and exhibits a melting profile when the difference is present in the first nucleic acid that is distinguishable from a melting profile of the second nucleic acid;

(c) amplifying the selected segment of first nucleic acid and the corresponding segment of the second nucleic acid by polymerase chain reaction in the presence of effective amounts of primers and probe to result in an amplified selected segment and an amplified corresponding segment, at least a portion thereof having the primer and probe hybridized thereto with the fluorogenic resonance energy transfer pair in resonance energy transfer relationship;

(d) illuminating the amplified selected segment and the amplified corresponding segment with the labeled primer and probe hybridized thereto with a selected wavelength of light to elicit fluorescence by the fluorogenic resonance energy transfer pair;

(e) measuring fluorescence emission as a function of temperature to determine in a first melting profile of said probe melting from the amplified selected segment of first nucleic acid and a second melting profile of said probe melting from the amplified corresponding segment of second nucleic acid; and

(f) comparing the first melting profile to the second melting profile, wherein a difference therein indicates the presence of the difference in the sample nucleic acid.

41. The method of claim 40 wherein the resonance energy transfer pair comprises fluorescein as the donor and a member selected from the group consisting of Cy5 and Cy5.5 as the acceptor.

42. The method of claim 40 wherein the donor and the acceptor are coupled to the labeled primer and probe such that when both the labeled primer and probe are hybridized to the amplified selected segment or the amplified corresponding segment, the donor and acceptor are separated by no more than about 15 nucleotide residues.

43. The method of claim 42 wherein the donor and the acceptor are coupled to labeled primer and probe such that when the labeled primer and probe are hybridized to the amplified selected segment or the amplified corresponding segment, the donor and acceptor are separated by about 4-6 nucleotide residues.

44. A method of detecting heterozygosity at a selected locus in the genome of an individual, wherein the genome comprises a mutant allele and a corresponding reference allele, each comprising the selected locus, comprising the steps of:

(a) obtaining sample genomic DNA from the individual;

(b) providing a pair of oligonucleotide primers configured for amplifying, by polymerase chain reaction, a first selected segment of the mutant allele and a second selected segment of the corresponding reference allele wherein both the first and second selected segments comprise the selected locus;

(c) providing a pair of oligonucleotide probes, one of said probes being labeled with an acceptor fluorophore and the other probe being labeled with a donor fluorophore of a fluorogenic resonance energy transfer pair such that upon hybridization of the two probes with the amplified first and second selected segments one of the probes spans the selected locus and exhibits a first melting profile with the amplified first selected segment that is distinguishable from a second melting profile with the amplified second selected segment;

(d) amplifying the first and second selected segments of sample genomic DNA by polymerase chain reaction in the presence of effective amounts of probes to result in amplified first and second selected segments, at least a portion thereof having both the probes hybridized thereto with the fluorogenic resonance energy transfer pair in resonance energy transfer relationship;

(e) illuminating the amplified first and second selected segments having the probes hybridized thereto with a selected wavelength of light to elicit fluorescence by the donor and acceptor;

(f) measuring a fluorescence emission as a function of temperature to determine a first melting profile of said one of the probes melting from the amplified first selected segment and a second melting profile of said one of the probes melting from the amplified second selected segment; and

(g) comparing the first melting profile to the second melting profile, wherein distinguishable melting profiles indicate heterozygosity in the sample genomic DNA.

45. The method of claim 44 wherein the fluorogenic resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

46. The method of claim 44 wherein the donor and the acceptor are coupled to the probes such that when the probe and reference oligonucleotide are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by no more than about 25 nucleotide residues.

47. The method of claim 46 wherein the donor and the acceptor are coupled to the probes such that when the probes are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by about 0-5 nucleotide residues.

48. The method of claim 47 wherein the donor and the acceptor are coupled to the probes such that when the probes are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by about 0-2 nucleotide residues.

49. The method of claim 48 wherein the donor and the acceptor are coupled to the probes such that when the probes are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by 1 nucleotide residue.

