Fluorescence polarization detection of nucleic acids

• US 20030219754A1
• Filed: 05/23/2002
• Published: 11/27/2003
• Est. Priority Date: 05/23/2002
• Status: Abandoned Application

• Current Assignee: Perkinelmer Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Perkinelmer Life Sciences Incorporated (Revvity, Inc.)
• Inventors: Boeckman, Faye A., Hungate, Eric A., Oleksy, Jerome E., Rencs, Erik V.
• Application Number:
  US10/155,285
• Publication Number:
  US 20030219754A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6484 Optical fibres
• 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 21/6445 Measuring fluorescence polarisation
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

The apparatus and method described herein detect fluorescence polarization (FP) during a nucleic acid reaction such as PCR amplification or isothermal amplification. Fluorescence polarization can be concurrently detected in multiple samples. In addition, multiple different fluorophores can be used for detect different sequences within a sample during the same reaction.

Claims

1. An apparatus comprising:

a sample carrier that comprises spatially distinct nucleic acid samples;

a light source configured to concurrently excite fluorescent compounds, located in a plurality of spatially distinguishable areas within a first region of the sample carrier, with polarized light;

a detection system configured to concurrently detect emitted light from the fluorescent compounds in each of the areas of the plurality in the first region; and

a thermal control unit configured to regulate the temperature of the sample carrier.

2. The apparatus of claim 1 wherein the thermal control unit is configured to cyclically heat and cool the carrier during a reaction course.

3. The apparatus of claim 1 wherein the sample carrier is immobilized relative to one or both of the detection system and the light source.

4. The apparatus of claim 1 wherein the sample carrier comprises a plurality of physically bounded areas.

5. The apparatus of claim 4 wherein each area of the plurality comprises a container within the sample carrier.

6. The apparatus of claim 1 wherein the detection system is further configured to detect emitted light of a first polarity and emitted light of a second polarity.

7. The apparatus of claim 6 wherein the first and second polarities are approximately orthogonal to each other.

8. The apparatus of claim 6 wherein the first polarity is parallel to the polarity of the polarized light from the light source, and the second polarity is non-parallel to the polarity of the polarized light from the light source.

9. The apparatus of claim 6 wherein the detection system is configured to detect the first and second polarity light concurrently.

10. The apparatus of claim 6 wherein the detection system comprises a first and second detector.

11. The apparatus of claim 9 wherein the detection system has a single detector, and the first and second polarity light are projected onto different regions of the detector.

12. The apparatus of claim 6 wherein the detection system is further configured to detect the first and second polarity light sequentially.

13. The apparatus of claim 12 wherein the detection system comprises a polarizer that is controlled to enable sequential detection of the first and second polarity light.

14. The apparatus of claim 1 wherein the detection system is further configured to distinguish polarized light from a first fluorophore from polarized light from a second fluorophore.

15. The apparatus of claim 6 wherein the first region comprises all physically distinct samples of the sample carrier.

16. The apparatus of claim 1 wherein the apparatus further comprises a scanning mirror.

17. The apparatus of claim 16 wherein the scanning mirror reflects excitation light from a light source.

18. The apparatus of claim 16 wherein the scanning mirror reflects emitted light from the sample carrier to a detector.

19. The apparatus of claim 1 wherein the light path emanating from the light source is parallel to the incident light path into the detector.

20. The apparatus of claim 19 wherein the detector is positioned between the light source and an imageable surface of the sample carrier.

21. The apparatus of claim 1 wherein the light path from the light source to the sample carrier surface or the light path from an imageable surface of the sample carrier to the detector is oblique with respect to the imageable surface.

22. The apparatus of claim 1 wherein the light source is further configured to excite a second region and the detection system is further configured to detect emitted light from the second region.

23. The apparatus of claim 1 wherein the detection system is configured to detect light in a plane parallel to the polarized light from the light source.

24. The-apparatus of claim 1 wherein the detection system and light source are in signal communication to enable a temporal delay between excitation and detection.

