Methods for forensic DNA quantitation

• US 10,538,804 B2
• Filed: 01/19/2017
• Issued: 01/21/2020
• Est. Priority Date: 06/15/2009
• Status: Active Grant

• Current Assignee: ANDE Corp.
• Original Assignee: ANDE Corp.
• Inventors: Tan, Eugene, Selden, Richard F., Turingan, Rosemary S.
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US15/410,399
• Publication Number:
  US 20170204453A1
• Time in Patent Office: 1,097 Days
• Field of Search: None
• 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 a human genomic double stranded DNA target in a sample fluid containing or suspected of containing said target, comprising:

combining in a microfluidic channel (i) the sample fluid and (ii) at least one molecular beacon probe capable of binding to at least one repetitive human Alu sequence said probe further comprising a signaling moiety;

to form a test fluid, wherein said target in the sample fluid has not been amplified;

heating said test fluid to denature the double stranded DNA and cooling said test fluid to facilitate binding of the molecular beacon probe to said target so that the molecular beacon probe becomes bound to said target;

locating the test fluid in a detector region in the microfluidic channel;

detecting the signaling moiety; and

quantifying said target in the sample fluid within 1 minute of combining the sample fluid and the molecular beacon probe.

2. The method of claim 1, wherein the quantifying step comprises determining the concentration of said target in the test fluid.

3. The method of claim 1, wherein the molecular beacon probe has a unique specificity for said at least one repetitive human Alu sequence.

4. The method of claim 1, wherein the locating step comprises flowing the test fluid to the detector region in the microfluidic channel.

5. The method of claim 4, wherein the locating step comprises flowing the test fluid through the detector region of the microfluidic channel during detection.

6. The method of claim 1, wherein the locating step comprises maintaining the test fluid stationary at a region in the microfluidic channel and moving a detector into alignment with the region to transform the region into the detector region.

7. The method of claim 1, wherein the microfluidic channel is located in a biochip comprising a plurality of microfluidic channels and areas for sample manipulation;

and wherein the method further comprises after the quantifying step, a step comprising directing a selected quantity of the sample fluid to an area of the biochip, wherein said selected quantity is determined, at least in part, based on the results of the quantifying step.

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

9. The method of claim 8, wherein the microfluidic channel is contained in a biochip and wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

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

11. The method of claim 1, wherein said target, if present, has a concentration less than 1 nanogram per microliter or is present in a total amount in the sample fluid of less than 1 nanogram.

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

13. The method of claim 1, wherein the sample fluid further comprises non-human, non-target nucleic acid.

14. The method of claim 1 wherein the quantifying step comprises determining the total amount of the target in the test fluid.

15. The method of claim 1 wherein the molecular beacon probe comprises SEQ ID NO:

5 or SEQ ID NO;

6.

16. A method for quantifying a human genomic double stranded DNA target in a sample fluid containing or suspected of containing said target, comprising:

combining in a microfluidic channel (i) the sample fluid and (ii) at least one molecular beacon probe capable of binding to at least one repetitive human Alu sequence, said probe further comprising a signaling moiety to form a test fluid, wherein said target in the sample fluid has not been amplified;

heating said test fluid to denature the double stranded DNA and cooling said test fluid to facilitate binding of the molecular beacon probe to said target so that the molecular beacon probe becomes bound to said target;

locating the test fluid in a detector region in the microfluidic channel;

detecting the signaling moiety; and

quantifying said target in the sample fluid within 1 minute of combining the sample fluid and the molecular beacon probe;

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

17. The method of claim 16, wherein the quantifying step comprises determining the concentration of said target in the test fluid.

18. The method of claim 16, wherein the quantifying step comprises determining the total amount of said target in the test fluid.

19. The method of claim 16, wherein the molecular beacon probe has a unique specificity for said at least one repetitive human Alu sequence.

20. The method of claim 16, wherein the locating step comprises flowing the test fluid to the detector region in the micro fluidic channel.

21. The method of claim 20, wherein the locating step comprises flowing the test fluid through the detector region of the micro fluidic channel during detection.

22. The method of claim 16, wherein the locating step comprises maintaining the test fluid stationary at a region in the microfluidic channel and moving a detector into alignment with the region to transform the region into the detector region.

23. The method of claim 16, wherein the microfluidic channel is located in a biochip comprising a plurality of microfluidic channels and areas for sample manipulation;

and wherein the method further comprises after the quantifying step, a step comprising directing a selected quantity of the sample fluid to an area of the biochip, wherein said selected quantity is determined, at least in part, based on the results of the quantifying step.

24. The method of claim 16, wherein detecting the signaling moiety comprises irradiating the signaling moiety with a laser.

25. The method of claim 24, wherein the microfluidic channel is contained in a biochip and wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

26. The method of claim 16, wherein detecting the signaling moiety comprises detecting fluorescence.

27. The method of claim 16, wherein the sample fluid comprises a forensic sample.

28. The method of claim 27, wherein the sample fluid further comprises non-human, non-target nucleic acid.

29. The method of claim 16 wherein the molecular beacon probe comprises SEQ ID NO:

5 or SEQ ID NO;

6.

