Methods and devices for digital PCR
Methods of conducting a nucleic acid reaction, including methods for performing digital PCR using a “droplet-in-oil” technology, may include at least partially segmenting a starting sampled 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 a sample comprising a starting number of copies of a target nucleic acid;
segmenting the sample into a set of sample droplets using a continuous channel flow of immiscible carrier fluid through a plurality of channels, wherein at least 50% of the set of sample droplets do not contain copies of a target nucleic acid;
while individual droplets of the set are wrapped in the immiscible carrier fluid, thermal cycling the droplets through a plurality of temperatures thereby allowing a copy of target nucleic acid, if present, to be amplified in each droplet;
after thermal cycling the set of sample droplets to a near-saturation level of amplification of the droplets in which a copy of target nucleic acid is present, flowing the set of sample droplets separately past a detector by the immiscible carrier fluid, whereby the detector detects a presence or absence of an amplified target nucleic acid in each droplet within the set of sample droplets by measuring fluorescence;
calculating a total number of droplets detected to contain amplified target nucleic acid; and
calculating the starting number based on the calculated total number of droplets detected to contain amplified target nucleic acid.
2. The method of claim 1 , wherein a volume of each droplet of the set of sample droplets ranges from 0.1 pl to 500 nl.
3. The method of claim 2 , wherein the volume of each droplet of the set of sample droplets ranges from 10 nl to 500 nl.
4. The method of claim 3 , wherein the volume of each droplet of the set of sample droplets ranges from 30 nl to 350 nl.
5. The method of claim 1 , wherein the sample contains at least 10 copies of the target nucleic acid and at least 10% of the sample is segmented.
6. The method of claim 1 , wherein the set of sample droplets has 5 droplets-50 droplets.
7. The method of claim 1 , wherein the set of sample droplets has 100 or fewer droplets, each having a volume of 500 nl or less.
8. The method of claim 1 , wherein fewer than 50% of droplets in the set of sample droplets are detected to contain amplified target nucleic acid.
9. The method of claim 1 , further comprising performing reverse transcription of mRNA to prepare the sample.
10. The method of claim 1 , wherein the sample contains one genome equivalent of nucleic acid or less.
11. The method of claim 1 , wherein the channels comprises a capillary having a circular cross-section.
12. The method of claim 1 , wherein the droplets of the set of sample droplets are spherical.
13. The method of claim 1 , wherein the target nucleic acid is DNA.
14. The method of claim 13 , wherein the DNA is genomic DNA.
15. The method of claim 1 , wherein the sample further comprises one or more primers.
16. The method of claim 1 , wherein the set of sample droplets contains one or more primers.
17. The method of claim 1 , wherein at least 90% of the set of sample droplets do not contain copies of a target nucleic acid.
18. The method of claim 1 , wherein at least 99% of the set of sample droplets do not contain copies of a target nucleic acid.