25. An apparatus comprising:

a plurality of spatially distinguishable reaction samples, each comprising amplification reagents that include a nucleic acid primer that is attached to a fluorophore;

an amplification control unit that is configured to control conditions of the reaction samples for nucleic acid amplification;

a fluorescence polarization monitor that is configured to concurrently monitor fluorescence polarization associated with each reaction sample of the plurality.

26. The apparatus of claim 25 wherein the fluorescence polarization monitor comprises a source of polarized light and a detector that can concurrently monitor emitted light from each sample of the plurality of reaction samples.

27. The apparatus of claim 26 wherein the detector is configured to concurrently monitor emitted light of a predetermined polarity.

28. The apparatus of claim 27 wherein the detector is configured to sequentially detect light of a first polarity and light of a second polarity, the light of the first polarity being parallel to the plane of the polarized light from the source.

29. The apparatus of claim 28 wherein the first and second polarities are orthogonal to each other.

30. The apparatus of claim 25 wherein at least some of the samples comprise a second nucleic acid primer that is attached to a second fluorophore that is spectrally distinguishable from the first fluorophore, and the detector comprises optical filters that can distinguish emitted light from the first and second fluorophore.

31. A method comprising:

providing a plurality of spatially distinct nucleic acid samples and amplification reagents that comprises a fluorophore attached to a nucleic acid primer;

concurrently amplifying each sample of the plurality; and

during the amplifying, concurrently detecting fluorescence polarization information associated with-the fluorophore from each sample of the plurality.

32. The method of claim 31 wherein the detecting comprises detecting fluorescence polarization information at at least a plurality of instances during the amplifying.

33. The method of claim 31 wherein the amplifying comprises thermal cycles and the detecting comprises detecting fluorescence polarization information at at least one instance for each cycle.

34. The method of claim 33 wherein the at least one instance for each cycle is at a predetermined temperature of the cycle.

35. The method of claim 31 wherein the amplifying and detecting are effected by an apparatus comprising

a light source configured to concurrently excite the fluorophores, located in a plurality of the spatially distinct samples, with polarized light;

and

a detection system configured to concurrently detect emitted light from the fluorophores in each of the spatially distinct samples of the plurality.

36. The method of claim 31 wherein the amplifying comprises PCR amplification.

37. The method of claim 36 wherein the PCR amplification comprises exponential amplification.

38. The method of claim 36 wherein the PCR amplification comprises linear amplification.

39. The method of claim 31 wherein the detecting comprises exciting the fluorophore with polarized excitation light and detecting emitted light in a first predetermined plane.

40. The method of claim 39 wherein the first predetermined plane is parallel to the plane of the polarized excitation light.

41. The method of claim 40 wherein the detecting further comprises detecting emitted light in a second predetermined plane.

42. The method of claim 41 wherein the emitted light in the first and second predetermined planes are detected concurrently.

43. The method of claim 41 wherein the emitted light in the first and second predetermined planes are detected at separate times.

44. The method of claim 35 wherein each of the samples is disposed in a separate address of a sample carrier.

45. The method of claim 44 wherein the sample carrier is stationary relative to the light source and/or detection system throughout the amplifying.

46. A method comprising:

providing a reaction mixture that include a nucleic acid sample, amplification reagents, and a fluorescent probe that is bindable to double-stranded nucleic acid and has at least a 10-fold preference for double-stranded nucleic acid relative to single-stranded nucleic acid;

amplifying each sample of the plurality; and

during the amplifying, detecting fluorescence polarization information associated with the fluorescent probe at at least a plurality of instances.

47. The method of claim 46 wherein the fluorescent probe is an intercalating dye.

48. The method of claim 47 wherein the dye is Sybr Green or ethidium bromide.

49. The method of claim 46 wherein a plurality of reaction mixtures having different nucleic acid samples are provided, and the mixtures are concurrently amplified and concurrently detected.

