Microfluidic nucleic acid analysis

• US 8,871,446 B2
• Filed: 10/02/2003
• Issued: 10/28/2014
• Est. Priority Date: 10/02/2002
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hong, Jong Wook, Studer, Vincent, Anderson, W. French, Quake, Stephen R., Leadbetter, Jared
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US10/678,946
• Publication Number:
  US 20050053952A1
• Time in Patent Office: 4,044 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0809 rectangular shaped
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Nucleic acid from cells and viruses sampled from a variety of environments may purified and expressed utilizing microfluidic techniques. In accordance with one embodiment of the present invention, individual or small groups of cells or viruses may be isolated in microfluidic chambers by dilution, sorting, and/or segmentation. The isolated cells or viruses may be lysed directly in the microfluidic chamber, and the resulting nucleic acid purified by exposure to affinity beads. Subsequent elution of the purified nucleic acid may be followed by ligation and cell transformation, all within the same microfluidic chip. In one specific application, cell isolation, lysis, and nucleic acid purification may be performed utilizing a highly parallelized microfluidic architecture to construct gDNA and cDNA libraries.

Claims

1. A method of obtaining nucleic acid from a cell sample, the method comprising:

(a) providing a sample comprising a plurality of cells suspended in solution;

(b) physically isolating one or more cells of the sample within a microfluidic flow channel by a process that comprises flowing the solution partially through a first and a second valve in the microfluidic flow channel while the first and second valves are open, and then closing the first and second valves such that a part of the solution containing the one or more cells is isolated therebetween;

(c) exposing the one or more cells in the part of the solution that was isolated between the first and the second valves in step (b) to a lysis chemical while said cell(s) are valved off from other cells in the sample, thereby releasing nucleic acid from the isolated cells; and

(d) amplifying at least a portion of a nucleic acid released in step (c), wherein steps (b), (c) and (d) are performed in a microfluidic device.

2. The method of claim 1 wherein only a portion of the nucleic acid is amplified.

8. The method according to claim 1 wherein step (c) comprises introducing the lysis chemical through a cross-flow injector structure, wherein the cross-flow injector structure comprises the intersection of a first microfluidic flow channel and a second microfluidic flow channel.

9. The method according to claim 1 wherein the first and second valves are closed in step (b) by actuation of a deflectable membrane.

10. The method according to claim 9 wherein the deflectable membrane is actuated by a control line.

11. The method according to claim 1 wherein step (c) comprises mixing cells and/or virions in the part of the solution that was isolated in step (b) with the lysis chemical in a loop channel in the microfluidic device.

20. The method of claim 1, comprising simultaneously closing a plurality of valves in the microfluidic flow channel so as to separate the sample into a plurality of isolated segments of about the same size.

21. The method of claim 20, comprising separately and simultaneously lysing biological elements from each of said isolated segments.

22. The method of claim 1, wherein at least a portion of the nucleic acid is amplified in step (d) by polymerase chain reaction (PCR).

23. The method of claim 1, wherein the heterogeneous biological elements comprise viruses.

24. The method of claim 1, wherein the heterogeneous biological elements comprise cells.

25. The method of claim 1, wherein the heterogeneous biological elements comprise bacteria.

26. The method of claim 1, wherein the heterogeneous biological elements comprise eukaryotic cells.

3. A method of characterizing phylogenetic, gene, or functional diversity exhibited by a specific environment comprising a plurality of biological elements, the method comprising:

(a) providing a sample from the environment comprising heterogeneous biological elements suspended in solution, wherein said elements are cells and/or virions;

(b) physically isolating an individual biological element or a subset of the biological elements in a microfluidic flow channel by a process that comprises flowing the sample partially through a first and a second valve in the microfluidic flow channel while the first and second valves are open, and then closing the first and second valves such that a part of the sample containing the individual biological element or the subset of the biological elements is isolated therebetween;

(c) exposing the individual biological element or the subset of biological elements in the part of the sample that was isolated between the first and the second valves in step (b) to a lysis chemical while valved off from other such elements in the sample, thereby releasing nucleic acid from the biological element(s);

(d) amplifying at least a portion of the nucleic acid, and

(e) identifying at least one of phylogenetic, gene, and functional diversity of the amplified portion of the purified nucleic acid, wherein steps (b), (c) and (d) are performed in a microfluidic device.

