WO2020218551A1 - ゲルカプセル方式による１細胞ゲノムライブラリーからの配列スクリーニング法 - Google Patents

ゲルカプセル方式による１細胞ゲノムライブラリーからの配列スクリーニング法 Download PDF

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WO2020218551A1
WO2020218551A1 PCT/JP2020/017790 JP2020017790W WO2020218551A1 WO 2020218551 A1 WO2020218551 A1 WO 2020218551A1 JP 2020017790 W JP2020017790 W JP 2020017790W WO 2020218551 A1 WO2020218551 A1 WO 2020218551A1
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cell
cells
capsule
amplification
drug
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English (en)
French (fr)
Japanese (ja)
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正人 細川
春子 竹山
西川　洋平
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ｂｉｔＢｉｏｍｅ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present disclosure relates to the application of individual analysis of single cell or cell-like structures using the encapsulation method.
  • Genome sequence information is basic information for understanding living organisms, and genome decoding enables understanding of the diversity and function of microorganisms related to lineage, evolution, diseases, and biogeochemical cycle. For this reason, whole-genome analysis is indispensable for understanding the function of refractory culture.
  • metagenomic analysis in which various microbial genomes are collectively extracted from a sample and sequenced, has been the mainstream.
  • each cell is first divided and then processing is started. Since only the genetic information derived from a single cell is directly acquired, for example, the calculation difficulty for reconstructing the genomic information is overwhelmingly easier than the metagenomic analysis. However, highly accurate cell manipulation and nucleic acid reaction are required to decipher the extremely small amount of intracellular genome sequence without omission.
  • the steps of 1-cell genome analysis can be roughly divided into (1) separation of 1 cell of microorganism, (2) lysis of microorganism, (3) whole genome amplification, and (4) sequence analysis of amplified genome.
  • the reaction system of several tens of microliters used in the conventional molecular biology experiment is inappropriate for precisely handling a very small sample of one cell according to the above flow.
  • the genome decoding rate remained at about 30%. Since it is difficult to completely prevent contamination from aerosols and the like in an open experimental environment, a clean experimental environment dedicated to single-cell genome analysis experiments is required. Another problem is that the throughput of the reaction system is extremely low with respect to the need to execute the sequence from each cell, and expensive dispensing robots and the like have been used for experimental operations.
  • nucleic acid amplification and detection can be performed as it is, and have completed the present disclosure.
  • Examples of embodiments of the present disclosure include: (Item 1) (A) A step of encapsulating two or more cells or cell-like structures together with a first agent for each cell or cell-like structure.
  • (B) A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each cell or cell-like structure or a substance derived thereto together with a second agent.
  • (D) A step of carrying out a reaction based on the second drug on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived thereto, or a capsule containing them, is encapsulated for each cell or cell-like structure or a substance derived thereto together with a drug X.
  • X is an integer of 3 or more.
  • (B) A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each cell or cell-like structure or a substance derived thereto together with a second agent.
  • (D) A step of carrying out a reaction based on the second drug on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived thereto, or a capsule containing them, is encapsulated for each cell or cell-like structure or a substance derived thereto together with a drug X.
  • X is an integer of 3 or more.
  • (D') A method for analyzing a cell or cell-like structure comprising the step of analyzing the cell or cell-like structure with the desired agent.
  • (Item 3) The method according to any one of the above items, wherein (E) to (G) do not exist, and the first drug and the second drug are drugs required for nucleic acid amplification.
  • (Item 4) The method according to any one of the above items, wherein the desired drug is a drug required for nucleic acid amplification.
  • the single cell or single cell-like structure is encapsulated by flowing the suspension of the single cell or single cell-like structure into a microchannel and shearing the suspension with oil.
  • the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
  • the solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • the first amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the second amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the one cell or cell-like structure and the first label are encapsulated in the droplet.
  • (Item 23) The method according to any one of the above items, wherein the nucleic acid contained in the cell or cell-like structure in A) is genomic DNA.
  • (Item 24) The method according to any one of the above items, which comprises amplifying a species-specific sequence or a sequence stored in a specific species in the analysis of C).
  • (Item 25) A system for analyzing cells or cell-like structures [X] A drug for encapsulating two or more cells or cell-like structures or a substance derived from them, or a capsule containing them, and / or an encapsulating drug storage unit for storing them.
  • [Y] A desired drug used for analysis and / or an analytical drug storage unit for storing them, A system comprising [Z] means for encapsulation and [W] means for performing analysis with the desired agent, if desired.
  • the system of item 25 further comprising the features of any one or more of the above items.
  • (Item 26) A kit for providing capsules for analyzing cells or cell-like structures.
  • [X] An agent for encapsulating two or more cells or cell-like structures or substances derived from them, or capsules containing them.
  • [Y] With the desired drug used for analysis A kit that includes.
  • the kit of item 26 further comprising the features of any one or more of the above items.
  • a method of analyzing cells or cell-like structures (A) A step of encapsulating two or more cells or cell-like structures together with a drug for nucleic acid amplification for each cell or cell-like structure. (B) A step of performing the first nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and (C) A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each of the cell or cell-like structure or a substance derived thereto together with a drug for nucleic acid amplification.
  • (D) A step of performing a second nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived from the cell or cell-like structure obtained in D), or a capsule containing them, is prepared for each cell or cell-like structure or a substance derived from the cell or cell-like structure.
  • nucleic acid amplification, detection, selection, etc. can be performed while holding individual single biological units (for example, "single cell") as a set, and when it is desired to acquire rare bacterial data, host DNA When a large amount of such substances are mixed, data on only specific bacteria is required, and it can be applied when evaluation of diversity is unnecessary.
  • single biological units for example, "single cell”
  • the probability of catching all rare microbial species is high.
