Nucleic acid amplification with continuous flow emulsion

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.

1 .- 9 . (canceled) 
     
     
         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 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.   
     
     
         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 expose to alternating processes of heating and cooling at a temperature and time sufficient to amplify the one or more species of nucleic acid templates by polymerase chain reaction. 
     
     
         12 . The apparatus according to  claim 10 , wherein the orifice is sized between 50 μm to 300 μm. 
     
     
         13 . The apparatus according to  claim 10 , wherein the orifice is sized to approximately 150 μm. 
     
     
         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 μm to about 800 μm. 
     
     
         15 . The apparatus according to  claim 10 , wherein an intersection area of the orifice and the first conduit includes a diameter of about 400 μm. 
     
     
         16 . The apparatus according to  claim 10 , further comprising collecting means for collecting amplified copies of the one or more species of 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 aqueous microreactors have an average size of between approximately 50 to approximately 250 μm in diameter. 
     
     
         23 . The apparatus according to  claim 10 , wherein the one or more species of 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 , wherein each bead has a diameter of between approximately 2 μm to approximately 100 μm. 
     
     
         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 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.   
     
     
         27 . The apparatus according to  claim 26 , wherein the orifice delivers the plurality of water-in-oil 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 μm and 600 μm. 
     
     
         29 . The apparatus according to  claim 27 , wherein the narrowed region includes a diameter of approximately 300 μm. 
     
     
         30 . The apparatus according to  claim 26 , wherein the orifice includes a diameter between approximately 60 μm and about 300 μm. 
     
     
         31 . The apparatus according to  claim 26 , wherein the orifice includes a diameter of approximately 120 μm. 
     
     
         32 . The apparatus according to  claim 26 , wherein the aqueous microreactors have an average size of between approximately 50 to approximately 250 μm in diameter. 
     
     
         33 . The apparatus according to  claim 26 , wherein the one or more species of 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 each bead has a diameter of between approximately 2 μm to approximately 100 μm. 
     
     
         35 . The apparatus according to  claim 34 , wherein each bead is a sepharose bead. 
     
     
         36 . (canceled) 
     
     
         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 μm and about 50 μm. 
     
     
         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 a syringe pump, comprising:
 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.   
     
     
         41 . The method according to  claim 40 , wherein the actuator is capable of moving the plunger between about 5 μm and about 50 μm. 
     
     
         42 . The method according to  claim 40 , wherein the predetermined frequency is between about 1 Hz and 500 Hz. 
     
     
         43 . The emulsion generator according to  claim 37 , further comprising:
 a magnetically-attractive mixing element disposed in the body 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 . The emulsion generator according to  claim 43 , further comprising:
 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 43 , 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 μm and about 50 μm. 
     
     
         49 . The syringe pump according to  claim 47 , wherein the predetermined frequency is between about 1 Hz and 500 Hz. 
     
     
         50 . The syringe pump according to  claim 47 , further comprising:
 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 to 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.