4. The method of claim 3 wherein genetic diversity is identified by sequence analysis.

5. The method of claim 3 further comprising:

observing morphology of the physically isolated individual biological element or subset of biological elements prior to lysis; and

correlating the genetic diversity with the observed morphology.

12. The method according to claim 3 wherein step (c) comprises introducing the lysis chemical through a cross-flow injector structure, wherein the cross-flow injector structure comprises the intersection of a first microfluidic flow channel and a second microfluidic flow channel.

13. The method according to claim 3 wherein the individual biological element or the subset of biological elements is isolated within the microfluidic channel in step (b) through the actuation of a deflectable membrane.

14. The method according to claim 13 wherein the deflectable membrane is actuated by a control line.

15. The method according to claim 3 wherein step (c) comprises mixing cells and/or virions in the part of the sample that was isolated in step (b) with the lysis chemical in a loop channel in the microfluidic device.

27. The method of claim 3, comprising simultaneously closing a plurality of valves in the microfluidic flow channel so as to separate the sample into a plurality of isolated segments.

28. The method of claim 27, comprising separately and simultaneously lysing biological elements from each of said isolated segments.

29. The method of claim 3, wherein at least a portion of the nucleic acid is amplified in step (d) by polymerase chain reaction (PCR).

30. The method of claim 3, wherein the heterogeneous biological elements comprise viruses.

31. The method of claim 3, wherein the heterogeneous biological elements comprise cells.

32. The method of claim 3, wherein the heterogeneous biological elements comprise bacteria.

33. The method of claim 3, wherein the heterogeneous biological elements comprise eukaryotic cells.

6. A method of obtaining genetic information regarding one or more biological elements contained in a complex environmental sample, the method comprising:

(a) providing an environmental sample comprising heterogeneous biological elements suspended in solution, wherein said elements are cells and/or virions;

(b) physically isolating an individual biological element or a subset of the biological elements in a microfluidic flow channel by a process that comprises flowing the sample partially through a first and a second valve in the microfluidic flow channel while the first and second valves are open, and then closing the first and second valves such that a part of the sample containing the individual biological element or the subset of the biological elements is isolated therebetween;

(c) exposing the individual biological element or the subset of biological elements in the part of the sample that was isolated between the first and the second valves in step (b) to a lysis chemical while valved off from other such elements in the sample to expose nucleic acid present therein;

(d) amplifying at least a portion of the nucleic acid, and

(e) identifying genetic information represented by the amplified nucleic acid wherein steps (b), (c) and (d) are performed in a microfluidic device.

7. The method of claim 6 further comprising:

transforming a cell with the amplified portion of the purified nucleic acid ligated into an appropriate vector; and

culturing the transformed cell to clone the amplified sequence.

16. The method according to claim 6 wherein step (c) comprises introducing the lysis chemical through a cross-flow injector structure, wherein the cross-flow injector structure comprises the intersection of a first microfluidic flow channel and a second microfluidic flow channel.

17. The method according to claim 6 wherein the individual biological element or the subset of biological elements is isolated within the microfluidic channel in step (b) through the actuation of a deflectable membrane.

18. The method according to claim 17 wherein the deflectable membrane is actuated by a control line.

19. The method according to claim 6 wherein step (c) comprises mixing cells and/or virions in the part of the sample that was isolated in step (b) with the lysis chemical in a loop channel in the microfluidic device.

34. The method of claim 6, comprising simultaneously closing a plurality of valves in the microfluidic flow channel so as to separate the sample into a plurality of isolated segments.

35. The method of claim 34, comprising separately and simultaneously lysing biological elements from each of said isolated segments.

36. The method of claim 6, wherein at least a portion of the nucleic acid is amplified in step (d) by polymerase chain reaction (PCR).

37. The method of claim 6, wherein the heterogeneous biological elements comprise viruses.

38. The method of claim 6, wherein the heterogeneous biological elements comprise cells.

39. The method of claim 6, wherein the heterogeneous biological elements comprise bacteria.

40. The method of claim 6, wherein the heterogeneous biological elements comprise eukaryotic cells.