  • the present disclosure can also identify microbial species in advance and select samples for transition to analysis such as whole genome sequences.
  • the present disclosure can also selectively select or eliminate certain microorganisms to perform effective genomic or other analyses by preliminarily examining the presence or absence of specific specific genes.
  • FIG. 1 is a diagram showing a step of encapsulating a single cell or cell-like structure in a droplet and performing primary amplification. After the primary amplification, the nucleic acid in the gel capsule is stained using a fluorescent DNA intercalator such as DAPI.
  • FIG. 2 is a diagram showing a step of reencapsulating a gel capsule in a droplet.
  • the PCR primer and the PCR reaction solution to which the fluorescently labeled probe is added are also re-encapsulated.
  • the PCR reaction solution is an example, and other than this, a LAMP reaction, a SmartAmp method, and an RPA (recombinase polymerase) amplification method are also possible.
  • FIG. 3 is a diagram showing PCR amplification (or other technique) and gene detection.
  • a label such as Taqman primer
  • Gene detection can also be performed by using a DNA-binding fluorescent intercalator and a primer for specific gene detection in combination.
  • FIG. 4 is a diagram showing the stages of secondary amplification. The droplets to be subjected to the secondary amplification can be selected based on the results of staining and gene detection after the primary amplification. Sorting is also possible with flow cytometry, fluorescence microscopy, and microfluidic devices.
  • FIG. 5 is a diagram showing the steps of confirmation by PCR and library preparation.
  • FIG. 6 is a schematic diagram showing the overall flow of the sequence screening method from the 1-cell genome library by the gel capsule method.
  • FIG. 7 is a diagram showing a gel capsule at the time of gene detection.
  • FIG. 7A shows staining of gel capsules with DAPI and
  • FIG. 7B shows staining of gel capsules with FAM.
  • Two types of fluorescence are shown: droplets that do not show fluorescence, droplets that show only DAPI-derived fluorescence (blue) bound to DNA amplified by MDA, and DAPI-derived fluorescence and probe-derived fluorescence (FAM: green). Three types of droplets can be confirmed.
  • an encapsulation technique is used to perform another or the same reaction on a set that has been reacted with respect to a set that is individually separated and generated. And regarding the technology that enables efficient screening. Specifically, it can be applied to gene sequence analysis and can be specifically detected and selected from amplified polynucleotides prepared in parallel from various microorganisms by referring to the gene sequence.
  • cell refers to a particle that contains a molecule that carries the genetic information and is any particle that can be replicated (whether or not it is possible alone).
  • the term “cell” as used herein includes cells of unicellular organisms, bacteria, cells derived from multicellular organisms, fungi and the like.
  • cell-like structure refers to any particle containing a molecule having genetic information.
  • cell-like structures include organelles such as mitochondria, cell nuclei, and chloroplasts, and viruses.
  • gel refers to a colloidal solution (sol) in which a polymer substance or colloidal particles interact with each other to form a network structure as a whole and contain a large amount of a liquid phase as a solvent or a dispersion medium. A state in which fluidity is lost.
  • gelling means changing a solution into a “gel” state.
  • capsule refers to a capsule-like substance capable of holding cells or cell-like structures therein, and when it is made of gel, it is referred to as a gel-like fine particle. It is a shaped structure.
  • encapsulation refers to the process of encapsulating cells or cell-like structures in a capsule.
  • gel encapsulation refers to a process of encapsulating cells or cell-like structures in a gel capsule.
  • the "gel capsule” refers to a gel-like fine particle structure capable of holding a cell or a cell-like structure therein.
  • gene analysis means examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample.
  • the gene analysis can include those that utilize a nucleic acid amplification reaction.
  • Examples of gene analysis including these include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restriction enzyme fragment length polymorphism, repeat number polymorphism), expression analysis, fluorescence quenching probe ( Quenching Probe: Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR and the like can be mentioned.
  • single cell level refers to genetic information contained in one cell or cell-like structure or information on other biomolecules, as opposed to genetic information contained in another cell or cell-like structure. Or, it means that the processing is performed in a state of being distinguished from the information of other biomolecules. For example, when amplifying a polynucleotide at the "single biological unit level” or “single cell level”, the polynucleotide in one single biological unit or cell or cell-like structure, and another single organism, respectively. Each amplification takes place with the polynucleotides in the unit, or other cell or cell-like structure, distinguishable.
  • single cell analysis refers to genetic information or other biomolecule information contained in one cell or cell-like structure, and genetic information or other information contained in another cell or cell-like structure. It refers to analysis in a state that is distinguished from the information on biomolecules.
  • genetic information refers to information on a nucleic acid encoding a gene or other information contained in one cell or cell-like structure, and refers to the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid. Including yield.
  • nucleic acid information refers to information on nucleic acids contained in one cell or cell-like structure, and includes the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid yield.
  • identity refers to sequence similarity between two nucleic acid molecules. Identity can be determined by comparing positions in each sequence that can be aligned for comparison.
  • the disclosure comprises (A) encapsulating two or more cells or cell-like structures, together with a first agent, for each cell or cell-like structure, and (B). A step of performing a reaction based on the first drug on the cell or cell-like structure in a capsule, and (C) the cell or cell-like structure or a substance derived from the substance, or a capsule containing them, the cell. Alternatively, a step of encapsulating each cell-like structure or a substance derived thereto together with a second drug, and (D) a step of performing a reaction based on the second drug on the cell or cell-like structure in the capsule.
  • X is an integer of 3 or more
  • F if necessary, a reaction based on the Xth drug is carried out on the cell or cell-like structure in a capsule.
  • a method for analyzing a cell or a cell-like structure including the steps to be performed.
  • X is an integer of 3 or more, and an arbitrary integer is selected.