50. A method of detecting heterozygosity at a selected locus in the genome of an individual, wherein the genome comprises a mutant allele and a corresponding reference allele, each comprising the selected locus, comprising the steps of:

(a) obtaining sample genomic DNA from be individual;

(b) providing a pair of oligonucleotide primers configured for amplifying, by polymerase chain reaction, a first selected segment of the mutant allele and a second selected segment of the corresponding reference allele wherein both the first and second selected segments comprise the selected locus;

(c) providing an oligonucleotide probe, wherein one of said primers and the probe are each labeled with one member of a fluorescence energy transfer pair comprising a donor fluorophore and an acceptor fluorophore, and wherein the labeled probe hybridizes to the amplified first and second selected segments such that the probe spans the selected locus and exhibits a first melting profile with the amplified first selected segment that is distinguishable from a second melting profile with the amplified second selected segment;

(d) amplifying the first and second selected segments of sample genomic DNA by polymerase chain reaction in the presence of effective amounts of tee primers and probe to result in amplified first and second selected segments, at least a portion thereof having both the labeled probe hybridized thereto with the labeled probe and primer comprising the fluorogenic resonance energy transfer pair in resonance energy transfer relationship;

(e) illuminating the amplified first and second selected segments having the labeled probe hybridized thereto with light to elicit fluorescence by the donor and acceptor;

(f) measuring a fluorescence emission as a function of temperature to determine a first melting profile of said probe melting from the amplified first selected segment and a second melting profile of said probe melting from the amplified second selected segment; and

(g) comparing the first melting profile to the second melting profile, wherein distinguishable melting profiles indicate heterozygosity in the sample genomic DNA.

51. The method of claim 50 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

52. The method of claim 50 wherein the donor and the acceptor are coupled to the labeled primer and probe such that when the labeled probe is hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by no more than about 15 nucleotide residues.

53. The method of claim 52 wherein the donor and the acceptor are coupled to the labeled primer and probe such that when the labeled probe is hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by about 4-6 nucleotide residues.

54. A method of determining completion of a polymerase chain reaction in a polymerase chain reaction mixture comprising (1) a nucleic acid wherein the nucleic acid or a polymerase-chain-reaction-amplified product thereof consists of two distinct complementary strands, (2) two oligonucleotide primers configured for amplifying by polymerase chain reaction a selected segment of the nucleic acid to result in an amplified product, and (3) a DNA polymerase for catalyzing the polymerase chain reaction, comprising the steps of:

(a) adding to the mixture (1) an effective amount of an oligonucleotide probe labeled with a resonance energy transfer donor or a resonance energy transfer acceptor of a fluorogenic resonance energy transfer pair, wherein the probe is configured for hybridizing to the amplified product under selected conditions of temperature and monovalent ionic strength, and (2) an effective amount of a reference oligonucleotide labeled with the donor or the acceptor, with the proviso that as between the probe and reference oligonucleotide one is labeled with the donor and the other is labeled with the acceptor, wherein the reference oligonucleotide is configured for hybridizing to the amplified product under the selected conditions of temperature and monovalent ionic strength such that the donor and the acceptor are in resonance energy transfer relationship when both the probe and the reference oligonucleotide hybridize to the amplified product;

(b) amplifying the selected segment of nucleic acid by polymerase chain reaction to result in the amplified product, at least a portion thereof having both the probe and the reference oligonucleotide hybridized thereto with the fluorogenic resonance energy transfer pair in resonance energy transfer relationship; and

(c) illuminating the amplified product having the probe and reference oligonucleotide hybridized thereto with a selected wavelength of light for eliciting fluorescence by the fluorogenic resonance energy pair and monitoring fluorescence emission and determining a cycle when the fluorescence emission reaches a plateau phase, indicating the completion of the reaction.

55. The method of claim 54 wherein the fluorogenic resonance energy transfer pair is fluorescein and a member selected from the group consisting of Cy5 and Cy5.5.

