Nucleic acid amplification with continuous flow emulsion

• US 20050227264A1
• Filed: 01/28/2005
• Published: 10/13/2005
• Est. Priority Date: 01/28/2004
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 CORPORATION
• Inventors: Leamon, John H., Nobile, John R., Lee, William L.
• Granted Patent: US 7,927,797 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 25/312 with Venturi elements; Details thereof
• CPC Class Codes: B01F 25/314 wherein additional components are introduced at the circumference of the conduit
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00664 Three-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2400/0439 ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• 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 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/15 Gradients
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 35/1095 : for supplying the samples to flow-through analysers for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00

Abstract

Embodiments of the present invention are directed to methods and devices/systems for amplifying genetic material and may include providing a water-in-oil emulsion in a continuous flow. The emulsion may include a plurality of water droplets comprising microreactors. Each of the plurality of microreactors may include a single bead capable of capturing a nucleic acid template, a single species nucleic acid template and sufficient reagents to amplify the copy number of the nucleic acid template. The method also includes flowing the emulsion across a first temperature zone and a second lower temperature zone to thermally process the microreactors to amplify the nucleic acid template by polymerase chain reaction.

Claims

1. A method for amplifying genetic material comprising:

providing a water-in-oil emulsion in a continuous flow wherein the emulsion comprises a plurality of water droplets comprising microreactors, wherein a plurality of the microreactors each include one or more species of nucleic acid templates, and sufficient reagents to amplify the copy number of one of the nucleic acid templates; and

thermally processing the emulsion by flowing it across stationary controlled temperature zones to amplify nucleic acid templates by polymerase chain reaction.

2. The method according to claim 1, wherein each microreactor contains one or more beads capable of capturing a nucleic acid template.

3. The method according to claim 1, wherein one or more of the beads capture amplified copies of the nucleic acid template.

4. The method according to any of claims 1-3, further comprising breaking the emulsion to retrieve one or more of the amplified nucleic acid templates.

5. The method according to any of claims 1-3, further comprising filtering the water-in-oil emulsion to collect one or more of the amplified nucleic acid templates.

6. The method according to claim 1, wherein the microreactors have an average size of between approximately 50 to approximately 250 μ

m in diameter.

7. The method according to claim 1, wherein the nucleic acid templates are selected from the group consisting of genomic DNA, cDNA, episomal DNA, BAC DNA, and YAC DNA.

8. The method according to claim 2, wherein the bead has a diameter of between approximately 2 microns to approximately 100 microns.

9. The method according to claim 2, wherein the bead is selected from the group consisting of a sepharose bead, a solid bead and a monodisperse bead.

10. An apparatus for amplifying genetic material comprising:

at least one fluid delivery device;

at least one first temperature zone to cycle a plurality of microreactors each including one or more species nucleic acid templates to a first temperature;

at least one second temperature zone to cycle the plurality of microreactors to second temperature lower than the first temperature;

a first conduit for flowing at least a stream of oil therein from a first reservoir; and

a second conduit for flowing at least a water based PCR solution including nucleic acid templates and PCR reagent from a second reservoir out of an orifice and into the first conduit creating a water-in-oil emulsion, wherein the PCR solution upon entering the first conduit comprises a plurality of droplets comprising the microreactors for performing polymerase chain reactions, wherein a plurality of the microreactors each include one or more species of nucleic acid template.

11. The apparatus for amplifying genetic material according to claim 10, wherein downstream of the orifice, a length of the first conduit from a starting position to an ending position is arranged relative to at least the first and second temperature zones such that the length of the first conduit is exposed to alternating processes of heating and cooling at a temperature and time sufficient to amplify any of the nucleic acid templates by polymerase chain reaction.

12. The apparatus according to claim 10, wherein the orifice is sized between 50 μ

m to 300 μ

m.

13. The apparatus according to claim 10, wherein the orifice is sized to approximately 150 μ

tm.

14. The apparatus according to claim 10, wherein an intersection area of the orifice and the first conduit includes a diameter of between greater than 50 μ

m to about 800 μ

m.

15. The apparatus according to claim 10, wherein an intersection area of the orifice and the first conduit includes a diameter of about 400 μ

m.

16. The apparatus according to claim 10, further comprising collecting means for collecting one or more of the amplified nucleic acid templates from the first conduit downstream of the heating and cooling sources.

17. The apparatus according to claim 16, wherein the collecting means comprises a filter.

18. The apparatus according to claim 10, wherein the first and second temperature zones are circumferentially arranged on opposed sides of a curved surface, wherein the length of the first conduit is helically wound around the curved surface to provide alternating portions of the length of first conduit adjacent either the first temperature zone or the second temperature zone.

19. The apparatus according to claim 18, wherein the curved surface comprises a mandrel.

20. The apparatus according to claim 10, wherein the temperature zones are arranged along opposed heating and cooling linear surfaces, respectively, wherein the length of first conduit is wound along the opposed surfaces such that portions of the length of first conduit are alternately exposed to the heating and cooling surfaces a plurality of times.

21. The apparatus according to claim 20, where the linear surfaces are substantially vertical or horizontal.

22. The apparatus according to claim 10, wherein the microreactors have an average size of between approximately 50 to approximately 250 μ

m in diameter.

23. The apparatus according to claim 10, wherein the nucleic acid templates are selected from the group consisting of genomic DNA, cDNA, episomal DNA, BAC DNA, and YAC DNA.

24. The apparatus according to claim 10, further the nucleic acid templates are captured onto beads, wherein the beads have a diameter of between approximately 2 microns to approximately 100 microns.

25. The apparatus according to claim 24, wherein the bead is selected from the group consisting of a sepharose bead, a solid bead and a monodisperse bead.