  • (E) to (G) are optional steps, and if they do not exist, the embodiment is encapsulated twice. It is very difficult to analyze a large number of samples simply by encapsulating two or more cells or cell-like structures with a first drug and performing a reaction based on the first drug on the cells or cell-like structures. Although it may be difficult, the cells or cell-like structures that have undergone a reaction based on the first drug, or substances derived from the cells, or capsules containing them, are encapsulated together with the second drug, and the second drug is used.
  • the reaction of one drug and the second drug is reacted with different types of drugs, the first drug is used for primary screening, which is then used with the second and / or third to X drugs. Further (secondary or higher) screening can be performed.
  • the disclosure provides an aspect that applies once a single cell or cell-like structure has been prepared or processed.
  • (C') two or more cells or cell-like structures or substances derived thereto, or capsules containing them, are desired agents for each cell or cell-like structure or substances derived thereto.
  • a method for analyzing a cell or cell-like structure which comprises a step of encapsulating with and (D') analyzing the cell or cell-like structure using the desired agent.
  • the method of the present disclosure it is possible to select a cell or cell-like structure containing and / or not containing the gene of interest from a large amount of samples and subject it to analysis. For example, if you want to obtain only data on rare bacteria, if a large amount of host DNA is mixed and the proportion of target cells or cell-like structures is low, data on only specific bacteria is required, and evaluation of diversity is required. This disclosure is useful when is not required.
  • each capsule when the capsules are further encapsulated, each capsule may be made of a different material or the same material.
  • the capsule of step (A) may be a gel capsule.
  • the agent may be an agent required for nucleic acid amplification.
  • the drug may be a nucleic acid probe, or a drug that identifies a protein such as an antibody or other antigen.
  • the plurality of (first to X) agents may be independently the same or different.
  • it may contain a specific nucleic acid degrading agent, a modifier, a label, various enzymes, and the like.
  • the analysis in the step (H) is to analyze the capsules, and it may or may not be analyzed for each encapsulation. If it is performed in the middle, it can perform a function such as screening, and if it is finally performed, an analysis that also serves as an evaluation can be performed, but the present invention is not limited to these.
  • the analysis of step (H) may or may not include nucleic acid amplification.
  • the analysis of step (H) may analyze the sequencing of the nucleic acids contained and / or amplified, the yield of total nucleic acids, the presence or absence of specific sequences, the yield of nucleic acids of specific sequences. ..
  • the analysis of step (D) may be an analysis targeting proteins, sugar chains, lipids and the like.
  • a next-generation sequencer (NGS) or the like can be used for sequencing, and a whole genome sequence may be obtained.
  • the desired drug may be a drug required for nucleic acid amplification.
  • the desired agent may be an agent for detecting a particular molecule.
  • agents for detecting a particular molecule include probes, antibodies, intercalators, tags, radioactive substances, fluorescent dye-binding nucleotides, fluorescently labeled proteins and the like.
  • the disclosure is a method of analyzing a cell or cell-like structure.
  • B A step of performing the first nucleic acid amplification reaction on the cell or cell-like structure in a capsule
  • C A step of encapsulating the cell or cell-like structure or a substance derived thereto, or a capsule containing them, for each of the cell or cell-like structure or a substance derived thereto together with a drug for nucleic acid amplification.
  • (D) A step of performing a second nucleic acid amplification reaction on the cell or cell-like structure in a capsule, and (E) If necessary, the cell or cell-like structure or a substance derived from the cell or cell-like structure obtained in D), or a capsule containing them, is prepared for each cell or cell-like structure or a substance derived from the cell or cell-like structure.
  • the disclosure A) selects two or more cells or cell-like structures by a single cell or structure unit and includes the cells or cell-like structures.
  • the step A) is also referred to as primary amplification and can be performed by the method described below.
  • the present disclosure provides a method of amplifying a polynucleotide in a cell.
  • This amplification method uses a sample containing two or more cells or cell-like structures (including, for example, viruses, organs (Mt, Nuc), etc.) and uses the cells or cell-like structures as one cell or structure.
  • a step of encapsulating the droplets unit by unit, a step of gelling the droplets to form gel capsules, and a step of immersing the gel capsules in one or more lysis reagents to lyse the cells or cell-like structures.
  • the amplification method of the present disclosure can individually amplify a genome at the so-called single cell level or a gene assembly similar thereto.
  • the amplification method of the present disclosure realizes individual genome amplification by a very simple method, and therefore, in units of 100 units, 1000 units, 10,000 units, 100,000 units, or more.
  • Genomic information about cells can be obtained at one time, and therefore can be used as a library.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • any embodiment detailed in the following (droplet generation) section or other sections can be adopted.
  • the cells or cell-like structures that can be targeted in the amplification methods of the present disclosure are any number of two or more, eg, 10 or more, 50 or more, 100 or more, 500 or more. , 1000 or more, 5000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1 million or more, 5 million or more, 10 million or more.
  • the amplification method of the present disclosure can target a larger number of cells than using conventional single cell reaction systems, such as 0.2 mL, 1.5 mL microtube reaction systems.
  • any cell or cell-like structure described in the section can be adopted.
  • cells can be targeted.
  • cell-like structures are targeted, among which viruses or organelles such as mitochondria and nuclei can be targeted.
  • the sample containing the provided cells or cell-like structures may be provided in any form.
  • select an appropriate medium including buffers, salts, nutrients, other components, etc.
  • any cell or cell-like structure selected from the section (cells and cell-like structures). can do.
  • any component may be used as long as it is a component suitable for droplet generation. It is preferable that the component is also suitable for gelation. Examples of such components include, but are not limited to, buffer solutions such as PBS, Tris-HCl, TE, and HEPES, as well as sterile water, seawater, artificial seawater, and various liquid media.
  • a detergent-free medium such as water or buffer may be preferred.
  • any embodiment described in the section can be adopted for encapsulation of cells or cell-like structures in droplets of cells or structure units one by one.
