Droplet-based nucleic acid amplification method and apparatus
View Patent ↗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.
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 μL.
6. The method of claim 1 wherein the amplification-ready droplet has a volume which ranges from about 1 mL to about 1 μL.
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.