Droplet-based nucleic acid amplification method and apparatus

• US 7,851,184 B2
• Filed: 12/15/2006
• Issued: 12/14/2010
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Paik, Philip Y., Pollack, Michael G.
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): Wilder; Cynthia B
• Application Number:
  US11/639,490
• Publication Number:
  US 20090291433A1
• Time in Patent Office: 1,460 Days
• Field of Search: 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• 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: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention relates to a droplet-based nucleic acid amplification method and apparatus. According to one embodiment, a method of amplifying a nucleic acid in a biological sample is provided, wherein the method includes: (a) providing a system comprising a droplet microactuator electronically coupled to and controlled by a processor capable of executing instructions, the droplet microactuator comprising: (i) a sample potentially comprising a target nucleic acid; (ii) a substrate comprising electrodes for conducting droplet operations; and (iii) one or more temperature control means arranged in proximity with one or more of the electrodes for heating a region of the droplet microactuator such that a droplet can be transported into the region for heating; (b) using droplet operations to combine on the droplet microactuator one or more amplification reagent droplets and one or more sample droplets to yield an amplification-ready droplet; and (c) thermal cycling the amplification-ready droplet sufficient to result in amplification of a target nucleic acid when present in the amplification-ready droplet.

Claims

1. A method of amplifying a nucleic acid in a sample, the method comprising:

(a) providing a system comprising;

(i) one or more sample droplets potentially comprising a target nucleic acid and at least partially surrounded by a filler fluid comprising an oil;

(ii) one or more amplification reagent droplets comprising reagents for amplifying the target nucleic acid;

(iii) a droplet microactuator comprising a substrate comprising electrodes for conducting electrowetting-mediated droplet operations on the substrate;

(iv) a processor capable of executing instructions and electronically coupled to and controlling the electrodes;

(v) one or more temperature control means arranged for heating a region of the substrate such that a droplet on the substrate comprising the electrodes can be transported by the droplet operations into the region for heating; and

(vi) a magnet arranged to immobilize magnetically responsive beads in a droplet on the substrate during a droplet operation mediated by the electrodes;

(b) combining, by conducting the droplet operations on the substrate comprising the electrodes, the one or more amplification reagent droplets and the one or more sample droplets to yield an amplification-ready droplet;

(c) thermal cycling the amplification-ready droplet sufficient to result in amplification of a target nucleic acid when present in the amplification-ready droplet thereby yielding an amplified droplet, the thermal cycling comprising transporting, by conducting the droplet operations on the substrate comprising the electrodes, the amplification-ready droplet into and out of proximity with the region for heating;

(d) merging, by conducting the droplet operations on the substrate comprising the electrodes, the amplified droplet comprising amplified target nucleic acid with a droplet comprising one or more magnetically responsive beads having an affinity for the amplified nucleic acid yielding a merged droplet thereby immobilizing amplified nucleic acid on the one or more magnetically responsive beads; and

(e) executing a droplet-based washing protocol to separate unbound components of the merged droplet from the one or more magnetically responsive beads, wherein the droplet-based washing protocol comprises;

(i) merging, by conducting the droplet operations on the substrate comprising the electrodes, a wash droplet with the droplet comprising the one or more magnetically responsive beads to yield a merged droplet;

(ii) immobilizing the one or more magnetically responsive beads in the merged droplet using the magnet; and

(iii) separating, b conducting the substrate comprising the electrodes, a droplet comprising unbound components and lacking the immobilized beads from the merged droplet thereby carrying away the unbound components from the beads.

2. The method of claim 1 wherein step 1(b) comprises:

(a) transporting one or more amplification reagent droplets from a reservoir on the droplet microactuator;

(b) transporting one or more sample droplets from a reservoir on the droplet microactuator; and

(c) merging one or more amplification reagent droplets with one or more sample droplets to yield the amplification-ready droplet.