  • a microchannel is used to flow a suspension of cells or cell-like structures into the microchannel and shear the suspension to create individual cells or cell-like structures.
  • Encapsulated droplets can be produced, and in addition to the explanation in (Droplet preparation), those skilled in the art can appropriately prepare and carry out components and parameters with reference to the representative examples exemplified in the examples. it can.
  • any embodiment described in the following (gelation) section can be adopted as the step of gelling the droplet to form a gel capsule.
  • gelation cools a droplet made so that the material of the droplet or droplet (eg, a sample containing a cell or cell-like structure) contains the material of a gel capsule.
  • the material of the droplet or droplet eg, a sample containing a cell or cell-like structure
  • gelation can be gelled by giving a stimulus such as light.
  • any material described in the following (gelation) section can be used.
  • the step of lysing a cell or cell-like structure can be realized by immersing a gel capsule in one or more lysing reagents, and any embodiment described in the following (dissolution) item. Can be adopted.
  • the polynucleotide in the cell is eluted into the gel capsule and retained in the gel capsule in a state where the substance binding to the polynucleotide is removed. It is important to be processed in.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • the lysate remaining in the gel capsule and the lysis reagent after the dissolution operation are passed through the gel capsule using an appropriate washing solution, and the inhibitor is transferred to the outside of the gel capsule. It may be desirable to release to.
  • the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations, such as amplification reactions, can be performed smoothly.
  • the step of amplifying a polynucleotide in a gel capsule can be realized by contacting the polynucleotide with an amplification reagent, and any embodiment defined in the following (amplification) can be adopted. it can.
  • the present disclosure may include encapsulating cells or cell-like structures in droplets, one cell or structural unit at a time, using a sample containing two or more cells or cell-like structures. Further, in the present disclosure, the device may include a droplet making section in which cells or cell-like structures are encapsulated in droplets one by one or in units of structures.
  • Droplet production can be performed using, for example, a microchannel.
  • the droplet making section may include a microchannel.
  • droplets encapsulating each cell or cell-like structure can be made. Shearing can be done at regular intervals. Shearing of the suspension can be done with oil.
  • oil for example, mineral oil (for example, light mineral oil), vegetable oil, silicone oil, fluorinated oil, etc. can be used.
  • the diameter of the droplet may be from about 1 to 250 ⁇ m, more preferably from about 10 to 200 ⁇ m, for example, the diameter of the droplet is about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about. It may be 30 ⁇ m, about 40 ⁇ m, about 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • the present disclosure may include the step of gelling a droplet to form a gel capsule.
  • the device may include a gel capsule generating unit that gels droplets to form gel capsules.
  • Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet.
  • the inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.
  • the diameter of the gel capsule may be about 1-250 ⁇ m, more preferably about 10-200 ⁇ m, eg, about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about 30 ⁇ m, about 40 ⁇ m, about. It may be 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • the diameter of the gel capsule may be the same as that of the droplet to be produced, but the diameter may change during gelation.
  • the material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
  • a photocurable resin for example, PEG-DA
  • the gel capsule may be a hydrogel capsule.
  • hydrogel refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.
  • DNA When extracting DNA from a large number of cells at once, DNA can be purified by phenol / chloroform extraction and ethanol precipitation.
  • the amount of the genetic substance per cell is very small, and it is necessary to individually convert the genetic substance into a nucleic acid-only state without loss.
  • nucleic acid purification is attempted by a general bulk scale procedure, the result is that no nucleic acid can be extracted or only nucleic acid derived from impurities can be extracted.
  • Loss of contamination and target genetic material is a major problem in single-cell experiments, but by using a gel capsule containing a single cell or cell-like structure, purified genetic material (eg, DNA) can be placed in the gel capsule. It can be retained in the cell, and the possibility of contamination of molecules from the outside can be eliminated. In addition, a large amount of one cell can be processed in parallel with a very simple operation. The steps of centrifuging the test tube containing the gelled droplets, removing the supernatant and replacing it with a cleaning solution can be performed. Alternatively, the gelled droplets can be filtered through a filter to remove the supernatant, the cleaning solution is passed through the mixture, and finally the gel capsules are collected. By using a gel capsule, the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations, such as amplification reactions, can be performed smoothly.
  • purified genetic material eg, DNA
  • a composition comprising a gel capsule or a material thereof may be provided. From the points described above or below, such compositions may be useful for amplifying nucleic acids in cells at the single cell level. Also, such compositions can be useful for making genomic libraries. In a further embodiment, a composition comprising a gel capsule or a material thereof and cells in a single cell state can be provided. From the points described above or below, such compositions may be useful for amplifying nucleic acids in cells at the single cell level. Also, such compositions can be useful for making genomic libraries. Such compositions can be useful for sequencing intracellular nucleic acids at the single cell level.
  • the present disclosure may include immersing a gel capsule in one or more lysis reagents to lyse the cell or cell-like structure. Further, in the present disclosure, the device may include a dissolution reagent immersion portion for immersing the gel capsule in the dissolution reagent. Upon lysis, the polynucleotide in the cell may be eluted in the gel capsule and retained in the gel capsule with the substance binding to the polynucleotide removed. Dissolving reagents include, for example, enzymes, surfactants, other denaturing agents, reducing agents and pH regulators, and combinations thereof can also be used.
  • a composition comprising a lysing reagent for amplifying nucleic acids in cells at the single cell level may be provided.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • the lysis reagent may be selected from at least one group consisting of lysozyme, achromopeptidase, protease, sodium dodecyl sulfate and potassium hydroxide.
