Microfluidic nucleic acid analysis
Nucleic acid from cells and viruses sampled from a variety of environments may purified and expressed utilizing microfluidic techniques. Individual or small groups of cells or viruses may be isolated in microfluidic chambers by dilution, sorting, and/or segmentation. The isolated cells or viruses may be lysed directly in the microfluidic chamber, and the resulting nucleic acid purified by exposure to affinity beads. Subsequent elution of the purified nucleic acid may be followed by ligation and cell transformation, all within the same microfluidic chip. Cell isolation, lysis, and nucleic acid purification may be performed utilizing a highly parallelized microfluidic architecture to construct gDNA and cDNA libraries.
1. A method for preparing a plurality of cDNA libraries from single cells in a microfluidic apparatus, wherein the apparatus comprises:
(a) a solid substrate;
(b) a cell separation and processing system embedded in the substrate that includes an arrangement of flow channels configured so that individual cells from a cell population can be singly isolated;
(c) one or more inlet channels in the substrate connected with the cell separation and processing system so that a reagent can be delivered through the inlet channel(s) and combined with cells isolated by the system; and
(d) one or more outlet channels in the substrate connected with the cell separation and processing system so that contents obtained by lysing isolated cells can be flowed through the outlet channel(s) and kept separate;
wherein the method comprises:
(1) receiving a sample of cells into the cell separation and processing system of the apparatus;
(2) processing the sample in the cell separation and processing system such that a plurality of single cells from the sample are fluidically isolated from all other cells in the sample, thereby producing isolated single cells;
(3) combining a lysing chemical or buffer with the isolated cells such that mRNA is liberated from each of at least some of the isolated single cells in a manner that keeps the mRNA from each of the isolated single cells separate; and
(4) reverse transcribing and amplifying the mRNA liberated in step (3) such that a separate cDNA library is formed from at least some of the isolated single cells;
thereby producing said plurality of cDNA libraries from single cells.
2. The method of claim 1 , wherein the method further comprises:
(5) flowing the separate cDNA libraries through the outlet channel(s) of the apparatus and keeping separate at least some of the cDNA libraries formed in step (4).
3. The method of claim 1 , wherein step (2) and step (3) are performed separately.
4. The method of claim 1 , wherein the cell separation and processing system is configured to singly isolate cells from the cell population by way of a plurality of valves in the system that are operable to close channels between single cells.
5. The method of claim 1 , wherein the cell separation and processing system is configured to singly isolate bacteria.
6. The method of claim 1 , wherein the cell separation and processing system is configured to singly isolate eukaryotic cells.
7. The method of claim 1 , wherein the cell separation and processing system comprises one or more mixing structures configured to actively mix a lysing agent delivered through the inlet channels with cells singly isolated by the cell separation and processing system.
8. The method of claim 7 , wherein the one or more mixing structures are rotary mixers.
9. The method of claim 1 , wherein step (3) comprises operating the apparatus so as to actively mix the lysis chemical or buffer with the single cells.
10. The method of claim 1 , wherein step (3) comprises operating the apparatus so as to diffusively mix the lysis chemical or buffer with the single cells.
11. The method of claim 1 , further comprising sequencing cDNA libraries from the single cells.
12. A method for preparing a plurality of nucleic acid libraries from single cells in a microfluidic apparatus, wherein the apparatus comprises:
(a) a solid substrate;
(b) a cell separation and processing system embedded in the substrate that includes an arrangement of flow channels configured so that individual cells from a cell population can be singly isolated;
(c) one or more inlet channels in the substrate connected with the cell separation and processing system so that a reagent can be delivered through the inlet channel(s) and combined with cells isolated by the system; and
(d) one or more outlet channels in the substrate connected with the cell separation and processing system so that contents obtained by lysing isolated cells can be flowed through the outlet channel(s) and kept separate;
wherein the method comprises:
(1) receiving a sample of cells into the cell separation and processing system of the apparatus;
(2) processing the sample in the cell separation and processing system such that a plurality of single cells from the sample are fluidically isolated from all other cells in the sample, thereby producing isolated single cells;
(3) combining a lysing chemical or buffer with the isolated cells such that nucleic acid is liberated from each of at least some of the isolated single cells in a manner that keeps the nucleic acid from each of the isolated single cells separate; and
(4) amplifying the nucleic acid liberated in step (3) such that a separate nucleic acid library is formed from at least some of the isolated single cells;
thereby producing said plurality of nucleic acid libraries from single cells.
13. The method of claim 12 , wherein the method further comprises:
flowing the separate nucleic acid libraries through the outlet channel(s) of the apparatus and keeping separate at least some of the nucleic acid libraries formed in step (4).
14. The method of claim 12 , wherein step (2) and step (3) are performed separately.
15. The method of claim 12 , wherein the cell separation and processing system is configured to singly isolate cells from the cell population by way of a plurality of valves in the system that are operable to close channels between single cells.
16. The method of claim 12 , comprising delivering a lysing agent through the inlet channels and actively mixing the lysing agent with singly isolated cells in the cell separation and processing system.
17. The method of claim 16 , wherein the one or more mixing structures are rotary mixers.
18. The method of claim 12 , wherein step (3) comprises operating the apparatus so as to actively mix the lysis chemical or buffer with the single cells.
19. The method of claim 12 , wherein step (3) comprises operating the apparatus so as to diffusively mix the lysis chemical or buffer with the single cells.
20. The method of claim 12 , further comprising sequencing nucleic acid or cDNA libraries from the single cells.