30. A method for quantifying of a human genomic double stranded DNA target in a sample fluid containing or suspected of containing said target, comprising:

providing a microfluidic device coupled to an electronic device comprising a detector comprising an laser, wherein the laser is integrated in the microfluidic device;

combining in a microfluidic channel of the microfluidic device (i) the sample fluid and (ii) at least one molecular beacon probe capable of binding to at least one repetitive human Alu sequence said molecular beacon probe further comprising a signaling moiety to form a test fluid, wherein said target in the sample fluid has not been amplified;

heating said test fluid to denature the double stranded DNA and cooling said test fluid to facilitate binding of the molecular beacon probe to said target so that the molecular beacon probe becomes bound to said target;

locating the test fluid in a detector region in the microfluidic channel positioned in operative proximity to the detector of the electronic device;

irradiating the signaling moiety using the integrated laser; and

quantifying said target in the sample fluid within 1 minute of combining the sample fluid and the molecular beacon probe.

31. The method of claim 30, wherein the quantifying step comprises determining the concentration of said target in the test fluid.

32. The method of claim 30, wherein the quantifying step comprises determining the total amount of said target in the test fluid.

33. The method of claim 30, wherein the molecular beacon probe has a unique specificity for said at least one repetitive human Alu sequence.

34. The method of claim 30, wherein the locating step comprises flowing the test fluid to the detector region in the microfluidic channel.

35. The method of claim 34, wherein the locating step comprises flowing the test fluid through the detector region of the microfluidic channel during detection.

36. The method of claim 30, wherein the locating step comprises maintaining the test fluid stationary at a region in the microfluidic channel and moving a detector into alignment with the region to transform the region into the detector region.

37. The method of claim 30, wherein the microfluidic channel is located in a biochip comprising a plurality of microfluidic channels and areas for sample manipulation;

and wherein the method further comprises after the quantifying step, a step comprising directing a selected quantity of the sample fluid to an area of the biochip, wherein said selected quantity is determined, at least in part, based on the results of the quantifying step.

38. The method of claim 30, wherein said target, if present, has a concentration less than 1 nanogram per microliter or is present in a total amount in the sample fluid of less than 1 nanogram.

39. The method of claim 30, wherein detecting the signaling moiety comprises detecting fluorescence.

40. The method of claim 30, wherein the sample fluid further comprises non-human, non-target nucleic acid.

41. The method of claim 30, wherein the sample is a forensic sample.

42. The method of claim 30 wherein the molecular beacon probe comprises SEQ ID NO:

5 or SEQ ID NO;

6.

43. A method for manipulating a human genomic double stranded DNA target in a sample fluid containing said target, comprising:

providing a micro fluidic device comprising a plurality of micro fluidic channels and areas for sample manipulation coupled to an electronic device comprising a detector;

combining in a micro fluidic channel of the microfluidic device (i) the sample fluid and (ii) at least one molecular beacon probe capable of binding to at least one repetitive human Alu sequence, said molecular beacon probe further comprising a signaling moiety to form a test fluid, wherein said target in the sample fluid has not been amplified;

heating said test fluid to denature the double stranded DNA and cooling said test fluid to facilitate binding of the molecular beacon probe to said target so that the molecular beacon probe becomes bound to said target;

locating the test fluid in a detector region in the microfluidic channel positioned in operative proximity to the detector of the electronic device;

quantifying said target in the sample fluid within 1 minute of combining the sample fluid and the molecular beacon probe; and

directing a selected quantity of the sample fluid to an area of the biochip, wherein said selected quantity is determined, at least in part, based on the results of the quantifying step.

44. The method of claim 43, wherein the quantifying step comprises determining the concentration of said target nucleic acid in the test fluid.

45. The method of claim 43, wherein the quantifying step comprises determining the total amount of said target nucleic acid in the test fluid.

46. The method of claim 43, wherein the molecular beacon probe has a unique specificity for said at least one repetitive human Alu sequence.

47. The method of claim 43, wherein the locating step comprises flowing the combined fluid to the detector region in the microfluidic channel.

48. The method of claim 43, wherein the locating step comprises flowing the combined fluid through the detector region of the microfluidic channel during detection.

49. The method of claim 43, wherein the locating step further comprises maintaining the combined fluid stationary at the detector region in the microfluidic channel and moving the detector of the electronic device into alignment with the detector region.

50. The method of claim 43, wherein said target, if present, has a concentration less than 1 nanogram per microliter or is present in a total amount in the sample fluid of less than 1 nanogram.

51. The method of claim 43, further comprising the step of detecting the signaling moiety by irradiating the signaling moiety with a laser.

52. The method of claim 51, wherein the microfluidic channel is contained in a biochip and wherein the laser is a component of a system coupled to the biochip comprising the microfluidic channel.

53. The method of claim 43, further comprising the step of detecting the signaling moiety by detecting fluorescence.

54. The method of claim 43, wherein the sample fluid further comprises non-human, non-target nucleic acid.

55. The method of claim 43, wherein the sample is a forensic sample.

56. The method of claim 43 wherein the molecular beacon probe comprises SEQ ID NO:

5 or SEQ ID NO;

6.