Methods for forensic DNA quantitation

• US 9,550,985 B2
• Filed: 06/15/2010
• Issued: 01/24/2017
• Est. Priority Date: 06/15/2009
• Status: Active Grant

• Current Assignee: ANDE Corp.
• Original Assignee: NetBio, Inc.
• Inventors: Selden, Richard F, Tan, Eugene, Turingan, Rosemary S.
• Primary Examiner(s): Dauner, Joseph G
• Application Number:
  US12/816,370
• Publication Number:
  US 20110008785A1
• Time in Patent Office: 2,415 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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: C12N 15/1017 by filtration, e.g. using filters, frits, membranes
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 15/1484 microstructural devices
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G02B 27/10 Beam splitting or combining systems polarising systems G02B27/28; mixing and splitting light signals using optical waveguides G02B6/28; beam shaping, e.g. changing the cross-sectional area, by dividing or superposing multiple light beams G02B27/0905
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Described herein are methods and devices for nucleic acid quantification and, in particular, to microfluidic methods and devices for nucleic acid quantification. In certain embodiments methods of quantifying a target nucleic acid without the need for amplification are provided. The methods involve, in some embodiments, allowing a binding agent to become immobilized with respect to the target nucleic acid. In some cases, the binding agent comprises a signaling moiety that can be used to quantify the amount of target nucleic acid. In another aspect, the quantification can be carried out rapidly. For example, in certain embodiments, the quantification can be completed within 5 minutes. In yet another aspect, samples containing a low amount of target nucleic acid can be quantified. For instance, in some cases, samples containing less than 100 nanograms per microliter may be quantified. Also described are devices and kits for performing such methods, or the like.

Claims

1. A method for quantifying an unamplified double stranded target nucleic acid in a sample fluid containing or suspected of containing the unamplified double stranded target nucleic acid, comprising:

combining in a microfluidic channel located in a biochip, said biochip further comprising a plurality of microfluidic channels and active areas for sample manipulation, at least a portion of the sample fluid and a probe fluid containing a binding agent comprising a signaling moiety to form a test fluid;

heating said test fluid to denature the unamplified double stranded target nucleic acid and cooling said test fluid to bind the binding agent to the denatured unamplified target nucleic acid;

maintaining the test fluid stationary in the microfluidic channel at a detector region;

detecting the signaling moiety in the test fluid at said detector region; and

quantifying the denatured unamplified target nucleic acid in the test fluid based on the detected signaling moiety;

wherein the denatured unamplified target nucleic acid in the test fluid is quantified within 1 minute of combining the portion of the sample fluid and said probe fluid.

2. The method of claim 1, wherein quantifying the denatured unamplified target nucleic acid comprises determining the concentration of the denatured unamplified target nucleic acid in the test fluid.

3. The method of claim 1, wherein the binding agent is a specific binding agent having unique specificity for the denatured unamplified target nucleic acid.

4. The method of claim 3, wherein the specific binding agent hybridizes to the denatured unamplified target nucleic acid.

5. The method of claim 4, wherein the specific binding agent is a molecular beacon probe.

6. The method of claim 1, wherein detecting the signaling moiety comprises irradiating the signaling moiety with a laser.

7. The method of claim 6, wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

8. The method of claim 1, wherein detecting the signaling moiety comprises detecting fluorescence.

9. The method of claim 1, wherein the unamplified double stranded target nucleic acid, if present,

has a concentration less than 1 nanogram per microliter in the sample fluid or is present in a total amount of less than 1 nanogram in the sample fluid.

10. The method of claim 1, wherein the sample fluid comprises a forensic sample.

11. The method of claim 10, wherein the unamplified double stranded target nucleic acid is a human nucleic acid.

12. The method of claim 11, wherein the sample fluid further comprises a non-human, non-target nucleic acid.

13. The method of claim 11, wherein the unamplified double stranded target nucleic acid is the human Alu repetitive element.

14. A method for quantifying an unamplified double stranded target nucleic acid in a forensic sample fluid containing or suspected of containing the unamplified double stranded target nucleic acid, comprising:

combining in a microfluidic channel located in a biochip, said biochip further comprising a plurality of microfluidic channels and active areas for sample manipulation, at least a portion of the forensic sample fluid and a probe fluid containing a binding agent comprising a signaling moiety to form a test fluid;

heating said test fluid to denature the unamplified double stranded target nucleic acid and cooling the test fluid to bind the binding agent to the denatured unamplified target nucleic acid;

maintaining the test fluid stationary in the microfluidic channels at a detector region;

detecting the signaling moiety in the test fluid at said detector region; and

quantifying the denatured unamplified target nucleic acid in the test fluid based on the detected signaling moiety;

wherein the denatured unamplified target nucleic acid in the test fluid is quantified within 1 minute of combining the portion of the forensic sample fluid and the probe fluid.

