Two-dimensional cell array device and apparatus for gene quantification and sequence analysis

Patent metadata

Field	Value
Publication number	US-10030240-B2
Application number	US-201314771339-A
Country	US
Kind code	B2
Filing date	Mar 12, 2013
Priority date	Mar 12, 2013
Publication date	Jul 24, 2018
Grant date	Jul 24, 2018

Abstract

In order to conduct gene expression analysis of a number of genes in a number of cells, it has been necessary to separate cells, extract genes therefrom, amplify nucleic acids, and perform sequence analysis. However, separation of cells imposes damages on the cells, and it requires the use of an expensive system. Gene expression analysis in each cell can be carried out with high accuracy by arranging a pair of structures comprising a cell trapping section and a nucleic acid trapping section in a vertical direction to extract individual genes in relevant cells, synthesizing cDNA in the nucleic acid trapping section, amplifying nucleic acids, and analyzing the sequences using a next-generation sequencer.

First claim

The invention claimed is:

1. A device for nucleic acid extraction comprising:
an upper reaction region comprising a cell inlet, an upper inlet, and an upper outlet;
a cell trapping section for immobilizing a single cell;
a fluid channel in which an extraction solution for extracting a nucleic acid from the cell flows downward through the cell trapping section;
a nucleic acid trapping section connected to the cell trapping section via the fluid channel and located downstream of the cell trapping section, the nucleic acid trapping section being capable of immobilizing the extracted nucleic acid;
a fluid channel that discharges the solution after nucleic acid extraction from the nucleic acid trapping section in a direction opposite from the cell trapping section; and
a porous array sheet with a pore that connects the nucleic acid trapping region to a lower reaction region,
wherein the lower reaction region comprises a lower outlet,
wherein the cell trapping section, the two fluid channels, and the nucleic acid trapping section are paired in a vertical direction and a plurality of the pairs are arranged in a planar direction, and
wherein the nucleic acid trapping section comprises a DNA probe for nucleic acid trapping, and the DNA probe comprises a cell recognition tag sequence.

2. The device for nucleic acid extraction according to claim 1 , wherein the nucleic acid trapping section comprises beads on which the DNA probe for nucleic acid trapping is immobilized.

3. The device for nucleic acid extraction according to claim 2 , wherein the DNA probe for nucleic acid trapping comprises a sequence for identifying a position on a chip.

4. The device for nucleic acid extraction according to claim 2 , wherein the DNA probe for nucleic acid trapping comprises separate sequences for the trapped nucleic acid molecules.

5. The device for nucleic acid extraction according to claim 4 , wherein the upper inlet introduces an enzyme for reverse transcription of RNA trapped by the nucleic acid trapping section.

6. The device for nucleic acid extraction according to claim 1 , wherein the nucleic acid trapping section comprises a porous membrane comprising the DNA probe for nucleic acid trapping immobilized on pores.

7. The device for nucleic acid extraction according to claim 1 , wherein the cell trapping section comprises a substance immobilized thereon that chemically binds to a substance on a cell surface.

8. The device for nucleic acid extraction according to claim 1 , wherein a region immediately downstream of the cell trapping section is made of an optically transparent material.

9. The device for nucleic acid extraction according to claim 1 , wherein the nucleic acid trapping section is provided in a region immediately downstream of the cell trapping section.

10. The device for nucleic acid extraction according to claim 1 , wherein the nucleic acid trapping section is provided in a region other than the region immediately downstream of the cell trapping section.

11. An apparatus for nucleic acid treatment, comprising: the device for nucleic acid extraction according to claim 1 ; and wherein the upper inlet introduces a reagent for constructing a cDNA library into the device for nucleic acid extraction.

12. An apparatus for nucleic acid treatment, comprising: the device for nucleic acid extraction according to claim 1 ; a reagent for constructing a cDNA library; and wherein the upper inlet introduces the reagent for nucleic acid amplification into the device for nucleic acid extraction.

13. An apparatus for nucleic acid treatment, comprising: the device for nucleic acid extraction according to claim 1 ; and a microscope section for observing cells trapped by the cell trapping section under a differential interference microscope, phase contrast microscope, Raman microscope, or Coherent Raman microscope.

14. A method for extracting a nucleic acid from a cell using a device for nucleic acid extraction comprising an upper reaction region comprising a cell inlet, an upper inlet, and an upper outlet;
a cell trapping section for immobilizing a single cell;
a fluid channel in which an extraction solution for extracting a nucleic acid from the cell flows downward through the cell trapping section;
a nucleic acid trapping section connected to the cell trapping section via the fluid channel and located downstream of the cell trapping section, the nucleic acid trapping section being capable of immobilizing the extracted nucleic acid;
a fluid channel that discharges the solution after nucleic acid extraction from the nucleic acid trapping section in a direction opposite from the cell trapping section; and
a porous array sheet with a pore that connects the nucleic acid trapping region to a lower reaction region,
wherein the lower reaction region comprises a lower outlet,
wherein the cell trapping section, the two fluid channels, and the nucleic acid trapping section are paired in a vertical direction and a plurality of the pairs are arranged in a planar direction, and
wherein the nucleic acid trapping section comprises a DNA probe for nucleic acid trapping, and the DNA probe comprises a cell recognition tag sequence,
the method comprising:
bringing cells into contact with the cell trapping section so as to allow the cell trapping section to trap each cell;
allowing the extraction solution for extracting the nucleic acid from the cell to flow downward through the cell trapping section in the fluid channel;
immobilizing the extracted nucleic acid to the nucleic acid trapping section via the DNA probe comprising the cell recognition tag sequence; and
discharging the solution after nucleic acid extraction from the nucleic acid trapping section in the direction opposite from the cell trapping section through the fluid channel.

Assignees

• Hitachi Ltd

Inventors

• Shirai Masataka
• Kambara Hideki
• Taniguchi Kiyomi
• Tanabe Maiko

Classifications

• B01L2300/0636

  Integrated biosensor, microarrays · CPC title

• B01L2200/0631

  Purification arrangements, e.g. solid phase extraction [SPE] · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• B01L2200/0668

  Trapping microscopic beads · CPC title

• B01L2200/10

  Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept · CPC title