106. The method of claim 55 wherein the fluorogenic resonance energy transfer pair is fluorescein and Cy5.5.

56. A method of determining completion of a polymerase chain reaction in a polymerase chain reaction mixture comprising (1) a nucleic acid wherein the nucleic acid or a polymerase-chain-reaction-amplified product thereof consists of two distinct complementary strands, (2) two oligonucleotide primers configured for amplifying by polymerase chain reaction a selected segment of the nucleic acid to result in an amplified product, and (3) a DNA polymerase for catalyzing the polymerase chain reaction, comprising the steps of:

(a) adding to the mixture an effective amount of a nucleic-acid-binding fluorescent dye;

(b) amplifying the selected segment of nucleic acid by polymerase chain reaction in the mixture to which the nucleic-acid-binding fluorescent dye has been added to result in the amplified product with nucleic-acid-binding fluorescent dye bound thereto;

(c) illuminating amplified product with nucleic-acid-binding fluorescent dye bound thereto with light for eliciting fluorescence therefrom;

(d) monitoring fluorescence emission; and

(e) determining a cycle when the fluorescence emission reaches a plateau phase, indicating the completion of the reaction;

wherein the monitoring fluorescence emission step consists of monitoring an amplification depend emission of the fluorescent dye.

57. The method of claim 56 wherein the nucleic-acid-binding fluorescent dye is a member selected from the group consisting of SYBR™

GREEN I, ethidium bromide, pico green, acridine orange, thiazole orange, YO-PRO-1, and chromomycin A3.

58. The method of claim 57 wherein the double-strand-specific fluorescent dye is SYBR™

GREEN I.

59. A method of determining a concentration of a selected nucleic acid template by competitive quantitative polymerase chain reaction comprising the steps of:

(a) in a reaction mixture comprising;

(i) effective amounts of each of a pair of oligonucleotide primers configured for amplifying, in a polymerase chain reaction, a selected segment of the selected template and a corresponding selected segment of a competitive template to result in amplified products thereof,

(ii) an effective amount of an oligonucleotide probe labeled with a resonance energy transfer donor or a resonance energy transfer acceptor of a fluorogenic resonance energy transfer pair, wherein the probe is configured for hybridizing to the amplified products such that the probe melts from the amplified product of the selected template at a melting temperature that is distinguishable from the melting temperature at which the probe melts from the amplified product of the competitive template,

(iii) an effective amount of a reference oligonucleotide labeled with the donor or the acceptor, with the proviso that as between the probe and reference therefor;

oligonucleotide one is labeled with the donor and the other is labeled with the acceptor, wherein the reference oligonucleotide is configured for hybridizing to the amplified products such that the donor and the acceptor are in resonance energy transfer relationship when both the probe and the reference oligonucleotide hybridize to the amplified products;

amplifying, by polymerase chain reaction, an unknown amount of the selected template and a known amount of the competitive template to result in the amplified products thereof;

(b) illuminating the reaction mixture with a selected wavelength of light for eliciting fluorescence by the fluorogenic resonance energy transfer pair and determining a fluorescence emission as a function of temperature as the temperature of the reaction mixture is changed to result in a first melting curve of the probe melting from the amplified product of the selected template and a second melting curve of the probe melting from the competitive template;

(c) converting the first and second melting curves to first and second melting peaks and determining relative amounts of the selected template and the competitive template from such melting peaks; and

(d) calculating the concentration of the selected template based on the known amount of the competitive template and the relative amounts of selected template and competitive template.

60. The method of claim 59 wherein the reference oligonucleotide is one of the pair of oligonucleotide primers.

61. The method of claim 60 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

62. The method of claim 60 wherein the donor and the acceptor are coupled to the probe and reference oligonucleotide such that when the probe and reference oligonucleotide are hybridized to the amplified segment, the donor and acceptor are separated by no more than about 25 nucleotide residues.

63. The method of claim 62 wherein the donor and the acceptor are coupled to the probe and the reference oligonucleotide such that when the probe and reference oligonucleotide are hybridized to the amplified segment, the donor and acceptor are separated by about 0-5 nucleotide residues.