15. The method of claim 14, wherein quantifying the denatured unamplified target nucleic acid comprises determining the concentration of the denatured unamplified target nucleic acid in the test fluid.

16. The method of claim 14, wherein quantifying the denatured unamplified target nucleic acid comprises determining the total amount of the denatured unamplified target nucleic acid in the test fluid.

17. The method of claim 14, wherein the binding agent is a specific binding agent having unique specificity for the denatured unamplified target nucleic acid.

18. The method of claim 17, wherein the specific binding agent hybridizes to the denatured unamplified target nucleic acid.

19. The method of claim 18, wherein the specific binding agent is a molecular beacon probe.

20. The method of claim 14, wherein the unamplified double stranded target nucleic acid, if present, has a concentration less than 1 nanogram per microliter in the forensic sample fluid or is present in a total amount-of less than 1 nanogram in the forensic sample fluid.

21. The method of claim 14, wherein detecting the signaling moiety comprises irradiating the signaling moiety with a laser.

22. The method of claim 21, wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

23. The method of claim 14, wherein detecting the signaling moiety comprises detecting fluorescence.

24. The method of claim 14, wherein the unamplified double stranded target nucleic acid is a human nucleic acid.

25. The method of claim 24, wherein the sample fluid further comprises a non-human, non-target nucleic acid.

26. The method of claim 24, wherein the unamplified double stranded target nucleic acid is the human Alu repetitive element.

27. A method for quantifying an unamplified double stranded target nucleic acid in a sample fluid containing or suspected of containing the unamplified double stranded target nucleic acid and also containing a contaminating non-human nucleic acid, comprising:

combining in a microfluidic channel located in a biochip, said biochip further comprising a plurality of microfluidic channels and active areas for sample manipulation, at least a portion of the sample fluid and a probe fluid containing a binding agent comprising a signaling moiety to form a test fluid;

heating said test fluid to denature the unamplified double stranded target nucleic acid and cooling the test bind the binding agent to the denatured unamplified target nucleic acid;

maintaining the test fluid stationary in the microfluidic channel at a detector region;

detecting the signaling moiety in the test fluid at said detector region; and

quantifying the denatured unamplified target nucleic acid in the test fluid based on the detected signaling moiety in the test fluid;

wherein the denatured unamplified target nucleic acid in the test fluid is quantified within 1 minute of combining the portion of the sample fluid and the probe fluid.

28. The method of claim 27, wherein quantifying the denatured unamplified target nucleic acid comprises determining the concentration of the denatured unamplified target nucleic acid in the test fluid determining the concentration of the unamplified target nucleic acid in the sample fluid.

29. The method of claim 27, wherein quantifying the denatured unamplified target nucleic acid comprises determining the total amount of the denatured unamplified target nucleic acid in the test fluid.

30. The method of claim 27, wherein the binding agent is a specific binding agent having unique specificity for the denatured unamplified target nucleic acid.

31. The method of claim 30, wherein the specific binding agent hybridizes to the denatured unamplified target nucleic acid.

32. The method of claim 31, wherein the specific binding agent is a molecular beacon probe.

33. The method of claim 27, wherein the unamplified double stranded target nucleic acid, if present, has a concentration less than 1 nanogram per microliter in the sample fluid or is present in a total amount of less than 1 nanogram in the sample fluid.

34. The method of claim 27, wherein detecting the signaling moiety comprises irradiating the signaling moiety with a laser.

35. The method of claim 34, wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

36. The method of claim 27, wherein detecting the signaling moiety comprises detecting fluorescence.

37. The method of claim 27, wherein the unamplified double stranded target nucleic acid is a human nucleic acid.

38. The method of claim 37, wherein the sample fluid further comprises a non-human, non-target nucleic acid.

39. The method of claim 37, wherein the unamplified double stranded target nucleic acid is the human Alu repetitive element.