3. The method of claim 2 further comprising:

(a) loading one or more amplification reagent droplets into a reservoir on the droplet microactuator by flowing the one or more amplification reagent droplets into the reservoir from a locus exterior to the droplet microactuator; and

/or

(b) loading one or more sample droplets into a reservoir on the droplet microactuator by flowing the one or more amplification reagent droplets into the reservoir from a locus exterior to the droplet microactuator.

4. The method of claim 1 further comprising:

(a) loading one or more amplification reagent droplets onto the droplet microactuator by flowing the one or more amplification reagent droplets onto the droplet microactuator from a locus exterior to the droplet microactuator; and

/or

(b) loading one or more sample droplets onto the droplet microactuator by flowing the one or more sample droplets onto the droplet microactuator from a locus exterior to the droplet microactuator.

5. The method of claim 1 wherein the amplification-ready droplet has a volume which ranges from about 1 mL to about 10 μ

L.

6. The method of claim 1 wherein the amplification-ready droplet has a volume which ranges from about 1 mL to about 1 μ

L.

7. The method of claim 1 wherein the amplification-ready droplet has a volume which is less than 100 mL.

8. The method of claim 1 further comprising detecting the presence or absence of amplified target nucleic acid in the amplification-ready droplet during or after thermal cycling.

9. The method of claim 1 further comprising dispensing a droplet from the amplification-ready droplet during thermal cycling.

10. The method of claim 1 further comprising quantifying amplified target nucleic acid in the amplification-ready droplet during or after thermal cycling.

11. The method of claim 10 further comprising extrapolating the quantity of target nucleic acid in the amplification-ready droplet prior to thermal cycling.

12. The method of claim 1 wherein step 1(c) is accomplished in the amplification-ready droplet in proximity to a heater by changing the temperature of the heater.

13. The method of claim 1 wherein the droplet operations of step 1(d) further comprise subjecting the amplified target nucleic acid to denaturing conditions to render it single-stranded prior to contact with the one or more magnetically responsive beads.

14. The method of claim 1 wherein the amplified target nucleic acid is biotinylated and the beads comprise streptavidin bound thereto.

15. The method of claim 1 further comprising repeating steps (e)(i) to (e)(iii) until a predetermined concentration of unbound substances is achieved.

16. The method of claim 1 further comprising a step (e)(iv) comprising releasing the immobilized beads.

17. The method of claim 16 further comprising repeating steps (e)(i) to (e)(iv) until a predetermined concentration of unbound substances is achieved.

18. The method of claim 1 further comprising executing droplet operations selected to effect an elution protocol for freeing immobilized nucleic acid from the one or more magnetically responsive beads.

19. The method of claim 1 further comprising quantifying amplified target nucleic after a predetermined number of cycles.

20. The method of claim 1 further comprising stopping the thermal cycling when:

(a) a predetermined quantity of target nucleic acid is detected;

or

(b) a predetermined quantity of target nucleic acid is not detected after a predetermined number of cycles.

21. The method of claim 1 further comprising transporting a droplet comprising the amplified nucleic acid for further processing.

22. The method of claim 1 further comprising detecting one or more target nucleic acids.

23. The method of claim 22 further comprising providing a user output indicating the presence or absence of one or more target nucleic acids.

24. The method of claim 22 wherein the target nucleic acid comprises a diagnostic nucleic acid.

25. The method of claim 1 wherein step (a) is preceded by flowing one or more reagents and/or one or more samples from a locus exterior to the droplet microactuator onto the droplet microactuator.

26. The method of claim 25 wherein the external reservoir is located on a cartridge which is coupled to the droplet microactuator in a manner which establishes a flow path from the external reservoir onto the substrate.

27. The method of claim 25 wherein the external reservoir is located on a cartridge which is coupled to the droplet microactuator in a manner which establishes a flow path from the external reservoir into a reservoir on the substrate.

28. The method of claim 1 further comprising repeatedly transporting by conducting the droplet operations on the substrate comprising the electrodes an amplification-ready droplet having a volume which does not exceed 50 nL through one or more heated zones on the substrate.

29. The method of claim 28 wherein the amplification-ready droplet is essentially free of surfactant.