64. The method of claim 63 wherein the donor and the acceptor are coupled to the probe and reference oligonucleotide such that when the probe and transfer oligonucleotide are hybridized to the amplified segment, the donor and acceptor are separated by about 0-2 nucleotide residues.

65. The method of claim 64 wherein the donor and the acceptor are coupled to the probe and reference oligonucleotide such that when the probe and transfer oligonucleotide are hybridized to the amplified segment, the donor and acceptor are separated by 1 nucleotide residue.

66. The method of claim 59 wherein the reference oligonucleotide is not one of the oligonucleotide primers.

67. The method of claim 66 wherein the resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

68. The method of claim 67 wherein the donor and the acceptor are coupled to the probe and the reference oligonucleotide such that when the probe and reference oligonucleotide are hybridized to the amplified segment, the donor and acceptor are separated by no more than about 15 nucleotide residues.

69. The method of claim 68 wherein the donor and the acceptor are coupled to the probe and the reference oligonucleotide such that when the probe and reference oligonucleotide are hybridized to the amplified segment, the donor and acceptor are separated by about 4-6 nucleotide residues.

70. A method of determining a concentration of a selected nucleic acid template in a polymerase chain reaction comprising the steps of:

(a) in a reaction mixture comprising;

(i) effective amounts of each of a first pair of oligonucleotide primers configured for amplifying, in a polymerase chain reaction, a selected first segment of the selected template to result in an amplified first product thereof,

(ii) effective amounts of each of a second pair of oligonucleotide primers configured for amplifying, in a polymerase chain reaction, a selected second segment of a reference template to result in an amplified second product thereof,

(iii) an effective amount of a nucleic-acid-binding fluorescent dye;

amplifying, by polymerase chain reaction, an unknown amount of the selected template to result in the amplified first product and a known amount of the reference template to result in the amplified second product thereof;

(b) illuminating the reaction mixture with a selected wavelength of light for eliciting fluorescence by the nucleic-acid-binding fluorescent dye;

(c) continuously monitoring through a series of amplification cycles the fluorescence emitted as a function of temperature and choosing one cycle to result in a melting curve of the amplified products wherein the first product and second product melt at different temperatures, wherein the monitoring consists of monitoring the amplification dependent fluorescence emitted as a function of temperature;

(d) converting the melting curves to melting peaks and determining relative amounts of the selected template and the reference template from such melting peaks; and

(e) calculating the concentration of the selected template based on the known amount of the reference template and the relative amounts of selected template and reference template.

71. The method of claim 70 wherein the nucleic-acid-binding fluorescent dye is selected from the group consisting of SYBR™

GREEN I, ethidium bromide, pico green, acridine orange, thiazole orange, YO-PRO-1, and chromomycin A3.

72. The method of claim 71 wherein the double-strand specific fluorescent dye is SYBR™

GREEN I.

73. A method of monitoring amplification of a selected template in a polymerase chain reaction that also comprises a positive control template comprising the steps of:

(a) in a reaction mixture comprising;

(i) effective amounts of each of a first pair of oligonucleotide primers configured for amplifying, in a polymerase chain reaction, a selected first segment of the selected template to result in an amplified first product thereof,

(ii) effective amounts of each of a second pair of oligonucleotide primers configured for amplifying, in a polymerase chain reaction, a selected second segment of the positive control template to result in an amplified second product thereof,

(iii) an effective amount of a nucleic-acid-binding fluorescent dye;

subjecting the selected template and the positive control template to conditions for amplifying the selected template and the positive control template by polymerase chain reaction; and

(b) illuminating the reaction mixture with a selected wavelength of light for eliciting fluorescence by the nucleic-acid-binding fluorescent dye and continuously monitoring the fluorescence emitted as a function of temperature during an amplification cycle of the polymerase chain reaction to result in a first melting curve of the amplified first product, if the selected template is amplified, and a second melting curve of the amplified second product, if the positive control template is amplified;

wherein obtaining of the second melting curve indicates that the polymerase chain reaction was operative, obtaining the first melting curve indicates that the selected first segment was amplifiable, and absence of the first melting curve indicates that the selected first segment was not amplifiable.