40. The method of claim 27, wherein the sample fluid is a forensic sample fluid.

41. A method for quantifying an unamplified double stranded target nucleic acid in a sample fluid containing or suspected of containing the unamplified double stranded target nucleic acid, comprising:

providing a microfluidic device said device further comprising a plurality of microfluidic channels and active areas for sample manipulation, and coupled to an electronic device comprising a detector comprising an integrated laser;

combining in a microfluidic channel of the microfluidic device at least a portion of the sample fluid and a probe fluid containing a binding agent comprising a signaling moiety to form a test fluid;

heating said test fluid to denature the unamplified double stranded target nucleic acid and cooling the test fluid to bind the binding agent to the denatured unamplified target nucleic acid to form a complex;

maintaining the test fluid stationary in the microfluidic channel at a detector region;

irradiating the signaling moiety using the integrated laser, wherein the complex is in solution in the test fluid positioned in operative proximity to the detector of the electronic device;

detecting the signaling moiety in the test fluid at said detector region; and

quantifying the denatured unamplified target nucleic acid in the test fluid based on the detected signaling moiety;

wherein the denatured unamplified target nucleic acid in the test fluid is quantified within 1 minute of combining the portion of the sample fluid and the probe fluid.

42. The method of claim 41, wherein quantifying the denatured unamplified target nucleic acid in the test fluid comprises determining the concentration of the denatured unamplified target nucleic acid in the test fluid.

43. The method of claim 41, wherein quantifying the denatured unamplified target nucleic acid in the test fluid comprises determining the total amount of the denatured unamplified target nucleic acid in the test fluid.

44. The method of claim 41, wherein the binding agent is a specific binding agent having unique specificity for the denatured unamplified target nucleic acid.

45. The method of claim 44, wherein the specific binding agent hybridizes to the denatured unamplified target nucleic acid.

46. The method of claim 45, wherein the specific binding agent is a molecular beacon probe.

47. The method of claim 41, wherein the unamplified double stranded target nucleic acid, if present, has a concentration less than 1 nanogram per microliter in the sample fluid or is present in a total amount of less than 1 nanogram in the sample fluid.

48. The method of claim 41, wherein detecting the signaling moiety comprises detecting fluorescence.

49. The method of claim 41, wherein the unamplified double stranded target nucleic acid is a human nucleic acid.

50. The method of claim 49, wherein the sample fluid further comprises a non-human, non-target nucleic acid.

51. The method of claim 49, wherein the unamplified double stranded target nucleic acid is the human Alu repetitive element.

52. The method of claim 41, wherein the sample fluid is a forensic sample fluid.

53. A method for directing a sample fluid containing or suspected of containing an unamplified double stranded target nucleic acid to an active area of a microfluidic device, comprising:

providing the microfluidic device comprising a plurality of microfluidic channels and active areas for sample manipulation coupled to an electronic device comprising a detector;

combining in a microfluidic channel of the microfluidic device a portion of the sample fluid and a probe fluid containing a binding agent comprising a signaling moiety to form a test fluid;

heating said test fluid to denature the double stranded nucleic acid and cooling the test fluid to bind the binding agent to the denatured unamplified target nucleic acid;

maintaining the test fluid stationary in the microfluidic channel at a detector region;

detecting the signaling moiety in the test fluid at said detector region in the microfluidic channel, wherein said detector region is positioned in operative proximity to the detector of the electronic device;

quantifying the denatured unamplified target nucleic acid in the test fluid based on the detected signaling moiety; and

directing a selected quantity of the sample fluid containing the unamplified double stranded target nucleic acid to the active area of the microfluidic device, wherein the selected quantity is determined, at least in part, based on the results of the quantifying step;

wherein the denatured unamplified target nucleic acid in the test fluid is quantified within 1 minute of combining the portion of the sample fluid and the probe fluid.

54. The method of claim 53, wherein the quantifying the denatured unamplified target nucleic acid comprises determining the concentration of the denatured unamplified target nucleic acid in the test fluid.

55. The method of claim 53, wherein quantifying the denatured unamplified target nucleic acid comprises determining the total amount of the denatured unamplified target nucleic acid in the test fluid.

56. The method of claim 53, wherein the binding agent is a specific binding agent having unique specificity for the denatured unamplified target nucleic acid.

57. The method of claim 56, wherein the specific binding agent hybridizes to the denatured unamplified target nucleic acid.

58. The method of claim 57, wherein the specific binding agent is a molecular beacon probe.

59. The method of claim 53, wherein the unamplified double stranded target nucleic acid, if present, has a concentration less than 1 nanogram per microliter in the sample fluid or is present in a total amount of less than 1 nanogram in the sample fluid.

60. The method of claim 53, wherein detecting the signaling moiety comprises irradiating the signaling moiety with a laser.

61. The method of claim 60, wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

62. The method of claim 53, wherein detecting the signaling moiety comprises detecting fluorescence.

63. The method of claim 53, wherein the unamplified target nucleic acid is a human nucleic acid.

64. The method of claim 63, wherein the sample fluid further comprises a non-human, non-target nucleic acid.

65. The method of claim 63, wherein the unamplified double stranded target nucleic acid is the human Alu repetitive element.