  • the only purpose is to detect the presence or absence of some sequences in a cell or cell-like structure, it is not always necessary to actively lyse the cell or cell-like structure, and physical stimulation or heat. It is also possible to perform detection based on the leakage of nucleic acid from cells or cell-like structures by stimulus. However, in order to obtain a large amount of information such as the entire genome from a single cell, it is preferable to actively destroy the cell or cell-like structure and isolate the genetic substance in the cell from the cell in a complete state. .. Further, when a gel capsule is used, thermal / mechanical irritation may lead to disintegration of the gel capsule, and it may be preferable to use a dissolving reagent.
  • a lytic reagent or a combination of lysing reagents that is strong to some extent.
  • Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.
  • a strong lysing reagent may inhibit a reaction such as DNA amplification, and is preferably sufficiently removed before the downstream reaction.
  • the gel capsule retains the genetic substance to be analyzed or amplified, so that the lytic reagent can be removed even in a single cell analysis in which the amount of the genetic substance is small. Therefore, it is possible to use a strong dissolving reagent or a combination of dissolving reagents. And the use of potent lysing reagents or combinations of lysing reagents allows for comprehensive nucleic acid amplification or genome analysis, regardless of the type of cell (including those with cell walls and other types of microorganisms). Can be.
  • the method may include removing the lysing reagent and / or contaminants from the gel capsule.
  • the dissolution reagent immersion portion includes means for removing the dissolution reagent and / or contaminants from the gel capsule.
  • the target molecule is part of a cell surface marker or nucleic acid and the goal is to detect its presence itself, the goal may be achieved even if the lysis operation is partial or undissolved. There is.
  • the genomic DNA when attempting to amplify the full length of genomic DNA, the genomic DNA usually has only one molecule in the cell, so that the cell or cell-like structure is completely dissolved and bound from the DNA. It is necessary to sufficiently remove proteins. As a result, even when a sample consisting of hundreds or more kinds of microorganisms such as intestinal microorganisms is targeted, all of them can be uniformly dissolved and whole genome amplification can be performed from all of them. It also makes it possible to prepare the library and finally obtain whole genome sequence information.
  • the present disclosure may include contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within a gel capsule.
  • the device may include an amplification reagent immersion portion for immersing the gel capsule in the amplification reagent.
  • the amplification reagent immersion portion may be provided with means for adjusting the temperature of the gel capsule, if necessary, after immersion in the amplification reagent.
  • Reactions involving heat treatment can lead to redissolution of gels (eg, agarose gels), which can disrupt the individualized shape and invalidate single cell isolation. is there.
  • an enzymatic reaction of about 60 degrees or less is desirable to maintain the gel droplet shape.
  • the constant temperature chain substitution amplification reaction is preferable in that it can be carried out within this temperature range and the entire genomic DNA can be amplified.
  • the enzyme used include phi29 polymerase, Bst polymerase, Aac polymerase, and recombinase polymerase.
  • a PCR primer for specifically detecting the organism and an oligonucleotide for detection such as Taqman probe.
  • the primer / probe can be prepared according to the oligonucleotide sequence design by general PCR, qPCR, or the like.
  • non-patent documents Li X, Zhang W, Wang C, Cui L, Yang CJ. Agarose droplet microfluidics for high-high lily parallell and effective Single 28 mulsion;
  • agarose By adding agarose to the PCR reaction solution as in 10.1039 / c0lc00145 g), it is possible to cool the droplets after PCR in the reaction step and gel-encapsulate the droplets again.
  • the gel capsule can be separated by using flow cytometry or the like.
  • the agarose skeleton (non-patent documents: Leng X, Zhang W, Wang C, Cui L, Yang CJ. ):. 2841-2843 doi:... 10.1039 / c0lc00145g) and beads (Novak R, Zeng Y, Shuga J, et al Single-cell multiplex gene detection and sequencing with microfluidically generated agarose emulsions Angew Chem Int Ed Engl 2011 It is also designed to be appropriately crosslinked with 50 (2): 390-395. Doi: 10.10012 / anie.201006089) so that the augmented by-product stays in the gel capsule. This makes it possible to select gel capsules using a fluorescent DNA-binding dye by retaining short-chain by-products in gel capsules.
  • RNA Ribonucleic acid
  • the purpose is to determine the type and expression level of a gene in an absolute (relative) manner. It is possible to quantify whether it is expressed only.
  • Treatment in gel capsules is advantageous for such amplification.
  • the cells or cell-like structures of interest in the present disclosure are not particularly limited, but are, for example, microorganisms (eg, bacteria, fungi, single cell animals), cells of multicellular organisms (eg, somatic cells, germ cells). , Cultured cells, tumor cells, animal cells, plant cells), intracellular organs (mitochonium, nucleus, chloroplast), viruses.
  • microorganisms eg, bacteria, fungi, single cell animals
  • cells of multicellular organisms eg, somatic cells, germ cells.
  • intracellular organs mitochonium, nucleus, chloroplast
  • RNA For cells of organisms with known genome sequences, which genes are expressed in them RNA In the case of analysis of an organism whose genome sequence and / or gene information is unknown, it is necessary to obtain information on the genome itself before RNA analysis. In that case, amplification of the genome sequence at the single cell level by the method of the present disclosure using gel capsules is advantageous.
  • a sample containing two or more cells or cell-like structures can be used.
  • the two or more cells may be derived from a plurality of organisms.
  • the sample include a microbial sample, a tissue sample, a mixed sample of a symbiotic microorganism and a host organism, and a sample containing microorganisms and cells taken from an animal / human sample.
  • the microbial sample include a bacterial flora sample, a sample containing two or less types of cells or cell-like structures, and a sample containing cells or cell structures other than bacteria such as fungi.
  • samples containing microorganisms and cells taken out from human samples include feces, saliva, sputum, surgical lavage fluid, blood, skin / body mucosa wipes and swabs, and can be used directly. , It may be used after performing an operation for separating cells and microorganisms.
