Gene analysis system

Patent metadata

Field	Value
Publication number	US-10876106-B2
Application number	US-201515553175-A
Country	US
Kind code	B2
Filing date	Apr 9, 2015
Priority date	Apr 9, 2015
Publication date	Dec 29, 2020
Grant date	Dec 29, 2020

Abstract

In order to interpret an arbitrary sequence region in many genes in many cells, it is necessary to degrade a nucleic acid into fragments and introduce a sequence that is different from one cell to another into each of the fragments. However, in the conventional configuration for analyzing many cells, there has been a problem that mixing of the degraded fragments among areas occurs before a tag sequence unique for each of the areas is introduced. The present invention provides a system for capturing a nucleic acid extracted from a cell in each of plural areas on a substrate and synthesizing a complementary DNA (cDNA) of the nucleic acid for each of the areas, wherein the system also includes a means for immediately introducing a tag sequence unique for each of the areas to the reaction product.

First claim

The invention claimed is:

1. A gene analysis system, comprising:
a substrate including a cell holding area configured to hold one cell arranged on the substrate;
a first probe that is placed on an inner surface of the cell holding area below the one cell, and has a nucleic acid capturing sequence;
a second probe that is placed on the inner surface of the same cell holding area below the one cell, and has a tag sequence which identifies the cell holding area and a reaction product capturing sequence,
wherein the nucleic acid capturing sequence of the first probe is configured to hybridize with a single strand nucleic acid extracted from the cell,
wherein the reaction product capturing sequence of the second probe is configured to hybridize with a reaction product which is synthesized from the single strand nucleic acid, the reaction product having a complementary sequence to the single strand nucleic acid,
wherein the second probe is further configured to hybridize with the reaction product and synthesize a tag sequence-introduced product having a complementary sequence to the reaction product, and which includes the tag sequence of the second probe which identifies the cell holding area,
wherein the reaction product capturing sequence of the second probe is different than the nucleic acid capturing sequence of the first probe, and
wherein the first probe comprises SEQ ID No. 1.

2. The system according to claim 1 , wherein the reaction product capturing sequence of the second probe is different than the nucleic acid capturing sequence of the first probe and wherein the second probe has the tag sequence which uniquely identifies the cell holding area in the substrate.

3. The system according to claim 1 , wherein the second probe further has at least one of a common sequence and a nucleic acid amplification correcting sequence.

4. The system according to claim 1 , wherein the first probe and the second probe are immobilized on a same carrier placed on the inner surface of the cell holding area.

5. The system according to claim 1 , wherein the first probe and the second probe are immobilized on the inner surface of cell holding area via a joint molecule.

6. The system according to claim 1 , wherein the first probe and the second probe are immobilized on different carriers placed on the inner surface of the cell holding area.

7. The system according to claim 1 , wherein the second probe comprises SEQ ID No. 2.

8. A gene analysis system, comprising:
a substrate including a cell holding area configured to hold one cell arranged on the substrate;
a first probe that is placed on an inner surface of the cell holding area below the one cell, and has a nucleic acid capturing sequence;
a second probe that is placed on the inner surface of the same cell holding area below the one cell, and has a tag sequence which identifies the cell holding area and a reaction product capturing sequence,
wherein the nucleic acid capturing sequence of the first probe is configured to hybridize with a single strand nucleic acid extracted from the cell,
wherein the reaction product capturing sequence of the second probe is configured to hybridize with a reaction product which is synthesized from the single strand nucleic acid, the reaction product having a complementary sequence to the single strand nucleic acid,
wherein the second probe is further configured to hybridize with the reaction product and synthesize a tag sequence-introduced product having a complementary sequence to the reaction product, and which includes the tag sequence of the second probe which identifies the cell holding area,
wherein the reaction product capturing sequence of the second probe is different than the nucleic acid capturing sequence of the first probe, and
wherein the first probe comprises SEQ ID No. 5, and the second probe comprises SEQ ID No. 2.

9. A gene analysis system, comprising:
a substrate including a cell holding area configured to hold one cell arranged on the substrate;
a first probe that is placed on an inner surface of the cell holding area below the one cell, and has a nucleic acid capturing sequence;
a second probe that is placed on the inner surface of the same cell holding area below the one cell, and has a tag sequence which identifies the cell holding area and a reaction product capturing sequence,
wherein the nucleic acid capturing sequence of the first probe is configured to hybridize with a single strand nucleic acid extracted from the cell,
wherein the reaction product capturing sequence of the second probe is configured to hybridize with a second reaction product which is synthesized from a first reaction product having a complementary sequence to the single strand nucleic acid, the second reaction product having a complementary sequence to the first reaction product,
wherein the second probe is further configured to hybridize with the second reaction product and synthesize a tag sequence-introduced product having a complementary sequence to the second reaction product and the tag sequence of the second probe which identifies the cell holding area,
wherein the reaction product capturing sequence of the second probe is different than the nucleic acid capturing sequence of the first probe, and
wherein the nucleic acid capturing sequence of the first probe comprises SEQ ID No. 9.

10. The system according to claim 9 , wherein the reaction product capturing sequence of the second probe is different than the nucleic acid capturing sequence of the first probe and wherein the second probe has the tag sequence which uniquely identifies the cell holding area in the substrate.

11. The system according to claim 9 , wherein the second probe further has at least one of a common sequence and a nucleic acid amplification correcting sequence.

12. The system according to claim 9 , wherein the first probe and the second probe are immobilized on a same carrier placed on the inner surface of the cell holding area.

13. The system according to claim 9 , wherein the first probe and the second probe are immobilized on the inner surface of cell holding area via a joint molecule.

14. The system according to claim 9 , wherein the first probe and the second probe are immobilized on different carriers placed on the inner surface of the cell holding area.

15. The system according to claim 9 , wherein the second probe comprises SEQ ID No. 2.

16. A gene analysis system, comprising:
a substrate including a cell holding area configured to hold one cell arranged on the substrate;
a first probe that is placed on an inner surface of the cell holding area below the one cell, and has a nucleic acid capturing sequence;
a second probe that is placed on the inner surface of the same cell holding area below the one cell, and has a tag sequence which identifies the cell holding area and a reaction product capturing sequence,
wherein the nucleic acid capturing sequence of the first probe is configured to hybridize with a single strand nucleic acid extracted from the cell,
wherein the reaction product capturing sequence of the second probe is configured to hybridize with a second reaction product which is synthesized from a first reaction product having a complementary sequence to the single strand nucleic acid, the second reaction product having a complementary sequence to the first reaction product,
wherein the second probe is further configured to hybridize with the second reaction product and synthesize a tag sequence-introduced product having a complementary sequence to the second reaction product and the tag sequence of the second pro

Assignees

• Hitachi Ltd

Inventors

• Tanabe Maiko
• Shirai Masataka
• Sakai Tomoyuki

Classifications

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12M1/00

  Apparatus for enzymology or microbiology · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12N15/1096

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