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:
within a first conduit having a circular cross section having a maximum dimension, flowing a sample including a first plurality of aqueous droplets comprising a single molecule of a target nucleic acid sequence and a second plurality of aqueous droplets containing no molecules of the target nucleic acid sequence, the aqueous droplets being separated within the conduit by an immiscible fluid that is immiscible with the aqueous droplets, the aqueous droplets having a maximum dimension that is less than the maximum dimension of the first conduit;
amplifying the target nucleic acid sequence in each of the first plurality of aqueous droplets to form an amplicon;
while flowing the aqueous droplets within a second conduit, using a fluorescent signal detector to detect and identify the first plurality of aqueous droplets by detecting fluorescence from the first plurality of aqueous droplets.
2. A method comprising:
sequentially contacting an aqueous sample fluid in a conduit with a 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, the aqueous sample fluid comprising a target nucleic acid sequence, wherein a first plurality of the discrete volumes contains at least one molecule of the target nucleic acid sequence and a second plurality of the discrete volumes contains no molecules of the target nucleic acid sequence;
amplifying the target nucleic acid sequence in one or more of the first plurality of discrete volumes to form an amplicon; and
in a second conduit, detecting a fluorescence signal from the amplicon in each of the one or more first plurality of discrete volumes; and
based on the detecting, discriminating between the first plurality of discrete volumes and the second plurality of discrete volumes.
3. The method of claim 2 , wherein the contacting comprises continuously flowing at least one of the aqueous sample fluid and the non-aqueous spacing fluid into the conduit.
4. The method of claim 2 , further comprising sorting the second plurality of discrete volumes from the first plurality of discrete volumes.
5. The method of claim 2 , wherein less than 37% of the discrete volumes comprise a single molecule of the target nucleic acid sequence.
6. The method of claim 5 , wherein 1% or more of the discrete volumes comprise a single molecule of the target nucleic acid sequence.
7. The method of claim 5 , wherein 10% or more of the discrete volumes comprise a single molecule of the target nucleic acid sequence.
8. The method of claim 1 , wherein less than 37% of aqueous droplets comprise a single molecule of the target nucleic acid sequence.
9. The method of claim 8 , wherein 1% or more of aqueous droplets comprise a single molecule of the target nucleic acid sequence.
10. The method of claim 8 , wherein 10% or more of aqueous droplets comprise a single molecule of the target nucleic acid sequence.
11. The method of claim 1 , wherein the aqueous droplets and the immiscible fluid form a plurality of emulsified droplets, the method further comprising:
dispensing emulsified droplets from an end of the conduit and into one or more containers; and
thermal cycling the container and the emulsified droplets in a thermal cycler.
12. The method of claim 11 , wherein the containers comprises wells of a multiwell plate.
13. The method of claim 11 , wherein at least some of the emulsified droplets have a volume that is less than about 50 nanoliters.
14. The method of claim 11 , wherein at least some of the emulsified droplets have a volume of between about 1 femtoliter and 1 nanoliter.
15. The method of claim 11 , further comprising, for at least some of the first plurality of aqueous droplets, detecting with a detector the amplicon or a derivative thereof in a second conduit.
16. The method of claim 11 , wherein the detector is a fluorescent signal detector.
17. The method of claim 11 , further comprising, after thermal cycling in the thermal cycler, running the sample through a capillary.
18. The method of claim 11 , further comprising, after thermal cycling in the thermal cycler, running the sample through on a capillary electrophoretic sequencer.