  • the microorganisms that can be targeted are not limited to, but are not limited to, eubacteria, Escherichia coli, bacilli, indigo bacteria, cocci, bacilli, spiral bacteria, gram-negative bacteria, gram-positive bacteria, archaea, fungi, etc. Can be mentioned.
  • Bacteria that can be targeted by the present disclosure for example, Negibacteria, Eobacteria, Deinococci, Deinococci, Deinococcales, Thermales, Chloroflexi, Anaerolineae, Anaerolineales, Caldilineae, Chloroflexales, Herpetosiphonales, Thermomicrobia, Thermomicrobiales, Sphaerobacterales, Ktedonobacteria, Ktedonobacterales, Thermogemmatisporales, Glycobacteria, Cyanobacteria , Gloeobacterophyceae, Gloeobacterales, Nostocophyceae, Synechococcophycidae, Synechococcales, Nostocophycidae, Chroococcales, Oscillatoriales, Nostocales, Pseudanabaenales, Spirochaetes, Spirochaetes, Spirochaetales, Fibrobacteres
  • Encapsulation The process of individually encapsulating cells or cell-like structures containing nucleic acids, or substances derived from them, together with necessary drugs, is also simply referred to as encapsulation.
  • the object to be encapsulated may be one amplified by primary amplification or may be a purified product of another reaction.
  • the object of encapsulation may be an amplified nucleic acid, a gel capsule containing the amplified nucleic acid, a cell or a cell-like structure containing the amplified nucleic acid.
  • the object to be reencapsulated has the polynucleotide in a state where the substance binding to the polynucleotide has been removed, or if the polynucleotide is amplified while maintaining the gel state in the gel capsule, gelation is performed in the encapsulation. It does not have to be, and it does not have to contain a gelling material.
  • the diameter of the capsule may be about 1-250 ⁇ m, more preferably about 10-200 ⁇ m, for example, the diameter of the capsule is about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about 30 ⁇ m, It may be about 40 ⁇ m, about 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • the diameter of the capsule may increase as the number of encapsulations increases.
  • each capsule when encapsulating the capsules for the second and subsequent times, each capsule may be made of different materials, some may overlap, or all may be made of the same material. You may.
  • the agent required for analysis may be the agent required for nucleic acid amplification.
  • amplification of nucleic acid derived from a single cell or cell-like structure can be performed by any method, but simply one cell or cell-like structure from a large number of cells or cell-like structures.
  • a step of encapsulating cells or cell-like structures one by one in a droplet a step of gelling the droplet to generate a gel capsule, and a step of forming the gel capsule 1
  • a substance in which a polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted into the gel capsule and binds to the genomic DNA or the portion thereof.
  • One cell by method comprising the step of retaining the polynucleotide in the gel capsule in a removed state and the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule.
  • an amplification reagent to amplify the polynucleotide in the gel capsule.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • step A) steps of selecting two or more cells or cell-like structures by a single cell or structure unit and amplifying the nucleic acids contained in the cells or cell-like structures.
  • a sample containing two or more cells or cell-like structures is used to enclose one cell or component in a droplet, and b) the droplet is gelled to form a gel capsule.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • the droplet is a single cell or single cell by flowing a suspension of single cell or single cell-like structures into a microchannel and shearing the suspension with oil. It can be made by encapsulating a suspension of similar structures.
  • the gel capsule may be a hydrogel capsule.
  • the material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
  • Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet.
  • the inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.
  • the gel capsule may be a hydrogel capsule.
  • hydrogel refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.
  • Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • a lysing reagent or a combination of lysing reagents that is strong to some extent.
  • Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.
  • the agent required for analysis may be a second amplification reagent.
  • the number of agents required for analysis may be one or more.
  • the first amplification reagent and the second amplification reagent are identical.
  • the first amplification reagent and the second amplification reagent are different.
  • the amplification reagents used may all be the same or all different.
  • the amplification reagents used may be partially identical.
  • the first amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the second amplification reagent is a reagent for amplification of genomic DNA or a portion thereof or a reagent for amplification of a specific sequence.
  • the reagent for specific sequence amplification may be a reagent that amplifies a cell type-specific sequence or a species-specific sequence, or a reagent that amplifies a sequence stored in a specific species.
  • the reagent for specific sequence amplification may be a reagent that amplifies a marker gene.
  • one cell or cell-like structure and a first label may be encapsulated in the droplet.
  • one cell or cell-like structure and a second label may be encapsulated in step B).
  • one cell or cell-like structure and one or more labels may be encapsulated in a) and / or step B) above.
  • the first sign and the second sign are the same. In another embodiment, the first and second markers are different. In other embodiments, the labels used may all be the same or all may be different. In certain embodiments, the labels used may be only partially identical. In some embodiments, the label may indicate the yield of total nucleic acid. In other embodiments, the label may indicate the presence or absence of a particular sequence. In certain embodiments, the label may indicate the yield of nucleic acid of a particular sequence. In other embodiments, the label may be an agent for detecting a particular molecule. In certain embodiments, agents for detecting a particular molecule include probes, antibodies, intercalators, tags, radioactive substances, fluorescent dye-binding nucleotides, fluorescently labeled proteins and the like.
  • the nucleic acid contained in the cell or cell-like structure in step A) can be genomic DNA or a portion thereof.
  • step C) may include evaluating the label. In some embodiments, step C) may include evaluating a first label and / or a second label. In other embodiments, the yield of total nucleic acid, the presence or absence of a particular sequence, or the yield of a particular sequence of nucleic acid, as indicated by the label, may be assessed.
  • the encapsulated cells or cell-like structures may be selected. In one embodiment, selection may be based on the presence or absence of a particular gene sequence, the yield of a particular gene or the total nucleic acid yield. In some embodiments, it may be selected when there is a specific gene sequence, or it may be selected when there is no specific gene sequence. In some embodiments, it may be selected if the yield of the particular gene is greater than or equal to the baseline yield. In some embodiments, it may be selected if the total nucleic acid yield is greater than or equal to the reference yield.