66. The method of claim 53, wherein the sample fluid is a forensic sample fluid.

67. A method for quantifying an unamplified double stranded target nucleic acid in a forensic sample fluid containing or suspected of containing the unamplified double stranded target nucleic acid, comprising:

combining in a microfluidic channel located in a biochip, said biochip further comprising a plurality of microfluidic channels and active areas for sample manipulation, at least a portion of the forensic sample fluid and a probe fluid containing a molecular beacon probe comprising a signaling moiety to form a test fluid;

heating said test fluid to denature the double stranded nucleic acid and cooling the test fluid to hybridize the molecule beacon probe to the denatured unamplified target nucleic acid;

maintaining the test fluid stationary at a detector region,

detecting the signaling moiety in the test fluid at said detector region; and

quantifying the denatured unamplified target nucleic acid in the test fluid based on the detected signaling moiety;

wherein the denatured target nucleic acid in the test fluid is quantified within 1 minute of combining the portion of the forensic sample fluid and the probe fluid.

68. The method of claim 67, wherein quantifying the denatured unamplified target nucleic acid comprises determining the concentration of the denatured unamplified target nucleic acid in the test fluid.

69. The method of claim 67, wherein quantifying the denatured unamplified target nucleic acid comprises determining the total amount of the denatured unamplified target nucleic acid in the test fluid.

70. The method of claim 67, wherein the unamplified double stranded target nucleic acid, if present, has a concentration less than 1 nanogram per microliter in the forensic sample fluid or is present in a total amount of less than 1 nanogram in the forensic sample fluid.

71. The method of claim 67, wherein detecting the signaling moiety comprises irradiating the signaling moiety with a laser.

72. The method of claim 71, wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

73. The method of claim 67, wherein detecting the signaling moiety comprises detecting fluorescence.

74. The method of claim 67, wherein the unamplified double stranded target nucleic acid is a human nucleic acid.

75. The method of claim 74, wherein the forensic sample fluid further comprises a non-human, non-target nucleic acid.

76. The method of claim 74, wherein the unamplified double stranded target nucleic acid is the human Alu repetitive element.

77. A method for quantifying an unamplified double stranded target nucleic acid in a sample fluid containing or suspected of containing the unamplified double stranded target nucleic acid, comprising:

combining in a microfluidic channel located in a biochip, said biochip further comprising a plurality of microfluidic channels and active areas for sample manipulation, at least a portion of the sample fluid and a binding agent comprising a signaling moiety to form a test fluid;

heating said test fluid to denature the unamplified double stranded target nucleic acid and cooling the test fluid to bind the binding agent to the denatured unamplified target nucleic acid to form a complex;

maintaining the test fluid stationary in the microfluidic channel at a detector region;

detecting the signaling moiety of the complex in the test fluid at said detector region;

quantifying the denatured unamplified target nucleic acid in the test fluid based on the detected signaling moiety;

wherein the denatured unamplified target nucleic acid in the test fluid is quantified within 1 minute of combining the portion of the sample fluid and the probe fluid.

78. The method of claim 77, wherein quantifying the denatured unamplified target nucleic acid comprises determining the concentration of the denatured unamplified target nucleic acid in the test fluid.

79. The method of claim 77, wherein quantifying the denatured unamplified target nucleic acid comprises determining the total amount of the denatured unamplified target nucleic acid in the test fluid.

80. The method of claim 77, wherein the binding agent is a specific binding agent having unique specificity for the denatured unamplified target nucleic acid.

81. The method of claim 80, wherein the specific binding agent hybridizes to the denatured unamplified target nucleic acid.

82. The method of claim 81, wherein the specific binding agent is a molecular beacon probe.

83. The method of claim 77, wherein detecting the signaling moiety comprises irradiating the signaling moiety with a laser.

84. The method of claim 83, wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

85. The method of claim 77, wherein detecting the signaling moiety comprises detecting fluorescence.

86. The method of claim 77, wherein the sample fluid comprises a forensic sample.

87. The method of claim 86, wherein the unamplified double stranded target nucleic acid is a human nucleic acid.

88. The method of claim 87, wherein the sample fluid further comprises a non-human, non-target nucleic acid.

89. The method of claim 87, wherein the unamplified double stranded target nucleic acid is the human Alu repetitive element.

90. The method of claim 77, wherein the unamplified double stranded target nucleic acid, if present, has a concentration less than 1 nanogram per microliter in the sample fluid or is present in a total amount-of less than 1 nanogram in the sample fluid.