Nucleic acid separation and amplification

• US 8,017,340 B2
• Filed: 07/29/2010
• Issued: 09/13/2011
• Est. Priority Date: 12/23/2004
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Inventors: Collier, Gordon Bruce, MacLeod, Jason Andrew, Dicke, William Charles, Wood, John Allister, Nemeth, Attila Csaba, Miller, Cary James
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US12/846,609
• Publication Number:
  US 20100297708A1
• Time in Patent Office: 411 Days
• Field of Search: 435/6, 435/91.2, 536/25.4
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• 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 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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: B01L 3/502769 characterised by multiphase flow arrangements
• 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: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/0332 using permanent magnets
• CPC Class Codes: B03C 1/0335 using coils
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]

Abstract

The present invention relates to methods for the extraction of nucleic acids from cells, the amplification of segments of nucleic acid and the detection of nucleic acids, all in a convenient and portable manner. In one embodiment, a sample comprising cells containing nucleic acid is exposed to an aqueous mixture comprising a lytic reagent and one or more beads capable of binding the nucleic acid released from said cells to form a nucleic acid-bead complex. The nucleic acid-bead complex is passed through an immiscible liquid layer to separate the nucleic acid from the aqueous mixture. The one or more beads are magnetic, and the nucleic acid-bead complex is passed through and separated from the immiscible liquid layer with an applied magnetic field. The invention is particularly suited for use in point-of-care medical diagnostics testing.

Claims

1. A nucleic acid separation method, comprising:

exposing a sample comprising cells containing nucleic acid to an aqueous mixture comprising a lytic reagent and one or more beads capable of binding the nucleic acid released from said cells to form a nucleic acid-bead complex; and

passing the nucleic acid-bead complex through an immiscible liquid layer to separate the nucleic acid from the aqueous mixture,

wherein the one or more beads are magnetic, and the nucleic acid-bead complex is passed through and separated from the immiscible liquid layer with an applied magnetic field.

2. The method of claim 1, wherein the sample is a buccal sample.

3. The method of claim 1, wherein the sample is blood.

4. The method of claim 1, wherein the immiscible liquid layer comprises organic liquid.

5. The method of claim 1, wherein the immiscible liquid layer comprises wax.

6. The method of claim 1, wherein the immiscible liquid layer is heated to facilitate passage of the nucleic acid-bead complex.

7. A method of transferring nucleic acid, comprising:

contacting nucleic acid at a first location with one or more beads to form a nucleic acid-bead complex in a liquid; and

transporting the nucleic acid-bead complex to a second location separated from the first location by an intermediary layer, wherein said intermediary layer is immiscible with the liquid,

wherein the one or more beads are magnetic, and the nucleic acid-bead complex is passed through and separated from the intermediary layer with an applied magnetic field.

8. The method of claim 7, wherein the nucleic acid and one or more inhibitors of nucleic acid amplification processes are insoluble in the intermediary layer.

9. A nucleic acid separation method, comprising:

introducing a sample comprising biological cells through a first layer to a second layer comprising magnetic beads, wherein said first layer is contiguous with said second layer;

incubating the sample for sufficient time to permit lysis of the cells and form a nucleic acid-bead complex in said second layer; and

applying a magnetic field in proximity to the complex sufficient to move said complex from said second layer through said first layer, thereby separating the complex from the first layer and effectively filtering said complex.

10. The method of claim 9, wherein the sample is a buccal sample.

11. The method of claim 9, wherein the sample is blood.

12. The method of claim 9, wherein said first layer comprises a filtering medium.

13. The method of claim 9, wherein said first layer comprises organic liquid.

14. The method of claim 9, wherein said first layer comprises wax.

15. The method of claim 14, wherein said wax has a melting point between 25°

C. and 45°

C.

16. The method of claim 14, wherein said wax does not evaporate at 60°

C. to 90°

C.