  • reagents that specifically detect the presence or absence of a particular gene sequence include antibodies, probes, DNA-binding fluorescent dyes, fluorescent dye-binding nucleotides.
  • the yield of a specific gene or the total nucleic acid yield can be measured by absorbance measurement, fluorescence measurement, agarose gel electrophoresis, or microchip electrophoresis.
  • the method may include detecting nucleic acid having a particular sequence in a sample containing amplified nucleic acid derived from each cell.
  • the step of detecting a nucleic acid having a specific sequence may include amplifying and sequencing the nucleic acid having a specific sequence.
  • sorting may be performed by flow cytometry or the like.
  • a fluorescent DNA-binding dye (intercalator) that labels all nucleic acids with the first label is used.
  • a primer set for detecting a specific gene and a fluorescent DNA-binding dye (intercalator) or a fluorescent labeling position probe are used.
  • the fluorescent dyes used for the first and second labels do not have overlapping fluorescence wavelengths, and the fluorescence intensity can be measured by another channel in flow cytometry.
  • nucleic acid By introducing gel capsules containing the first and second labels into flow cytometry and selecting from the fluorescent signals of each gel capsule, a certain amount of nucleic acid is contained inside the gel capsule and a specific gene is included. Can be recovered. When selecting based on the yield of a specific sequence or the yield of total nucleic acid, the selection was performed by first detecting a gel capsule containing an amplified polynucleotide with a fluorescent intercalator, separating it into a plate by flow cytometry, and further separating it. Nucleic acid is reamplified from the polynucleotide of each gel capsule to prepare an amplified polynucleotide library.
  • the yield of the amplified nucleic acid is measured by absorptiometry, fluorescence measurement, agarose gel electrophoresis, and microchip electrophoresis, and the total amount of nucleic acid is measured.
  • a part of this amplified nucleic acid is used to perform specific gene amplification, gene sequencing or molecular weight determination, and the sequence information and yield of the amplified gene are evaluated, and each sample in the polynucleotide library is evaluated. Evaluate the presence or absence of a particular gene. With reference to the results of either or both of these, a sample is selected and transferred to another plate or the like.
  • the cell type-specific sequence, the species-specific sequence, or the sequence stored in the specific species may be amplified.
  • the marker gene may be amplified in the analysis of step C).
  • the present disclosure is, for example, a system for analyzing cells or cell-like structures, in which [X] two or more cells or cell-like structures or substances derived thereto, or capsules containing them are encapsulated. Drugs and / or encapsulated drug vaults for storing them, desired drugs used for [Y] analysis and / or analytical drug vaults for storing them, and [Z] means for encapsulation. And [W], as needed, provide a system comprising means for performing an analysis with the desired agent.
  • Examples of Y include reagents (polymerizer, primer set, etc.) for amplifying and decoding a specific gene
  • W includes, for example, reagents (fluorescent-labeled probe and intercalator) for detecting the amplified gene.
  • reagents fluorescent-labeled probe and intercalator
  • [Z] includes microfluidic devices, gelling agents, structures for encapsulation (cooling reaction tank, storage, etc.)
  • Examples include reagents (fluorescent-labeled probes and intercalators) for detecting amplified genes and measuring devices (flow cytometry).
  • kits The present disclosure is, for example, a kit for providing a capsule for analyzing a cell or a cell-like structure, which comprises [X] two or more cells or a cell-like structure or a substance derived thereto, or a substance thereof.
  • a kit comprising a drug for encapsulating the capsule and a desired drug for use in the [Y] analysis.
  • Y include reagents for amplifying and decoding a specific gene (polymerase, primer set, etc.) and reagents for detecting the amplified gene (fluorescent-labeled probe and intercalator).
  • a gel capsule containing an amplified and retained 1-cell genome-derived polynucleotide is prepared by the method described in the above section (Method for amplifying polynucleotide in cells). Suspended in a PCR solution containing a primer set corresponding to the gene region targeting the gel capsule containing the amplified polynucleotide and a PCR amplification detection indicator such as a fluorescent DNA intercalator or Taqman probe, and introduced the suspension into a microfluidic device. To do. Shear the suspension with oil to make droplets containing a single gel capsule.
  • PCR After collecting the droplets in an external container, perform a PCR operation.
  • the heat treatment of PCR disintegrates the gel capsule, the inner polynucleotide associates with the primer / enzyme in the PCR solution, and the polynucleotide is amplified in the droplet if the target gene is present.
  • the presence or absence of polynucleotide amplification can be confirmed and detected by the fluorescent color derived from the PCR amplification detection indicator.
  • the gel capsule containing the amplified polynucleotide is also labeled with a fluorescent DNA intercalator, and by using a PCR amplification detection indicator of another fluorescent color in the above means, the gel capsule can be obtained from a pattern of two or more fluorescent colors.
  • the droplet or the re-gelled droplet is selectively collected or eliminated, and the droplet containing the amplified polynucleotide containing the target gene is selectively contained in the containing solution.
  • Example 1 Encapsulation of cells and cell-like structures
  • a 1.5 cm coral branch obtained from Ishikawara in the sea area around Sesoko, Motobu-cho, Kunigami-gun, Okinawa was collected and filtered with a 0.22 ⁇ m diameter filter (DURAPORE membrane filter, GVWP04700, MERCK) to 5 mL of seawater. Collected in a 25 mL tube (2362-025, IWAKI) containing. After sufficiently crushing the coral branches using a scalpel (replacement blade scalpel holder (61-3813-28), replacement blade No. 10 (1-8545-11), AS ONE)), leave it on ice for 3 minutes. Then, large particles such as bone fragments were precipitated.