74. The method of claim 73 wherein the nucleic-acid-binding fluorescent dye is selected from the group consisting of SYBR™

GREEN I, ethidium bromide, pico green, acridine orange, thiazole orange, YO-PRO-1, and chromomycin A3.

75. The method of claim 74 wherein the double-strand specific fluorescent dye is SYBR™

GREEN I.

76. A method of analyzing nucleic acid hybridization comprising the steps of

(a) providing a mixture comprising a nucleic acid sample to be analyzed and a nucleic acid binding fluorescent entity;

and

(b) monitoring fluorescence while changing temperature at a rate of ≧

0.1°

C./second.

77. The method of claim 76 wherein the nucleic acid binding fluorescent entity is SYBR™

Green I.

78. The method of claim 76 wherein the nucleic acid binding fluorescent entity comprises a pair of oligonucleotide probes wherein one of the probes is labeled with a donor and the other probes is labeled with an acceptor of a fluorogenic resonance energy transfer pair.

79. The method of claim 78 wherein one of the probes is a primer for polymerase chain reaction amplification.

80. A method of quantitating an initial copy number of a sample containing an unknown amount of nucleic acid comprising the steps of

(a) amplifying by polymerase chain reaction at least one standard of known concentration in a mixture comprising the standard and a nucleic acid binding fluorescent entity;

(b) measuring fluorescence as a function of cycle number to result in a set of data points;

(c) fitting the data points to a given predetermined equation describing fluorescence as a function of initial nucleic acid concentration and cycle number;

(d) amplifying the sample containing the unknown amount of nucleic acid in a mixture comprising the sample and the nucleic acid binding fluorescent entity and monitoring fluorescence thereof; and

(e) determining initial nucleic acid concentration from the equation determined in step (c).

81. The method of claim 80 wherein the nucleic acid binding fluorescent entity is SYBR™

Green I.

82. The method of claim 80 wherein the nucleic acid binding fluorescent entity comprises a pair of oligonucleotide probes, one of which is labeled with a donor and the other of which is labeled with an acceptor of a fluorogenic resonance energy transfer pair.

83. The method of claim 82 wherein one of the probes is a primer for amplification.

91. A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps of

amplifying the target sequence by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding the probes, a thermostable polymerase, and primers for the targeted nucleic acid sequence to the biological sample to create an amplification medium, and thermally cycling the amplification medium between at least a denaturation temperature and all elongation temperature;

during at least one amplification cycle, illuminating amplification medium with light having a wavelength absorbed by the donor fluorophore and detecting fluorescent emission from the fluorescence energy transfer pair,

wherein the detecting consists of detecting an amplification dependent fluorescent emission.

92. A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps of

amplifying the target sequence by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding the probes, a thermostable polymerase and primers for the targeted nucleic acid sequence to the biological sample to provide an amplification medium and thermally cycling the amplification medium between at least a denaturation temperature and an elongation temperature;

illuminating the amplification medium with light having a wavelength absorbed by the donor fluorophore; and

monitoring temperature dependent fluorescence emissions from the fluorescence energy transfer pair during thermal cycling;

wherein the monitoring step consists of monitoring amplification dependent fluorescence emissions.

93. A method for detecting a target nucleic acid sequence of a biological sample, said method comprising the steps of

(a) adding to the biological sample an effective amount of a pair of oligonucleotide probes that hybridize to the target nucleic acid sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair, wherein an emission spectrum of the donor fluorophore and an absorption spectrum of the acceptor fluorophore overlap less than 25%, the acceptor fluorophore has a peak extinction coefficient greater than 100,000 M⁻

cm<sup>−

1</sup> and upon hybridization of the two probes, the donor and acceptor fluorophores are within 25 nucleotides of one another;

(b) illuminating the biological sample with a selected wavelength of light that is absorbed by said donor fluorophore; and

(c) detecting fluorescent emission of the biological sample;

wherein the illuminating and detecting steps occur during amplification and the detecting step consists of detecting the amplification dependent emission.

