METHOD FOR INTEGRALLY DETECTING NONDESTRUCTIVE MEASUREMENT INFORMATION AND GENOME-RELATED INFORMATION OF ONE CELL

• US 20200131562A1
• Filed: 05/02/2018
• Published: 04/30/2020
• Est. Priority Date: 05/02/2017
• Status: Active Grant

• Current Assignee: University of Tokyo
• Original Assignee: University of Tokyo
• Inventors: YACHIE, Nozomu, OTA, Sadao
• Granted Patent: US 11,584,951 B2
• Time in Patent Office: Days
• CPC Class Codes: C12M 1/00 Apparatus for enzymology or microbiology
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

The present invention provides a method for integrally detecting nondestructive measurement information and genome-related information of single cells.

More specifically, the present invention uses a method including:

• preparing a plurality of compartments containing single cell or a derivative thereof, a first bead(s), and a second bead(s) per compartment;
• detecting both nondestructive measurement information of single cell and imaging information of the first bead(s) and associating the nondestructive measurement information of single cell with the imaging information of the first bead(s) before preparation of each compartment or in each compartment;
• obtaining a hybridized complex;
• producing an amplified product derived from the hybridized complex; and
• integrally detecting nondestructive measurement information and genome-related information in single cell.

Claims

1. A method for integrally detecting nondestructive measurement information and genome-related information of single cells, the method comprising:

preparing a plurality of compartments containing a single cell or a derivative thereof, a first bead(s), and a second bead(s) per compartment,

wherein each first bead is

a particle cleavably linked to a first barcode nucleic acid corresponding to each imaging information or

an organism containing a first barcode nucleic acid corresponding to each imaging information, and

imaging information of the first bead(s) in each compartment can be clearly distinguished from each other, and

the second bead(s) is linked to a plurality of second barcode nucleic acids hybridizable with a genome-related nucleic acid corresponding to a cell genome or an expressed product thereof or the first barcode nucleic acid;

detecting both nondestructive measurement information of the single cell and imaging information of the first bead(s) and associating the nondestructive measurement information of the single cell with the imaging information of the first bead(s) before preparation of each compartment or in each compartment;

cleaving the first barcode nucleic acid from the associated first bead(s), and hybridizing each of the genome-related nucleic acid and the first barcode nucleic acid with the second barcode nucleic acid to obtain a hybridized complex;

producing an amplified product derived from the hybridized complex; and

integrally detecting nondestructive measurement information and genome-related information in the single cell using an expression pattern of the amplified product as an index.

2. The method according to claim 1, wherein

nondestructive measurement information of the single cell is detected using an expression pattern of a first amplified product derived from a hybridized complex of the first barcode nucleic acid with the second barcode nucleic acid as an index, and

genome-related information of the single cell is detected using an expression pattern of a second amplified product derived from a hybridized complex of the genome-related nucleic acid with the second barcode nucleic acid as an index.

3. The method according to claim 1, which is characterized by at least any one of (a) to (c):

(a) the number of the first beads per compartment is plural,

(b) the number of the second beads per compartment is one,

(c) the compartment is in a form of a well, a droplet, or a gel particle.

4. The method according to claim 1, wherein the genome-related nucleic acid is a genome DNA of the single cell, an RNA derived from a genome of the single cell or a cDNA thereof, or a nucleic acid probe specific to a protein expressed in the single cell.

5. The method according to claim 1, wherein the nondestructive measurement information is based on at least one piece of measurement information selected from color, fluorescence, size, shape, electromagnetic wave, transmission, phase, scattering, reflection, coherent Raman, Raman, and absorption spectrum.

6. The method according to claim 1, wherein each first barcode nucleic acid in the first bead(s) contains a first common barcode region which is common in the first bead(s) corresponding to same imaging information and a first hybridize region hybridizable with the second barcode nucleic acid.

7. The method according to claim 6, wherein sequence information of the first common barcode region becomes an index for identifying nondestructive measurement information of the single cell.

8. The method according to claim 1, wherein each of the plurality of second barcode nucleic acids linked to the second bead(s) comprises a second common barcode region which is in common with each other, a second unique barcode region which can be clearly distinguished from each other, and a second hybridize region hybridizable with the genome-related nucleic acid or the first barcode nucleic acid.

9. The method according to claim 8, wherein sequence information of the second common barcode region becomes an index for identifying the single cell or a derivative thereof existing in the compartment.

10. The method according to claim 8, wherein sequence information of the second unique barcode region becomes an index for identifying the genome-related nucleic acid.

11. The method according to claim 1, wherein the second barcode nucleic acid further comprises a PCR primer region.

12. The method according to claim 8, wherein the second hybridize region comprises the first hybridize region or a nucleic acid complementary to the genome-related nucleic acid.

