NUCLEIC ACID AMPLIFICATION WITH CONTINUOUS FLOW EMULSION
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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.
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Citations
52 Claims
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1-9. -9. (canceled)
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10. An apparatus for amplifying genetic material comprising:
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at least one fluid delivery device; at least one first temperature zone to cycle a plurality of aqueous microreactors formed from a water-in-oil emulsion to a first temperature; at least one second temperature zone to cycle the plurality of aqueous microreactors to a second temperature lower than the first temperature; a first conduit for flowing at least a continuous stream of oil therein from a first reservoir; and a second conduit for flowing at least a water based PCR amplification reaction mixture including one or more species of nucleic acid templates, a plurality of beads, and PCR reagents necessary for amplification of the one or more species of nucleic acid templates from a second reservoir out of an orifice and into the first conduit, thereby creating the water-in-oil emulsion in the continuous stream of oil, wherein the plurality of aqueous microreactors each include the one or more species of nucleic acid templates, a single bead capable of capturing the one or more nucleic acid templates, and sufficient PCR reagents to amplify the copy number of the one or more species of nucleic acid templates. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A cross-flow emulsification apparatus comprising:
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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 comprising one or more species of nucleic acid templates, a plurality of beads and PCR reagents necessary for amplification of the one or more species of nucleic acid template; and an orifice for delivering the PCR amplification reaction mixture from the second conduit into the first conduit, to form a plurality of water-in-oil droplets comprising aqueous microreactors, wherein the aqueous microreactors each include the one or more species of nucleic acid templates, a single bead capable of capturing the one or more nucleic acid templates, and sufficient PCR reagents to amplify the copy number of the one or more species of nucleic acid templates. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. (canceled)
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37. An emulsion generator comprising:
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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. - View Dependent Claims (38, 39, 43, 44, 45, 46)
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40. A method for substantially reducing clogging of a nozzle in a syringe pump, comprising:
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providing a syringe pump having at least one syringe including a body for dispensing a mixture of micron or less sized particles suspended in a medium, an actuator, a plunger having a plunger axis and an exit nozzle; 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. - View Dependent Claims (41, 42)
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47. A syringe pump comprising:
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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. - View Dependent Claims (48, 49, 50, 51, 52)
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Specification