Apparatus, system, and method using immiscible-fluid-discrete-volumes
Various embodiments of the teachings relate to a system or method for sample preparation or analysis in biochemical or molecular biology procedures. The sample preparation can involve small volume processed in discrete portions or segments or slugs, herein referred to as discrete volumes. A molecular biology procedure can be nucleic acid analysis. Nucleic acid analysis can be an integrated DNA amplification/DNA sequencing procedure.
1. A method comprising:
contacting a stream of aqueous sample fluid flowing in a first conduit with a stream of non-aqueous spacing fluid that is immiscible with the aqueous sample fluid to form discrete volumes of the aqueous sample fluid separated from one another by the non-aqueous spacing fluid, wherein the aqueous sample fluid comprises target nucleic acid, and wherein a first plurality of the discrete volumes contains at least one molecule comprising the target nucleic acid and a second plurality of the discrete volumes contains no molecules comprising the target nucleic acid;
amplifying the target nucleic acid in one or more of the first plurality of the discrete volumes to form an amplicon;
after amplifying the target nucleic acid, adding additional spacing fluid between consecutive discrete volumes of one or both of the first plurality of discrete volumes and the second plurality of discrete volumes, so as to adjust or control spacing between the consecutive discrete volumes;
in a second conduit, detecting a fluorescence signal from the amplicon in the one or more of the first plurality of the discrete volumes; and
based on the detecting, discriminating between the one or more of the first plurality of the discrete volumes and the second plurality of the discrete volumes.
2. The method of claim 1 , wherein the contacting comprises continuously flowing at least one of the aqueous sample fluid and the non-aqueous spacing fluid into the first conduit.
3. The method of claim 1 , further comprising separating the second plurality of the discrete volumes from the first plurality of the discrete volumes.
4. The method of claim 1 , wherein less than 37% of the first plurality of the discrete volumes comprise a single molecule comprising the target nucleic acid.
5. The method of claim 4 , wherein 1% or more of the first plurality of the discrete volumes comprise a single molecule comprising the target nucleic acid.
6. The method of claim 4 , wherein 10% or more of the first plurality of discrete volumes comprise a single molecule comprising the target nucleic acid.
7. The method of claim 1 , wherein the discrete volumes comprise droplets of the aqueous sample fluid.
8. The method of claim 1 further comprising:
dispensing the discrete volumes from the first conduit and into one or more containers, wherein the amplifying occurs while the discrete volumes are in the one or more containers.
9. The method of claim 8 , wherein the one or more containers comprise wells of a multi-well plate.
10. The method of claim 8 , wherein at least some of the discrete volumes have a volume that is less than about 50 nanoliters.
11. The method of claim 8 , wherein at least some of the discrete volumes have a volume of between about 1 femtoliter and 1 nanoliter.
12. The method of claim 1 , wherein the first and second conduits are in fluid communication with each other.
13. The method of claim 1 , further comprising sequencing the amplicon.
14. The method of claim 1 , further comprising running the amplicon through a capillary.
15. The method of claim 1 , further comprising running the amplicon through a capillary electrophoretic sequencer.
16. A method comprising:
forming droplets of an aqueous sample fluid separated from one another by a spacing fluid that is immiscible with the aqueous sample fluid, the aqueous sample fluid comprising a polynucleotide comprising a target nucleic acid sequence, wherein a first plurality of droplets contains at least one molecule of the polynucleotide comprising the target nucleic acid sequence per droplet and a second plurality of droplets contains no molecules of the polynucleotide comprising the target nucleic acid sequence;
performing an amplification assay on the polynucleotide in one or more of the first plurality of droplets to form an amplicon;
after performing the amplification assay, adding additional spacing fluid between consecutive aqueous droplets of one or both of the first plurality of aqueous droplets and the second plurality of aqueous droplets, so as to adjust or control the spacing between the consecutive aqueous droplets; and
monitoring for a fluorescence signal from one or more of the first plurality of droplets, the fluorescent signal indicative of a presence of the amplicon.
17. The method of claim 16 , wherein 1% or more of the droplets comprise a single molecule comprising the target nucleic acid sequence.
18. The method of claim 16 , wherein less than 37% of the droplets comprise a single molecule comprising the target nucleic acid sequence.
19. The method of claim 16 , wherein at least some of the droplets have a volume that is less than about 50 nanoliters.
20. The method of claim 16 , further comprising sequencing the amplicon.