Methods and devices for digital PCR
The invention provides methods of conducting a nucleic acid reaction, including methods for performing digital PCR using a “droplet-in-oil” technology. In the methods, the starting sampled is segmented at least partially into a set of sample droplets each containing on average about one or fewer copies of a target nucleic acid. The droplets are passed in a continuous flow of immiscible carrier fluid through a channel that passes through a thermal cycler, whereby the target is amplified. In one implementation, the droplets are about 350 nl each and the number of positively amplified droplets is counted at the near-saturation point.
1. A method of conducting a nucleic acid amplification reaction, the method comprising:
providing at least one starting sample comprising at least one nucleic acid;
segmenting at least part of the at least one starting sample to provide a set of sample droplets in a continuous flow of immiscible carrier fluid through a channel, each of said droplets of the set of sample droplets containing on average about one or fewer copies of a target nucleic acid;
merging the set of sample droplets with a second set of droplets having one or more primers to form a set of combined droplets;
passing the set of combined droplets through a plurality of thermal zones thereby allowing the target nucleic acid, if present, to be amplified in each droplet;
determining a Ct value of the target nucleic acid for at least one of the combined droplets;
detecting the presence or absence of, and/or determining the amount of, the amplified target nucleic acid in the droplets within the set of combined droplets while in the flow by analyzing the Ct value based on real-time amplification data;
determining the total number of combined droplets with amplified target nucleic acid; and
determining the starting copy number based on determining the total number of combined droplets with amplified target nucleic acid.
2. The method of claim 1 , wherein the volume of each droplet of the set of combined droplets is 0.1 pl-500 nl.
3. The method of claim 2 , wherein the volume of each droplet of the set of combined droplets is 10-500 nl.
4. The method of claim 3 , wherein the volume of each droplet of the set of combined droplets is 30-350 nl.
5. The method of claim 1 , wherein a portion of the sample representative of the entire sample is segmented into the droplets.
6. The method of claim 5 , wherein the starting sample contains at least 10 copies of the target nucleic acid and at least 10% of the sample is segmented.
7. The method of claim 5 , wherein the set of sample droplets contains 5-50 droplets.
8. The method of claim 5 , wherein the set of sample droplets from the starting sample contains 100 or fewer droplets, each of 500 nl or less.
9. The method of claim 1 , wherein at least 50% of droplets in the set of sample droplets do not contain the target-nucleic acid.
10. The method of claim 1 , wherein detecting the presence or absence of, and/or determining the amount of, the amplified target nucleic acid in the droplets is performed at multiple thermal cycles, thereby monitoring the amount of amplified target nucleic acid throughout the cycles.
11. The method of claim 1 , wherein fewer than 50% of droplets in the set of combined droplets are positively amplified.
12. The method of claim 1 , wherein the method comprises reverse transcription of mRNA to prepare the starting sample.
13. A method of processing a plurality of starting samples in parallel, each according to claim 1 , wherein at least some of the starting samples have (a) varying concentration of the target nucleic acid and/or (b) varying target nucleic acids.
14. The method of claim 13 , wherein the parallel processing comprises sets of combined droplets from different samples that form a train of alternating combined droplets in the continuous flow in the channel.
15. The method of claim 1 , wherein the droplets of the set of combined droplets remain wrapped in the immiscible carrier fluid while in the flow.
16. The method of claim 1 , wherein the channel comprises a capillary having a circular cross-section.
17. The method of claim 1 , wherein the droplets of the set of sample droplets are spherical.
18. A method of conducting a nucleic acid amplification reaction, the method comprising:
providing a starting sample containing approximately at least one nucleic acid;
segmenting at least part of the sample to provide a set of at least 10 sample droplets in a continuous flow of immiscible carrier fluid through a channel, each of said droplets of the set of sample droplets containing on average about one or fewer copies of a target nucleic acid;
merging the set of sample droplets with a second set of droplets having one or more primers to form a set of combined droplets;
passing the set of combined droplets through a plurality of thermal zones thereby allowing a target nucleic acid, if present in the combined droplets, to be amplified to near or above saturation level; and
determining a Ct value of the target nucleic acid for at least one of the combined droplets
detecting the presence or absence of, and/or determining the amount of, the amplified target nucleic acid in the combined droplets while in the flow by analyzing the Ct value based on real-time amplification data;
determining the total number of combined droplets with amplified target nucleic acid; and
determining the starting copy number based on determining the total number of combined droplets with amplified target nucleic acid.
19. The method of claim 1 further comprising flowing more than one sample.
20. The method of claim 1 , wherein the segmenting includes segmenting the sample into 1 pL-10 pL droplets.
21. The method of claim 1 , wherein the merging includes using a passive system to merge the droplets.
22. The method of claim 1 , further comprising determining the mass of the target nucleic acid.
23. The method of claim 22 , wherein the target nucleic acid is DNA.
24. The method of claim 23 , wherein the DNA is genomic DNA.
25. The method of claim 24 , wherein the mass of genomic DNA corresponds to copy numbers of target nucleic acid sequences.