26. A cross-flow emulsification apparatus comprising:

a first inlet for receiving an oil flow from a first conduit;

an outlet for directing a water-in-oil emulsion out of the apparatus;

a second inlet for receiving a water based PCR amplification reaction mixture, the mixture including nucleic acid templates and PCR reaction mixture; and

an orifice for delivering PCR reaction mixture from the second conduit into the first conduit, to form a plurality of water-in-oil droplets comprising microreactors, wherein a plurality of the microreactors each include one or more nucleic acid templates, and sufficient PCR amplification reaction mixture to produce a plurality of copies of nucleic acid template

27. The apparatus according to claim 26, wherein the orifice delivers the plurality of droplets into the first conduit at a narrowed region provided in the first conduit.

28. The apparatus according to claim 27, wherein the narrowed region includes a diameter between about 40 μ

m and 600 μ

m.

29. The apparatus according to claim 27, wherein the narrowed region includes a diameter of approximately 300 μ

m.

30. The apparatus according to claim 26, wherein the orifice includes a diameter between approximately 60 μ

m and about 300 μ

m.

31. The apparatus according to claim 26, wherein the orifice includes a diameter of approximately 120 μ

m.

32. The apparatus according to claim 26, wherein the microreactors have an average size of between approximately 50 to approximately 250 μ

m in diameter.

33. The apparatus according to claim 26, wherein the nucleic acid templates are selected from the group consisting of genomic DNA, cDNA, episomal DNA, BAC DNA, and YAC DNA.

34. The apparatus according to claim 26, wherein a plurality of the microreactors contain one of more beads, each bead having a diameter of between approximately 2 microns to approximately 100 microns.

35. The apparatus according to claim 34, wherein each bead is a sepharose bead.

36. An apparatus for amplifying genetic material comprising:

a water-in-oil emulsion in a continuous flow wherein the emulsion comprises a plurality of water droplets comprising microreactors, wherein a plurality of the microreactors include a single bead capable of capturing one or more nucleic acid templates, and sufficient reagents to amplify the copy number of the one or more nucleic acid templates; and

thermal processing means for thermally processing the emulsion to amplify nucleic acid templates by polymerase chain reaction.

37. An emulsion generator comprising:

an emulsion oil supply;

at least one syringe including a body and a plunger for dispensing a mixture for emulsifying into the emulsion oil;

a cross-flow emulsification device for emulsifying the mixture, the device including an input attached to the output of the syringe; and

a syringe pump including an actuator capable of oscillating the plunger of the at least one syringe micrometer distances at a predetermined frequency along a length of travel of the plunger within the syringe body of the at least one syringe.

38. The emulsion generator according to claim 37, wherein the actuator is capable of moving the plunger between about 5 μ

m and about 50 μ

m.

39. The emulsion generator according to claim 37, wherein the desired frequency is between about 1 Hz and 500 Hz.

40. A method for substantially reducing clogging of a nozzle in syringe pump, comprising:

providing a syringe pump having at least one syringe including a body, a plunger having a plunger axis and an exit nozzle, the body for dispensing a mixture of micron or less sized particles suspended in a medium; and

oscillating the plunger of the syringe along the axis of the plunger for micrometer distances at a predetermined frequency along a length of travel of the plunger within the syringe body.

41. The method according to claim 40, wherein the actuator is capable of moving the plunger between about 5 μ

m and about 50 μ

m.

42. The method according to claim 40, wherein the desired frequency is between about 1 Hz and 500 Hz.

43. An emulsion generator comprising:

an emulsion oil supply;

at least one syringe including a body and a plunger for dispensing a mixture for emulsifying into the emulsion oil;

a magnetically-attractive mixing element disposed in the body of the syringe;

a cross-flow emulsification device for emulsifying the mixture, the device including an input attached to the output of the syringe; and

a device capable of moving an external magnetic force axially along body of the syringe while in close proximity to the syringe body.

44. An emulsion generator comprising:

an emulsion oil supply;

at least one syringe including a body and a plunger for dispensing a mixture for emulsifying into the emulsion oil;

a magnetically-attractive mixing element disposed in the body of the syringe;

a cross-flow emulsification device for emulsifying the mixture, the device including an input attached to the output of the syringe; and

a rotating drum having a magnet helically wound along the surface of the drum, wherein the surface of the drum is positioned adjacent the body of the syringe.

45. The emulsion generator according to claim 44, wherein the magnet comprises a plurality of individual magnets helically spaced along the surface of the drum.

46. The emulsion generator according to claim 44, wherein the mixing element comprises a plastic coated metallic ball.

47. A syringe pump comprising:

an area for receiving at least one syringe, wherein the syringe includes a body and a plunger having a plunger axis, the syringe for dispensing a mixture for emulsification into an emulsion oil; and

an actuator capable of oscillating the plunger of the at least one syringe along the plunger axis micrometer distances at a predetermined frequency along a length of travel of the plunger within body of the at least one syringe.

48. The syringe pump according to claim 47, wherein the actuator is capable of moving the plunger between about 5 μ

m and about 50 μ

m.

49. The syringe pump according to claim 47, wherein the desired frequency is between about 1 Hz and 500 Hz.

50. A syringe pump comprising:

an area for receiving at least one syringe, wherein the syringe includes a body and a plunger having a plunger axis, the syringe for dispensing a mixture for emulsification into emulsion oil;

a magnetically attractive mixing element disposed in the body of the syringe; and

a rotating drum having a magnet helically wound along the surface of the drum, wherein the surface of the drum is positioned adjacent the body of the syringe.

51. The syringe pump according to claim 50, wherein the magnet comprises a plurality of individual magnets helically spaced along the surface of the drum.

52. The syringe pump according to claim 50, wherein the mixing element comprises a plastic coated metallic ball.