50. A method comprising:

providing a nucleic acid sample and amplification reagents that comprises a first fluorophore attached to a first nucleic acid primer and a second fluorophore attached to a second nucleic acid primer;

amplifying nucleic acid in the sample using the first and second primers; and

at at least a plurality of instances during the amplifying, detecting fluorescence polarization information associated with each of the fluorophores.

51. The method of claim 50 in which the first and second fluorophore have distinguishable absorption and/or emission spectra.

52. The method of claim 51 in which the detecting comprises sequentially detecting fluorescence polarization information of the first fluorophore at a first wavelength and information from the second fluorophore at a second wavelength.

53. The method of claim 50 in which the first and second primer hybridize to the same gene.

54. A method comprising:

providing a nucleic acid sample and amplification reagents that comprises a first fluorophore attached to a first nucleic acid primer, specific for a first nucleic acid species, and a second fluorophore attached to a second nucleic acid primer, specific for a second nucleic acid species;

amplifying nucleic acid in the sample using the first and second primers; and

at at least a plurality of instances during the amplifying, detecting fluorescence polarization information associated with each of the fluorophores.

55. The method of claim 54 in which the detecting comprises sequentially detecting fluorescence polarization information of the first fluorophore at a first wavelength and information from the second fluorophore at a second wavelength.

56. The method of claim 54 in which the first and second fluorophore are selected from the group consisting of:

a fluorescein, Texas Red, HEX, Cy3, Cy5, Cy5.5, Pacific Blue, a rhodamine, and Cy7.

57. The method of claim 54 in which at least four different labeled primers are used and detected.

58. An article of machine-readable medium, having embodied thereon instructions that cause a processor to effect a method comprising:

determining intensity values, wherein (i) each intensity value is determined as a function of a value representing fluorescence approximately perpendicular and a value approximately parallel to polarized excitation light, (ii) the fluorescence is detected from a fluorophore attached to a primer specific for a target nucleic acid, and (iii) each intensity value corresponds to an temporal instance during a nucleic acid amplification reaction;

extrapolating an initial intensity value from intensity values within an exponential region of the amplification reaction; and

inferring an initial concentration for the target nucleic acid.

59. The article of claim 58 wherein inferring comprises comparing an inferred initial intensity value for the target nucleic acid to a similarly inferred initial intensity value for a reference nucleic acid of known molecular concentration, and determining an estimated initial molecular concentration for the target nucleic acid.

60. An article of machine-readable medium, having embodied thereon instructions that cause a processor to effect a method comprising:

receiving a plurality of image maps, each map including information about detected light of a defined polarity at a plurality of imaged sites, each imaged site including a primer for amplification of a target nucleic acid; and

determining a value indicative of abundance of extended primers at each of the imaged sites.

61. The article of claim 60 wherein each of the imaged sites corresponds to a sample on a multi-sample carrier.

62. The article of claim 60 wherein the plurality of image maps comprise maps including information about detected light at different instances during a reaction.

63. The article of claim 62 wherein at least one of the imaged sites corresponds to a reference sample of known molecular concentration.

64. The article of claim 63 wherein the instructions further cause a processor to infer an initial concentration of target nucleic acid for at least some of the imaged sites from information for the imaged site that corresponds to a reference sample.

65. A database, stored on machine-readable medium, comprising:

data representing (a) fluorescence polarization assessments, (b) reaction samples, (c) temporal information; and

associations that relate each fluorescence polarization assessment to a reaction sample and a temporal value.

66. A database, stored on machine-readable medium, comprising:

a plurality of image maps, each map including information about detected light of a defined polarity at a plurality of imaged sites, wherein the detected light of each map is associated with a fluorophore and each map is associated with a temporal instance during an nucleic acid amplification reaction.

67. A system comprising:

an apparatus that comprises (1) an amplification control unit that is configured to control reaction conditions at a plurality of sites for nucleic acid amplification; and

(2) a fluorescence polarization monitor that is configured to concurrently monitor fluorescence polarization associated with each site of the plurality; and

a processor configured to receive information from the apparatus about the fluorescence polarization and infer initial values that correlate with concentration for a nucleic acid species at each site of the plurality.