Thermocycling device for nucleic acid amplification and methods of use
View Patent ↗The present invention provides thermocycling devices useful for amplification of nucleic acids in droplets. The thermocycling device utilizes the flow of one or more fluids through a main compartment at temperatures sufficient to conduct a polymerase chain reaction. Methods of amplifying nucleic acids in droplets are also provided.
1. A thermocycling device for amplifying nucleic acid in a droplet, the device comprising:
a reaction chamber comprising a plurality of droplets;
a first temperature-controlled fluid source comprising a fluid at a first temperature;
a second temperature-controlled fluid source comprising a fluid at a second temperature; and
a conduit fluidly coupled to the first and the second temperature-controlled fluid sources, and the reaction chamber;
wherein the device is adapted to immerse the plurality of droplets in the fluid at the first temperature within the reaction chamber and subsequently immerse the plurality of droplets in the fluid at the second temperature within the reaction chamber, wherein the reaction chamber is oriented so that a gravitational force causes the fluid at the first temperature and the fluid at the second temperature to exit the reaction chamber while maintaining the plurality of droplets in the reaction chamber.
2. The device of claim 1 , further comprising a third temperature-controlled fluid source at a third temperature, and the fluid at the first temperature is about 94°-100° Celsius, the fluid at the second temperature is about 50°-65° Celsius, and the fluid at the third temperature is about 68°-72° Celsius.
3. The device of claim 1 , wherein the conduit conducts the fluid of the first and second temperatures from said temperature-controlled fluid sources through the reaction chamber.
4. The device of claim 1 , wherein said reaction chamber comprises an inlet and an outlet, and said fluid at a first temperature and said fluid at a second temperature flows into said inlet and out of said outlet.
5. The device of claim 4 , wherein the conduit is fluidly coupled to the inlet and comprises a valve at one end for controlling flow from the first or second temperature-controlled fluid into the conduit.
6. The device of claim 1 , further comprising a droplet generator.
7. The device of claim 6 , wherein the droplet generator comprises a nucleic acid sample introduction unit and a unit for combining the sample with one or more PCR reagents.
8. The device of claim 6 , wherein the droplet generator comprises an injection orifice which connects a sample flow pathway to a channel comprising an immiscible carrier fluid.
9. The device of claim 6 , wherein the droplet generator comprises an inlet channel for flowing a sample fluid, an outlet channel, and two carrier fluid channels for flowing an immiscible carrier fluid, each of the channels intersecting at a junction, said inlet and outlet channels being perpendicular to the carrier fluid channels, and said inlet channel being narrower at a distal portion where it connects to the junction.
10. The device of claim 1 , further comprising a heating source in proximity to the first or second temperature-controlled fluid source.
11. The device of claim 10 , wherein the heating source is embedded within the device.
12. The device of claim 11 , wherein the heating source is a metal selected from the group consisting of tungsten and platinum.
13. The device of claim 10 , wherein the heating source is an external heating source.
14. The device of claim 10 , wherein the heating source is selected from the group consisting of a coil, a wire and a film.
15. The device of claim 1 , further comprising a detection module.
16. An apparatus for nucleic acid amplification comprising a plurality of the device of claim 1 .
17. A method of nucleic acid amplification, said method comprising the steps of:
a) providing a reaction chamber for housing a plurality of droplets;
b) flowing the plurality of droplets into the reaction chamber, each droplet comprising a single nucleic acid template, at least one primer and reagents sufficient for nucleic acid amplification;
c) flowing a first fluid having a first temperature into the reaction chamber and maintaining for a sufficient time to denature the nucleic acid template in the droplets, wherein the reaction chamber is oriented so that a gravitational force causes the first fluid to exit the reaction chamber while maintaining the droplets in the reaction chamber;
d) flowing a second fluid having a second temperature into the reaction chamber and maintaining for a sufficient time to anneal one or more of the PCR reagents to the nucleic acid template in the droplets, wherein the reaction chamber is oriented so that a gravitational force causes the second fluid to exit the reaction chamber while maintaining the droplets in the reaction chamber;
e) flowing a third fluid having a third temperature into the reaction chamber and maintaining for a sufficient time to extend the nucleic acid template in the droplets, wherein the reaction chamber is oriented so that a gravitational force causes the third fluid to exit the reaction chamber while maintaining the droplets in the reaction chamber.
18. The method of claim 17 , wherein said first fluid has a temperature range from 94°-100 20 Celsius, said second fluid has a temperature range from 50°-65° Celsius and said third fluid has a temperature range from 68°-72° Celsius.
19. The method of claim 17 , wherein steps c) through e) are repeated for one or more cycles.
20. The method of claim 19 , wherein steps c) through e) are repeated for 20-45 cycles.
21. The method of claim 17 , wherein said first, second and third fluids directly contact the one or more droplets.