Microfluidic nucleic acid analysis

Patent metadata

Field	Value
Publication number	US-9579650-B2
Application number	US-201414494284-A
Country	US
Kind code	B2
Filing date	Sep 23, 2014
Priority date	Oct 2, 2002
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

Abstract

Nucleic acid from cells and viruses sampled from a variety of environments may purified and expressed utilizing microfluidic techniques. In accordance with one embodiment of the present invention, 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. In one specific application, cell isolation, lysis, and nucleic acid purification may be performed utilizing a highly parallelized microfluidic architecture to construct gDNA and cDNA libraries.

First claim

What is claimed is:

1. A method of preparing a plurality of cDNA libraries from single cells, comprising:
obtaining a sample of cells;
isolating a plurality of single cells from the sample within one or more channels of a microfluidic device such that single cells are fluidically isolated from each other;
individually lysing at least some of the isolated single cells to liberate mRNA while keeping the mRNA from the single cells separate;
reverse transcribing the mRNA liberated from the single cells isolated within the microfluidic device to form cDNA; and
amplifying the cDNA, thereby obtaining the plurality of cDNA libraries from single cells.

2. A method of preparing a plurality of cDNA libraries from single cells, comprising:
obtaining a sample of cells;
isolating a plurality of single cells from the sample within one or more channels of a microfluidic device by segmentation;
lysing at least some often isolated single cells to produce isolated mRNA such that mRNA from each individual cell remains together and is kept separate from mRNA from all other cells in the sample; and
reverse transcribing and amplifying the mRNA from at least some of the single cells that were isolated within the microfluidic device to form cDNA, thereby obtaining the plurality of cDNA libraries from single cells.

3. The method of claim 1 , whereby mRNA from each individual cell remains together.

4. The method of claim 1 , further comprising performing a ligation reaction with the cDNA from the single cells.

5. The method of claim 1 or claim 2 , wherein amplifying comprises using different primers to amplify the cDNA from different isolated single cells.

6. The method of claim 2 , further comprising performing a ligation reaction with the cDNA from the single cells.

7. The method of claim 4 or claim 6 , wherein the ligation reaction comprises ligation to a host vector or plasmid.

8. The method of claim 4 or claim 6 , wherein ligated nucleic acid from the ligation reaction of the single cells is used to transform a host cell.

9. The method of any of claims 1 to 3 , wherein the plurality of single cells are fluidically isolated from each other and from other cells from the sample by closing a plurality of valves in the microfluidic device.

10. The method of claim 9 , whereby the plurality of single cells are fluidically isolated from each other in separate segments of the same channel.

11. The method of any of claims 1 to 3 , which includes determining an expression profile for the sample.

Assignees

• California Inst Of Techn

Inventors

• Hong Jong Wook
• Studer Vincent
• Anderson W French
• Quake Stephen R
• Leadbetter Jared

Classifications

• B01L3/50273 Primary

  characterised by the means or forces applied to move the fluids · CPC title

• B01L2300/1827

  using resistive heater · CPC title

• B01L2400/0481

  squeezing of channels or chambers · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• B01L3/502715 Primary

  characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces · CPC title