Microfluidic nucleic acid analysis

• US 9,579,650 B2
• Filed: 09/23/2014
• Issued: 02/28/2017
• Est. Priority Date: 10/02/2002
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hong, Jong Wook, Studer, Vincent, Anderson, W. French, Quake, Stephen R., Leadbetter, Jared
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/494,284
• Publication Number:
  US 20150238960A1
• Time in Patent Office: 889 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0809 rectangular shaped
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

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.

Claims

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.

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.

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.

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.

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