Gene analysis system

Patent metadata

Field	Value
Publication number	US-2018119135-A1
Application number	US-201515553175-A
Country	US
Kind code	A1
Filing date	Apr 9, 2015
Priority date	Apr 9, 2015
Publication date	May 3, 2018
Grant date	—

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

1 . A gene analysis system, comprising:
a single strand nucleic acid capturing means including a cell holding area arranged on a substrate, and a first probe that is placed in the cell holding area and has a sequence for capturing a single strand nucleic acid extracted from a cell; a reaction product synthesizing means for synthesizing a nucleic acid having a sequence that is the same as or complementary to the captured single strand nucleic acid as a reaction product in the cell holding area; a reaction product capturing means including a second probe that is placed in the cell holding area and has a sequence for capturing the reaction product; a tag sequence introducing means for synthesizing a tag sequence-introduced product having a sequence that is the same as or complementary to a nucleic acid of the reaction product, and having a tag sequence unique for the cell holding area; and a nucleic acid amplifying means for amplifying the tag sequence-introduced product.

2 . The system according to claim 1 , wherein
the reaction product synthesizing means is for synthesizing a nucleic acid complementary to the captured single strand nucleic acid as a reaction product in the cell holding area, and the tag sequence introducing means is for synthesizing a tag sequence-introduced product having a sequence complementary to a nucleic acid of the reaction product.

3 . The system according to claim 1 , wherein
the reaction product synthesizing means is for synthesizing a nucleic acid having a sequence complementary to the captured single strand nucleic acid as a first reaction product in the cell holding area, and then synthesizing a nucleic acid having a sequence complementary to a nucleic acid of the first reaction product as a second reaction product, and the tag sequence introducing means is for synthesizing a tag sequence-introduced product having a sequence complementary to a nucleic acid of the second reaction product.

4 . The system according to any one of claims 1 to 3 , wherein the first probe and the second probe have different sequences and wherein the second probe has a tag sequence unique for each cell holding area.

5 . The system according to any one of claims 1 to 3 , wherein the first probe and the second probe are the same probe, and have a tag sequence unique for each cell holding area.

6 . The system according to any one of claims 1 to 5 , wherein the second probe further has a common sequence that functions as a primer for amplifying a nucleic acid, and/or a nucleic acid amplification correcting sequence.

7 . The system according to any one of claims 1 to 6 , wherein the reaction product is synthesized using a primer including a sequence complementary to a part of the single strand nucleic acid.

8 . The system according to claim 7 , wherein the primer further includes a common sequence that functions as a primer for amplifying a nucleic acid.

9 . The system according to any one of claims 1 to 8 , wherein the first probe and/or the second probe is immobilized on the same carrier or on different carriers held in the cell holding area.

10 . The system according to any one of claims 1 to 9 , wherein the first probe and/or the second probe is immobilized on the cell holding area or a carrier held in the area via a joint molecule.

11 . The system according to any one of claims 6 to 10 , wherein the nucleic acid amplifying means is for performing amplification by an enzyme reaction using the common sequence and a sequence complementary to a sequence of the tag sequence-introduced product.

12 . A method for analyzing a gene, comprising:
a step of holding a cell in a sample in an area capable of holding a cell; a step of capturing a single strand nucleic acid extracted from the cell by hybridization with a first probe; a step of synthesizing a nucleic acid having a sequence the same as or complementary to the single strand nucleic acid as a reaction product; a step of capturing the reaction product by hybridization with a second probe that is the same as or different from the first probe, has a sequence for capturing the reaction product, and has a tag sequence unique for the area; a tag sequence introducing step of synthesizing a tag sequence-introduced product that has a sequence that is the same as or complementary to a nucleic acid of the reaction product and has the tag sequence unique for the cell holding area, with the second probe coupled thereto; and a step of amplifying the tag sequence-introduced product.

13 . A kit for analyzing a gene, for use in the method according to claim 12 , comprising:
a gene analysis device, including a substrate that includes one cell holding area or a plurality of cell holding areas, a first probe that is placed in the cell holding area and includes a sequence complementary to a single strand nucleic acid extracted from a cell, and a second probe that is placed in the cell holding area and has a sequence for capturing the reaction product; an enzyme and a reaction reagent to be used in the steps; and nucleotides for synthesizing a nucleic acid.

14 . The kit for analyzing a gene according to claim 13 , comprising:
a gene analysis device, including a substrate that includes one cell holding area or a plurality of cell holding areas, a first probe that is placed in the cell holding area and includes a sequence complementary to a single strand nucleic acid extracted from a cell, and a second probe that is placed in the cell holding area and has a sequence for capturing the reaction product; an enzyme and a reaction reagent to be used in the steps; and nucleotides for synthesizing a nucleic acid, wherein the kit includes a primer that can hybridize with a part of the single strand nucleic acid to synthesize a reaction product.

15 . The kit according to claim 13 or 14 , wherein the enzyme includes an enzyme for specifically adding a nucleic acid to an end of a reaction product.

Assignees

• Hitachi Ltd

Inventors

• Tanabe Maiko
• Shirai Masataka
• Sakai Tomoyuki

Classifications

• C12N15/1096

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

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q1/6886 Primary

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

• C12Q1/68

  involving nucleic acids · CPC title