94. A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps of

amplifying the target sequence by polymerase chain reaction in the presence of two linear nucleic acid probes capable of hybridizing to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase and primers for the targeted nucleic acid sequence to the biological sample and thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature;

exciting the sample with light at a wavelength absorbed by the donor fluorophore; and

monitoring the temperature dependent fluorescence from the fluorescence energy transfer pair.

95. The method of claim 94 wherein the donor and acceptor fluorophores are at a distance of about 0-5 nucleotides.

96. The method of claim 95 wherein the donor and acceptor fluorophores are at a distance of about 0-2 nucleotides.

97. The method of claim 96 wherein the donor and acceptor fluorophores are at a distance of 1 nucleotide.

98. A method of detecting heterozygosity at a selected locus in the genome of an individual, wherein the genome comprises a mutant allele and a corresponding reference allele, each comprising the selected locus, comprising the steps of:

(a) obtaining sample genomic DNA from the individual;

(b) providing a pair of oligonucleotide primers configured for amplifying, by polymerase chain reaction, a first selected segment of the mutant allele and a second selected segment of the corresponding reference allele wherein both the first and second selected segments comprise the selected locus;

(c) providing a pair of linear oligonucleotide probes capable of hybridizing with the mutant and reference alleles, one of said probes being labeled with an acceptor fluorophore and the other probe being labeled with a donor fluorophore of a fluoro(genic resonance energy transfer pair such that upon hybridization of the two probes with the amplified first and second selected segments one of the probes spans the selected locus and exhibits a first melting profile with the amplified first selected segment that is distinguishable from a second melting profile with the amplified second selected segment;

(d) amplifying the first and second selected segments of sample genomic DNA by polymerase chain reaction in the presence of effective amounts of probes to result in amplified first and second selected segments, at least a portion thereof having both the probes hybridized thereto with the fluorogenic resonance energy transfer pair in resonance energy transfer relationship;

(e) illuminating the amplified first and second selected segments having the probes hybridized thereto with a selected wavelength of light to elicit fluorescence by the donor and acceptor;

(f) measuring a fluorescence emission as a function of temperature to determine a first melting profile of said one of the probes melting from the amplified first selected segment and a second melting profile of said one of the probes melting from the amplified second selected segment; and

(g) comparing the first melting profile to the second melting profile, wherein distinguishable melting profiles indicate heterozygosity in the sample genomic DNA.

99. The method of claim 98 wherein the fluorogenic resonance energy transfer pair comprises fluorescein as the donor and Cy5 or Cy5.5 as the acceptor.

100. The method of claim 98 wherein the donor and the acceptor are coupled to the probes such that when the probe and reference oligonucleotide are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by no more than about 25 nucleotide residues.

101. The method of claim 100 wherein the donor and the acceptor are coupled to the probes such that when the probes are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by about 0-5 nucleotide residues.

102. The method of claim 101 wherein the donor and the acceptor are coupled to the probes such that when the probes are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by about 0-2 nucleotide residues.

103. The method of claim 102 wherein the donor and the acceptor are coupled to the probes such that when the probes are hybridized to the amplified first selected segment or the amplified second selected segment, the donor and acceptor are separated by 1 nucleotide residue.

107. An improved method of amplifying a target nucleic acid sequence of a biological sample, said method comprising the steps of

(a) adding to the biological sample an effective amount of a fluorescent label consisting of a nucleic-acid-binding fluorescent entity;

(b) amplifying the target nucleic acid sequence using polymerase chain reaction, comprising thermally cycling the biological sample using initial predetermined temperature and time parameters, and then

(i) illuminating the biological sample with a selected wavelength of light that is absorbed by said fluorescent entity during the polymerase chain reaction;

(ii) monitoring fluorescence from said sample to determine the optimal temperature and time parameters for the polymerase chain reaction; and

(iii) adjusting the initial temperature and time parameters in accordance with the fluorescence.