Chemical amplification based on fluid partitioning in an immiscible liquid

• US RE41,780 E1
• Filed: 05/09/2008
• Issued: 09/28/2010
• Est. Priority Date: 03/14/2003
• Status: Expired due to Term

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Inventors: Elkin, Christopher J., Colston, Bill W., Anderson, Brian L.
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US12/118,418
• Time in Patent Office: 872 Days
• Field of Search: 435/91.2, 435/287.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

Claims

1. An apparatus for nucleic acid amplification of a sample, comprising:

means for partitioning said sample into partitioned sections, wherein said means for partitioning said sample into partitioned sections comprises an injection orifice, and

means for performing PCR on said partitioned sections of said sample.

2. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said injection orifice is an injection orifice that produces microdroplets.

3. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said injection orifice is an injection orifice that injects said sample and a PCR reagent.

4. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for performing PCR on said partitioned sections of said sample comprises a continuous tube for circulating said partitioned sections of said sample through a heater to perform PCR.

5. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for performing PCR on said partitioned sections of said sample comprises a continuous tube for circulating said partitioned sections of said sample through a heater and cooler to perform PCR.

6. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for performing PCR on said partitioned sections of said sample comprises a pump, a continuous tube, and a heater.

7. The apparatus for nucleic acid amplification of a sample of claim 1 including means for detection and analysis of said partitioned sections of said sample comprising a laser and a detector.

8. The apparatus for nucleic acid amplification of a sample of claim 1 including means for detection and analysis of said partitioned sections of said sample comprising a blue laser and a detector.

9. The apparatus for nucleic acid amplification of a sample of claim 1 wherein said means for partitioning said sample into partitioned sections comprises means for separating said sample into immiscible slugs.

10. A method of nucleic acid amplification of a sample, comprising the steps of:

partitioning said sample into partitioned sections, wherein said step of partitioning said sample into partitioned sections comprises flowing said sample through an injection orifice into an immiscible carrier fluid, and

subjecting said partitioned sections of said sample to PCR.

11. The method of claim 10 wherein the nucleic acid amplification of a sample comprises PCR amplification of a DNA target.

12. The method of claim 11 wherein said partitioned sections contain, on average, a single template of a DNA target, and wherein said single template is amplified within said partitioned sections.

13. The method of claim 12 wherein said sample comprises multiple DNA targets, and wherein multiple partitioned sections have a single template of a different DNA target such that said single template is amplified within said multiple partitioned sections.

14. The method of claim 10, wherein the partitioned sections are passed by a detector to detect the amount of amplification.

15. The method of claim 14 wherein the detector is a light detector.

16. The method of claim 15 wherein an amount of amplification is indicated by fluorescence.

17. The method of claim 16 where a fluorophore dye is used.

18. The method of claim 15 wherein a laser is projected upon the partitioned sections as they pass between the laser and detector.

19. The method of claim 15 wherein the detector comprises a confocal imaging system.

20. The method of claim 15 wherein scattering profiles from the partitioned sections are used to eliminate background signals.

21. The method of claim 16 wherein the partitioned sections are probed for fluorescent signal at a rate of several thousand per second.

22. A nucleic acid amplification apparatus comprising

a microdroplet generator comprising an orifice, wherein said orifice connects a sample flow pathway to a channel or tube comprising an immiscible fluid, and wherein said channel or tube passes through a heating element.

23. The apparatus of claim 22 further comprising a cooler.

24. The apparatus of claim 22 wherein said microdroplet generator is capable of producing microdroplets with volumes in the picoliter range.

25. The apparatus of claim 22 wherein said microdroplet generator is capable of producing microdroplets having volumes of about 5×

10<sup>−

9</sup> liters to 1×

10<sup>−

12</sup> liters.

26. The apparatus of claim 22 wherein the immiscible fluid is mineral oil.

27. The apparatus of claim 22, further comprising a a pump for moving generated microdroplets in said immiscible fluid through the channel or tube.

28. The apparatus of claim 27 further comprising a pump for moving the microdroplets through the channel or tube.

29. The apparatus of claim 27 wherein the tube is a continuous tube.

30. The apparatus of claim 27 wherein the channel is a micromachined channel.

31. The apparatus of claim 28 wherein the pump for moving the microdroplets comprises a magnetohydrodynamic (MHD) element.

32. The apparatus of claim 27 wherein the channel or tube is heated and cooled.

33. The apparatus of claim 27 wherein the channel or tube extends through a heater and a cooler.

34. A nucleic acid amplification apparatus comprising:

a microdroplet generator comprising an orifice wherein said orifice connects a sample flow pathway to a channel or tube comprising an immiscible fluid, wherein said channel or tube passes through a heating element; and

wherein said apparatus further comprises a detector capable of detecting microdroplets in said immiscible fluid.

35. The apparatus of claim 34 wherein the detector is positioned such that generated microdroplets suspended in said immiscible fluid pass by the detector as they are moved through the channel or tube.

36. A method for nucleic acid amplification comprising:

producing microdroplets within an immiscible fluid in a channel or tube;

wherein the microdroplets comprise nucleic acids and components for performing nucleic acid amplification;

moving the microdroplets through the channel or tube; and

thermal cycling the microdroplets in the channel or tube to amplify the nucleic acids.

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

38. The method of claim 36 wherein the thermal cycling of the microdroplets comprises passing the microdroplets through a heater and a cooler.

39. The method of claim 36 wherein the thermal cycling of the microdroplets comprises heating and cooling the channel or tube comprising the microdroplets.

40. The method of claim 36 further comprising passing the microdroplets by a detector to detect an amount of amplification.

41. The method of claim 40 wherein the detector is a light detector.

42. The method of claim 41 wherein the amount of amplification is indicated by fluorescence.

43. The method of claim 42 where a fluorophore dye is used.

44. The method of claim 41 wherein a laser is projected upon the microdroplets as they pass between the laser and detector.

45. The method of claim 41 wherein the detector comprises a confocal imaging system.

46. The method of claim 41 wherein scattering profiles from the microdroplets are used to eliminate background signals.

47. A method comprising:

diluting a sample comprising a plurality of DNA targets and PCR reagents;

partitioning the sample into microdroplets in an immiscible fluid in a tube or channel of a microfluidic device, wherein a plurality of microdroplets containing a single template of the target DNA are formed; and

amplifying the target DNA in the microdroplets by heating and cooling such that a plurality of single templates within the microdroplets are amplified.

48. A method comprising:

a. performing PCR on a microdroplet suspended in an immiscible fluid in a microchannel, wherein said PCR comprises a plurality of cycles;

b. passing said microdroplet through said microchannel past a detector; and

c. detecting a PCR amplification product in said microdroplet.

49. The method of claim 48, wherein said microdroplet is isolated from a bulk solution, and whereby the number of PCR cycles needed to detect said amplication product in said microdroplet is less than the number of PCR cycles needed to detect amplication product in said bulk solution.

50. The method of claim 48, wherein said microdroplet is isolated from a bulk solution, and whereby the time needed for each cycle of PCR on said microdroplet is less than the time needed for each cycle of PCR in said bulk solution.

51. The method of claim 48 wherein the volume of said microdroplet is about 5×

10<sup>−

9</sup> liters to 1×

10<sup>−

12</sup> liters.