Microfluidic devices and methods of use in the formation and control of nanoreactors
The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. The invention provides for aqueous based emulsions containing uniquely labeled cells, enzymes, nucleic acids, etc., wherein the emulsions further comprise primers, labels, probes, and other reactants. An oil based carrier-fluid envelopes the emulsion library on a microfluidic device, such that a continuous channel provides for flow of the immiscible fluids, to accomplish pooling, coalescing, mixing, sorting, detection, etc., of the emulsion library.
1. A method for preparing nucleic acids for sequencing, the method comprising:
providing a first dispersed phase solution comprising primer-bound microbeads and primer pairs and a second dispersed phase solution comprising nucleic acid templates via respective first and second inlet channels in a coalescence module of a microfluidic device;
coalescing by the coalescence module droplets of the first and the second dispersed phase solution to generate nanoreactor droplets, a plurality of the droplets each comprising one of the primer-bound microbeads, at least one of the nucleic acid templates, and a label for amplified DNA;
amplifying the nucleic acids templates with the primer-bound microbeads and the primer pairs to generate bead-bound amplified nucleic acids;
separating using the label the droplets into a first population of the droplets that each contain the amplified DNA and a second population of the droplets that do not contain the amplified DNA;
breaking the first population of the droplets;
depositing the bead-bound amplified nucleic acids onto a solid support for sequencing.
2. The method of claim 1 , wherein the nucleic acid templates comprise DNA or RNA.
3. The method of claim 1 , wherein the nucleic acid templates comprise a whole genome.
4. The method of claim 1 , wherein a unique identifier is incorporated into the bead-bound amplified nucleic acids during amplification.
5. The method of claim 1 , wherein the microbeads comprise a polymer.
6. The method of claim 1 , wherein the droplets are surrounded by an immiscible carrier fluid.
7. The method of claim 1 , wherein amplifying comprises a polymerase chain reaction.
8. The method of claim 1 , wherein amplifying comprises flowing the droplets through a delay module to expose the droplets to thermocycling.
9. The method of claim 1 , wherein the depositing step includes depositing each bead of the bead-bound amplified nucleic acids into a well on the solid support of a detection module for sequencing.
10. The method of claim 9 , further comprising depositing into each well a reagent bead carrying enzymes required for pyrophosphate sequencing.
11. The method of claim 10 , further comprising sequencing the bead-bound amplified nucleic acids using a CCD camera.
12. The method of claim 1 , wherein the label comprises an oligonucleotide probe labeled with a dye and quencher wherein the oligonucleotide probe gets cleaved by polymerase during the amplifying step.
13. The method of claim 1 , wherein the label comprise a hairpin shaped oligonucleotide linked to a fluorophore and quencher, wherein when the oligonucleotide hybridizes to an amplicon the fluorophore is separated from the quencher, allowing the fluorophore to fluoresce.
14. The method of claim 1 , wherein the separating step uses fluorescence activated sorting.
15. The method of claim 1 , wherein the separating step includes detecting fluorescence from the droplets that each contain the amplified DNA.