Genetic analysis system

Patent metadata

Field	Value
Publication number	US-9862993-B2
Application number	US-201315028907-A
Country	US
Kind code	B2
Filing date	Oct 21, 2013
Priority date	Oct 21, 2013
Publication date	Jan 9, 2018
Grant date	Jan 9, 2018

Abstract

In order to decode arbitrary sequence regions for a large number of genes in a large number of cells, it is necessary to fragment the nucleic acids and introduced a sequence, which differs for each the cell, in the respective fragments. However, in conventional constructions for analyzing large numbers of cells, there was the problem that the cleaved fragments of different regions were intermingled before a tag sequence unique to each region could be introduced. The present invention is constructed to also comprise a genetic analysis system, when trapping nucleic acids extracted from a cell in multiple regions on a substrate and synthesizing and fragmenting the complementary DNA strands (cDNA) of the nucleic acids for each individual region, for immediately introducing a tag sequence unique to each of the regions into said fragments.

First claim

The invention claimed is:

1. A genetic analysis system comprising:
a substrate comprising one or more cell retention region(s) each capable of retaining a single-cell;
a first probe comprising a capturing sequence that comprises a sequence complementary to a portion of the nucleotide sequence of a single-strand nucleic acid extracted from the cell retained in the cell retention region and traps the extracted single-strand nucleic acid, and a tag sequence specific to each cell retention region, wherein the first probe is located in the cell retention region; and
a second probe comprising a cleaved fragment-complementary sequence that comprises a sequence complementary to a portion of the nucleotide sequence of a cleaved fragment resulting from the cleavage of a complementary strand synthesized by using the single-strand nucleic acid trapped by the first probe as a template, and forms base pairing with the cleaved fragment, and the tag sequence specific to each cell retention region, wherein the second DNA probe is located in the cell retention region.

2. The genetic analysis system according to claim 1 , wherein the first probe and the second probe each further comprise at least one of a common sequence and a nucleic acid amplification correction sequence.

3. The genetic analysis system according to claim 1 , wherein the cleaved fragment resulting from the cleavage of a complementary strand synthesized by using the single-strand nucleic acid as a template with a restriction enzyme.

4. The genetic analysis system according to claim 3 , wherein the cleaved fragment-complementary sequence comprises a sequence complementary to a cleaved end after the cleavage with the restriction enzyme.

5. The genetic analysis system according to claim 1 , wherein
the second probe further comprises a stem sense strand consisting of an arbitrary nucleotide sequence, and a stem antisense strand consisting of a nucleotide sequence complementary to the stem sense strand, wherein
any one of the stem sense strand and the stem antisense strand is located at the 3′ end of the second probe, and the other strand is located adjacent to the 3′-terminal side of the cleaved fragment-complementary sequence positioned at the 5′ end of the second probe, and both the strands are hybridized with each other within the second probe to form a stem structure.

6. The genetic analysis system according to claim 1 , wherein at least one of the first probe and the second probe is immobilized on a carrier retained on the surface of the cell retention region.

7. The genetic analysis system according to claim 6 , wherein at least one of the first probe and the second probe is immobilized on the carrier via a joint molecule.

8. The genetic analysis system according to claim 6 , wherein the first probe and the second probe are immobilized on the same carrier or different carriers.

9. The genetic analysis system according to claim 6 , wherein the 5′-terminal portion of at least one of the first probe and the second probe is immobilized on the carrier.

10. The genetic analysis system according to claim 6 , wherein a site other than the terminal portion of at least one of the first probe and the second probe is immobilized on the carrier.

11. The genetic analysis system according to claim 1 , wherein at least one or more of an existing density of the first probe and an existing density of the second probe per cell retention region is 5 pM or larger.

12. The genetic analysis system according to claim 1 , wherein the location of the second probe in the cell retention region is dissociable depending on change in environment.

13. The genetic analysis system according to claim 12 , wherein the environment is temperature or light.

14. A genetic analysis method comprising:
a first step of supplying multiple cells onto a substrate of a genetic analysis system according to claim 1 so that the cells are retained one by one in respective cell retention regions;
a second step of extracting a nucleic acid from each cell retained in the cell retention region in the first step, and capturing the single-strand nucleic acid by a first probe in the cell retention region;
a third step of using the first probe as a primer and the single-strand nucleic acid trapped in the second step as a template to synthesize a complementary strand thereof;
a fourth step of fragmenting the complementary strand synthesized in the third step, and capturing the cleaved fragment in the same cell retention region thereas; and
a fifth step of introducing a tag sequence to the trapped cleaved fragment on a cell retention region basis.

15. The genetic analysis method according to claim 14 , wherein the trapping of the cleaved fragment in the cell retention region in the fourth step is carried out via hybridization to the cleaved fragment-complementary sequence of the second probe.

Assignees

• Hitachi Ltd

Inventors

• Tanabe Maiko
• Kambara Hideki
• Shirai Masataka

Classifications

• C12Q2531/113

  PCR · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title

• B01L3/5085

  for multiple samples, e.g. microtitration plates · CPC title

• C12Q2545/114

  involving a quantitation step · CPC title

• C12Q2565/601

  being a microscope, e.g. atomic force microscopy [AFM] · CPC title