Tag-sequence-attached two-dimensional cDNA library device, and gene expression analysis method and gene expression analysis apparatus each utilizing same

Patent metadata

Field	Value
Publication number	US-11680286-B2
Application number	US-202117323311-A
Country	US
Kind code	B2
Filing date	May 18, 2021
Priority date	Jul 30, 2012
Publication date	Jun 20, 2023
Grant date	Jun 20, 2023

Abstract

The present invention relates to a method, a device, and an apparatus for analyzing the expression of a gene in single cells. Specifically, the present invention relates to: a device for gene expression analysis, characterized by including a support, in which a nucleic acid probe having a test nucleic acid capture sequence and a known sequence, and further containing a cell recognition tag sequence which differs depending on the difference in position on the surface of the support or in the vicinity of the surface thereof, and a common primer sequence having a known sequence is two-dimensionally distributed and immobilized on the surface of the support or in the vicinity of the surface thereof; and a method and an apparatus using the device for gene expression analysis.

First claim

The invention claimed is:

1. A gene expression analysis method, comprising:
a step of hybridizing a test nucleic acid to serve as a target to a nucleic acid probe in a support
wherein the support is a substrate through which a large number of pores penetrate,
wherein the interval between the pores is the size corresponding to a single cell or smaller than the size of a single cell,
wherein the nucleic acid probe is immobilized on an inner surface of the pores which penetrate the support, and wherein the nucleic acid probe comprises:
(a) a test nucleic acid capture sequence at its 3′ terminal side, wherein the test nucleic acid capture sequence is capable of hybridizing the test nucleic acid;
(b) a molecule recognition tag sequence, wherein the molecule recognition tag sequence has a known or unknown sequence of 5 to 30 bases to recognize the same test nucleic acid from a cell captured by the test nucleic acid capture sequence by collecting the same molecule recognition tag sequence in the sequencing data for the amplified product from the test nucleic acid captured by the test nucleic acid capture sequence;
(c) a cell recognition tag sequence, wherein the cell recognition tag sequence has a known sequence of 5 to 30 bases and differs depending on the difference in position in the pores whose planar location on the support is known in relation to the sequence of the cell recognition tag sequence; and
(d) a common primer sequence for amplification;
a step of synthesizing a complementary DNA strand to the test nucleic acid, thereby preparing a cDNA library constituted by the DNA complementary strand containing the tag sequence; and
a step of performing nucleic acid amplification of the whole or part of the cDNA library.

2. The method according to claim 1 , wherein the test nucleic acid to serve as a target is mRNA in individual cells constituting a biological tissue, and the cell recognition tag sequence is made to include a sequence which differs per region smaller than the size of the cell, whereby the cDNA library is prepared while keeping the in-plane positional information of the cell constituting the biological tissue.

3. The method according to claim 2 , wherein the biological tissue is a tissue section, and mRNA in individual cells constituting the tissue section is transferred to the support in which a nucleic acid probe containing a cell recognition tag sequence which differs per region is immobilized.

4. The method according to claim 1 , wherein the test nucleic acid to serve as a target is mRNA in multiple cells arranged in an array retained two-dimensionally, and the cell recognition tag sequence is made to include a sequence which differs per cell, whereby the cDNA library is prepared while keeping the positional information of the multiple cells.

5. The method according to claim 1 , wherein the nucleic acid probe further contains a transcription factor promoter sequence, and the nucleic acid amplification step includes a transcription reaction from a cDNA to a cRNA using the transcription factor.

6. The method according to claim 1 , further comprising a step of sequencing the amplified product.

Assignees

• Hitachi Ltd

Inventors

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

Classifications

• C12Q2565/601

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

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q2565/537

  characterised by the capture oligonucleotide acting as a primer · CPC title

• C12N15/1096

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

• C12Q2525/155

  incorporating/generating a new priming site · CPC title