Determination of cells using amplification
The present invention generally relates to microfluidics and, in particular, to systems and methods for determining cells using amplification. In one set of embodiments, cells are encapsulated within droplets and nucleic acids from the cells amplified within the droplets. The droplets may then be pooled together and the amplified nucleic acids can be determined using PCR or other suitable techniques. In some embodiments, techniques such as these can be used to detect relatively rare cells that may be present, e.g., if the droplets are amplified using conditions able to selectively amplify nucleic acids arising from the relatively rare cells.
1. A method, comprising:
encapsulating cells within a plurality of microfluidic droplets, including at least a first cell type and a second cell type, wherein the first cell type is present at a ratio of at least 1,000:1 of the first cell type relative to the second cell type;
lysing at least some of the cells within the plurality of droplets to release nucleic acids from the cells into the interior of the droplets;
within the interior of the droplets, applying conditions able to selectively amplify a target nucleic acid sequence suspected of being present within the nucleic acids released from the second cell type but not the first cell type;
combining the interiors of the droplets together to form a combined fluid; and
determining amplified nucleic acids contained within the combined fluid,
wherein determining the amplified nucleic acids comprises determining the amplified nucleic acids using PCR.
2. The method of claim 1 , wherein the cells comprise human cells.
3. The method of claim 1 , wherein the cells comprise cancer cells.
4. The method of claim 1 , wherein the cells comprise blood cells.
5. The method of claim 1 , wherein the cells comprise bacteria.
6. The method of claim 1 , wherein the target nucleic acid sequence is a DNA sequence.
7. The method of claim 1 , wherein applying conditions able to selectively amplify a target nucleic acid sequence comprises applying conditions able to cause amplification via PCR.
8. The method of claim 7 , wherein applying conditions able to selectively amplify a target nucleic acid sequence comprises applying conditions able to cause amplification via RT-PCR.
9. The method of claim 1 , wherein applying conditions able to selectively amplify a target nucleic acid sequence comprises adding a polymerase to at least some of the interior of the droplets.
10. The method of claim 1 , wherein at least some of the plurality of droplets comprises deoxyribonucleotides.
11. The method of claim 1 , wherein at least some of the plurality of droplets comprises primer.
12. The method of claim 1 , wherein applying conditions able to selectively amplify a target nucleic acid sequence comprises adding primer to at least some of the interior of the droplets.
13. The method of claim 1 , comprising applying conditions able to selectively amplify the target nucleic acid sequence by at least 10 3 times.
14. The method of claim 1 , wherein the droplets have a distribution of diameters such that no more than about 5% of the microfluidic droplets have a diameter less than about 90% or greater than about 110% of the overall average diameter of the microfluidic droplets.
15. The method of claim 1 , wherein combining the interiors of the droplets together to form a combined fluid comprises bursting the droplets to release the interiors of the droplets together.
16. The method of claim 1 , wherein combining the interiors of the droplets together to form a combined fluid comprises coalescing the droplets together.