18. A method for classifying a test cell, based on nondestructive measurement information in the test cell, using a classification model obtained based on the nondestructive measurement information and the genome-related information integrally detected by the method according to claim 1.

19. A method for obtaining nondestructive measurement information and genome-related information of single cells for a test substance, using the method for integrally detecting nondestructive measurement information and genome-related information of single cells according to claim 1, the method comprising:

including coexistence with a single cell or a derivative thereof, the first bead(s), and the second bead(s).

20. A method for screening a test substance, using the nondestructive measurement information and the genome-related information of the single cell for the test substance obtained by the method according to claim 19.

13. A system for integrally detecting nondestructive measurement information and genome-related information of single cells, the system comprising:

a compartment-preparing portion which prepares a plurality of compartments containing a single cell or a derivative thereof, a first bead(s), and a second bead(s) per compartment,

wherein each first bead is

a particle cleavably linked to a first barcode nucleic acid corresponding to each imaging information or

an organism containing a first barcode nucleic acid corresponding to each imaging information, and

imaging information of the first bead(s) in each compartment can be clearly distinguished from each other, and

the second bead(s) is linked to a plurality of second barcode nucleic acids hybridizable with a genome-related nucleic acid corresponding to a cell genome or an expressed product thereof or the first barcode nucleic acid;

a nondestructive measurement information- and imaging information-measuring portion which measures both nondestructive measurement information of the single cell and imaging information of the first bead(s) and associates the nondestructive measurement information of the single cell with the imaging information of the first bead(s) before preparation of each compartment or in each compartment;

a hybridized complex-forming portion which cleaves a first barcode nucleic acid corresponding to each imaging information from the associated first bead(s), and hybridizes each of the genome-related nucleic acid and the first barcode nucleic acid with the second barcode nucleic acid to obtain a hybridized complex;

an amplified product-producing portion which produces an amplified product derived from the hybridized complex; and

a nondestructive measurement information- and genome-related information-detecting portion which integrally detects nondestructive measurement information and genome-related information in the single cell using an expression pattern of the amplified product as an index.

14. The system according to claim 13, wherein the nondestructive measurement information- and imaging information-measuring portion comprises at least one selected from a microscope and a flow cytometer.

15. A combination of a first bead(s) and a second bead(s) for integrally detecting nondestructive measurement information and genome-related information of single cells, wherein

the first bead(s) is

a particle(s) cleavably linked to a first barcode nucleic acid corresponding to each imaging information or

an organism(s) containing a first barcode nucleic acid corresponding to each imaging information,

the second bead(s) is linked to a plurality of second barcode nucleic acids hybridizable with a genome-related nucleic acid corresponding to a cell genome or an expressed product thereof or the first barcode nucleic acid,

nondestructive measurement information of a single cell can be detected using an expression pattern of a first amplified product derived from a hybridized complex of the first barcode nucleic acid with the second barcode nucleic acid as an index, and genome-related information of the single cell can be detected using an expression pattern of a second amplified product derived from a hybridized complex of the genome-related nucleic acid with the second barcode nucleic acid as an index.

16. A detecting agent comprising a first bead(s), which is used together with a second bead(s) for integrally detecting nondestructive measurement information and genome-related information of single cells, wherein

the first bead(s) is

a particle(s) cleavably linked to a first barcode nucleic acid corresponding to each imaging information or

an organism(s) containing a first barcode nucleic acid corresponding to each imaging information,

the second bead(s) is linked to a plurality of second barcode nucleic acids hybridizable with a genome-related nucleic acid corresponding to a cell genome or an expressed product thereof or the first barcode nucleic acid,

nondestructive measurement information of a single cell can be detected using an expression pattern of a first amplified product derived from a hybridized complex of the first barcode nucleic acid with the second barcode nucleic acid as an index, and genome-related information of the single cell can be detected using an expression pattern of a second amplified product derived from a hybridized complex of the genome-related nucleic acid with the second barcode nucleic acid as an index.

17. A detecting agent comprising a second bead(s), which is used together with a first bead(s) for integrally detecting nondestructive measurement information and genome-related information of single cells, wherein

the first bead(s) is

a particle(s) cleavably linked to a first barcode nucleic acid corresponding to each imaging information or

an organism(s) containing a first barcode nucleic acid corresponding to each imaging information,

the second bead(s) is linked to a plurality of second barcode nucleic acids hybridizable with a genome-related nucleic acid corresponding to a cell genome or an expressed product thereof or the first barcode nucleic acid,

nondestructive measurement information of a single cell can be detected using an expression pattern of a first amplified product derived from a hybridized complex of the first barcode nucleic acid with the second barcode nucleic acid as an index, and genome-related information of a single cell can be detected using an expression pattern of a second amplified product derived from a hybridized complex of the genome-related nucleic acid with the second barcode nucleic acid as an index.