17. The method of claim 9, wherein said first layer comprises a material selected from the group consisting of docosane, tricosane, tricosaheneicosane and combinations thereof.

18. The method of claim 9, wherein said first layer comprises heneicosane.

19. The method of claim 9, wherein said first layer comprises silicone oil.

20. The method of claim 9, wherein said first layer comprises mesitylene.

21. The method of claim 9, further comprising the step of heating said first layer.

22. The method of claim 21, wherein said first layer is heated to a sufficient temperature to permit said first layer to melt and form a melted layer contiguous with said second layer.

23. The method of claim 22, further comprising the step of removing the heat from said first layer to permit said first layer to re-harden.

24. The method of claim 9, wherein said first layer prevents movement of the magnetic beads and other material in said second layer when said first layer is in a solid state.

25. The method of claim 9, wherein said first layer has a viscosity sufficiently low to permit passage of said complex from said second layer through said first layer when said first layer is in a liquid state.

26. The method of claim 9, wherein said second layer comprises a lytic buffer.

27. The method of claim 9, wherein said complex is formed via non-specific surface bonds.

28. The method of claim 9, wherein said complex is in the form of a pellet.

29. The method of claim 9, wherein said complex is isolated in a coating comprising the first layer while the second layer and remaining sample is isolated below said first layer.

30. The method of claim 9, wherein said magnetic beads are transferred to a means for nucleic acid amplification.

31. A method for extracting and amplifying nucleic acid, comprising:

introducing a sample comprising biological cells through a first layer to a second layer comprising magnetic beads, wherein said first layer is contiguous with said second layer;

incubating the sample for sufficient time to permit lysis of the cells and form a nucleic acid-bead complex in said second layer;

applying a magnetic field in proximity to the complex sufficient to move said complex from said second layer through and separated from said first layer, thereby removing said complex from said second and first layers; and

introducing said complex to a vessel containing a polymerase chain reaction (PCR) cocktail, wherein at least a portion of the nucleic acid elutes off said magnetic beads during a first heating cycle of the PCR.

32. The method of claim 31, wherein the sample is a buccal sample.

33. The method of claim 31, wherein the sample is blood.

34. The method of claim 31, wherein said first layer comprises a filtering medium.

35. The method of claim 31, wherein said first layer comprises organic liquid.

36. The method of claim 31, wherein said first layer comprises wax.

37. The method of claim 36, wherein said wax has a melting point from 25°

C. to 45°

C.

38. The method of claim 36, wherein said wax does not evaporate at 60°

C. to 90°

C.

39. The method of claim 31, wherein said first layer comprises a material selected from the group consisting of docosane, tricosane, tricosaheneicosane or a combination thereof.

40. The method of claim 31, wherein said first layer comprises heneicosane.

41. The method of claim 31, wherein said first layer comprises silicone oil.

42. The method of claim 31, wherein said first layer comprises mesitylene.

43. The method of claim 31, further comprising the step of heating said first layer.

44. The method of claim 43, wherein said first layer is heated to a sufficient temperature to permit said first layer to melt and form a melted layer contiguous with said second layer.

45. The method of claim 44, further comprising the step of removing the heat from said first layer to permit said first layer to re-harden.

46. The method of claim 31, wherein said first layer prevents movement of the magnetic beads and other material in said second layer when said first layer is in a solid state.

47. The method of claim 31, wherein said first layer has a viscosity sufficiently low to permit passage of said complex from said second layer through said first layer when said first layer is in a liquid state.

48. The method of claim 31, wherein said second layer comprises a lytic buffer.

49. The method of claim 31, wherein said complex is formed via non-specific surface bonds.

50. The method of claim 31, wherein said complex is in the form of a pellet.

51. The method of claim 31, wherein said complex is isolated in a coating comprising the first layer while the second layer and remaining sample is isolated below said first layer.

52. The method of claim 31, wherein water at a temperature of above, or about, 80°

C. is sufficient for elution.