Nucleic acid amplification with continuous flow emulsion

• US 7,927,797 B2
• Filed: 01/28/2005
• Issued: 04/19/2011
• Est. Priority Date: 01/28/2004
• Status: Expired due to Fees

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Nobile, John R., Lee, William L., Leamon, John H.
• Primary Examiner(s): Staples; Mark
• Application Number:
  US11/045,678
• Publication Number:
  US 20050227264A1
• Time in Patent Office: 2,272 Days
• Field of Search: None
• US Class Current: 435/6.16
• 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:

emulsifying an aqueous fluid comprising a reaction mixture and a plurality of suspended beads in a continuous flow of oil to form a plurality of aqueous microreactors that comprise a smaller dimension relative to a cross section of the continuous flow of oil, wherein a plurality of the microreactors each encapsulate a species of nucleic acid template, a bead capable of capturing the species of nucleic acid template, and sufficient reagents to amplify the species of nucleic acid template, and wherein the step of emulsifying comprises the steps of;

(i) continuously flowing the oil through a cross-flow emulsifier;

(ii) injecting the aqueous fluid into an injection tee operatively coupled to a vibratory anti-clogging device; and

(iii) applying a vibration to the injection tee from the vibratory anti-clogging device to prevent the suspended beads from clogging in a nozzle of the injection tee, thereby forming the aqueous microreactors;

directing the continuous flow of oil comprising the aqueous microreactors across a plurality of stationary controlled temperature zones to amplify the species of nucleic acid template by polymerase chain reaction and immobilize a plurality of the amplified copies on the bead in the aqueous microreactors; and

breaking the emulsion to retrieve one or more of the beads comprising the immobilized amplified species of nucleic acid template.

2. The method according to claim 1, further comprising filtering the continuous flow of oil to collect one or more of the beads comprising the immobilized amplified species of nucleic acid template.

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

m in diameter.

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

5. The method according to claim 1, wherein the beads have a diameter of between approximately 2 microns to approximately 100 microns.

6. The method according to claim 1, wherein the beads are selected from the group consisting of sepharose beads, solid beads, and monodisperse beads.

7. The method of claim 1, wherein a plurality of the aqueous microreactors are evenly distributed along a central area throughout the continuous flow.

8. The method of claim 1, wherein the dimension of the aqueous microreactors is 10 to 50 times smaller than the cross section of the continuous flow.

9. The method of claim 1, wherein the continuous flow is directed via a conduit through a thermal processing device comprising the stationary controlled temperature zones.

10. The method of claim 9, wherein the conduit is selected from the group consisting of a stainless steel conduit, a Polytetrafluoroethylene (PTFE) conduit, and a fused silica conduit.

11. The method of claim 9, wherein the thermal processing device comprises a mandrel, wherein the conduit is helically wound around the mandrel that comprises a first controlled temperature zone and a second controlled temperature zone.

12. The method of claim 9, wherein the thermal processing device comprises alternating sections of the conduit, wherein the sections of conduit are linear and a first section comprises a first controlled temperature zone and a second section comprises a second controlled temperature zone.

13. The method of claim 12, wherein the first and second sections of conduit are positioned in an opposed relationship.

14. The method of claim 1, wherein:

the aqueous microreactors are formed at a controlled rate.

15. The method of claim 14, wherein:

the controlled rate comprises a rate of droplet formation of 500 to 1000 emulsion droplets per second.

16. The method of claim 14, wherein:

the controlled rate comprises a rate of droplet formation of more than 1000 emulsion droplets per second.

17. The method of claim 1, wherein:

the continuous flow of oil flows at a consistent rate.

18. The method of claim 17, wherein:

the consistent rate includes a rate of about 1-10 mls/min.

19. The method of claim 17, wherein:

the consistent rate includes a rate of about 3 mls/min.

20. The method of claim 1, wherein:

the vibratory anti-clogging device is a piezo-electric actuator or an electromagnetic actuator.