  • DURAPORE membrane filter GVWP04700, MERCK
  • the supernatant was collected in a 1.5 mL tube (122-10, SSIbio) and centrifuged at 8,000 xg for 5 min (himac CF15RX, Koki Holdings). The supernatant was removed leaving the pellets, and 800 ⁇ L of seawater filtered through a 0.22 ⁇ m diameter filter (DURAPORE membrane filter, GVWP04700, MERCK) was added to suspend the pellets again. Next, a 250 xg centrifuge was performed for 5 min and the supernatant was collected in a new 1.5 mL tube.
  • DURAPORE membrane filter GVWP04700, MERCK
  • the concentration of bacterial cells in the prepared suspension was measured (microscope: CKX41, OLYMPUS, bacterial calculator A161,-5679-01, AS ONE), and ultra-low melting point agarose (A5030) was adjusted to a final concentration of 1.5%.
  • -10G, SIGMA-ALDRICH was added to prepare a mixed suspension of cells and cell-like structures used for gel capsule preparation (final bacterial cell concentration: 4.5 ⁇ 10 3 cells / ⁇ L).
  • a gel capsule having a diameter of 35 ⁇ m was prepared using the prepared mixed suspension of cells and cell-like structures. Subsequently, the gel capsule was immersed in a lytic reagent as a lysis reagent, a portion other than the object to be collected such as a cell wall of a cell was lysed inside the gel capsule, and genomic DNA was eluted into the gel capsule.
  • the gel capsule was immersed in Buffer D2 (QIAGEN), which is an aqueous solution containing potassium hydroxide, which is one of the lytic reagents, to dissolve the residual components and denature the genomic DNA.
  • Buffer D2 QIAGEN
  • the lytic test solution used in this example is lysozyme, achromopeptidase, proteinase K, sodium dodecyl sulfate, and Buffer D2.
  • Potassium hydroxide is also used in a normal DNA amplification reaction step, but since it also has a lytic effect, it is used as one of the lytic reagents in this example.
  • the eluted genomic DNA is not discharged from the gel capsule by the lytic reagent and is retained in the gel capsule.
  • the lytic reagent permeated into the gel capsule is also included in the contaminants.
  • Buffer D2 is added to proceed with the reaction, but a sufficient cleaning effect can be obtained by performing lysis operations with a plurality of reagents step by step and performing centrifugal cleaning in each step. Further, the cells may be lysed with each lytic reagent and then centrifuged.
  • the target genomic DNA can be collected by lysing the cells with a plurality of types of lytic reagents, and the lytic reagent and the poly of the lysed cells can be collected by centrifugation after immersion in the lytic reagent. Contaminants such as components other than nucleotides can be removed, and genomic DNA can be purified without inhibiting the subsequent genomic DNA amplification reaction.
  • the amplification reagent was added to the tube containing the gel capsule holding the denatured genomic DNA in the potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent.
  • the MDA (Multiple Replication Replication) method using phi29 DNA polymerase, which is a strand-substitution type DNA synthase was used.
  • the whole genome amplification reaction was carried out for 3 hours by immersing in the whole genome amplification reaction reagent REPLI-g Single Cell Kit (QIAGEN).
  • the amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2).
  • the gel capsule after whole genome amplification is purified by DPBS (Dulvecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) three times and washed, and then washed with DAPI (Dojin), which is a fluorescent DNA intercalator. Staining was performed using science). The stained gel capsules were washed again with DPBS (Fig. 1).
  • DPBS Dens Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific
  • Example 2-1 Droplet reencapsulation and gene amplification
  • the droplets produced in Example 1 were re-encapsulated.
  • the reagent for PCR was used as the reagent to be inserted at the time of re-encapsulation.
  • PCR reaction solution was prepared.
  • a primer designed for target gene detection and a Taqman probe prepared by Integrated DNA Technologies
  • the primers and probes used in this example were designed to specifically detect a part of the 16S rRNA gene (V3-V4 region) of a microorganism belonging to the genus Endozoicomonas, which is a microorganism coexisting in coral tissue (manufactured by Integrated). DNA Technologies) and, in the presence of the target sequence, exhibit green fluorescence from the FAM after the PCR reaction.
  • a DNA staining reagent SYBR Green, EvaGreen (registered trademark), etc.
  • a gel capsule was added to the prepared PCR reaction solution to prepare a PCR reaction solution in which the gel capsule was suspended.
  • microdroplets having a diameter of 50 ⁇ m (this diameter can be 40 to 60 ⁇ m, etc.) were prepared from a PCR reaction solution in which gel capsules were suspended, and microdroplets were prepared.
  • a gel capsule was encapsulated in the droplet.
  • the gel capsule concentration in the microdroplets at this time is preferably about 0.5 cell / drop. All the prepared droplets were collected in a PCR tube.
  • the recovered material was subjected to a PCR reaction using a thermal cycler.
  • the PCR reaction conditions were as follows: initial heat denaturation at 95 ° C. for 3 minutes, heat denaturation at 95 ° C. for 5 seconds, annealing and extension reaction at 60 ° C. for 30 seconds for 28 cycles, and storage at 4 ° C. after completion of the reaction.
  • the gel capsule encapsulated inside was thermally disintegrated, and the polynucleotide held in the capsule reacted with the primer / enzyme in the PCR solution.
  • the polynucleotide is amplified in the droplet, increasing the intensity of probe-derived fluorescence or DNA intercalator-derived fluorescence.
  • the droplets were observed under a fluorescence microscope to confirm the fluorescence in the droplets.
  • Example 2-2 Detection / selection without reencapsulation and gene amplification
  • the gel capsule prepared in Example 1 is immersed in a recombinase polymerase amplification reaction (RPA) reagent.
  • RPA recombinase polymerase amplification reaction
  • the RPA reagent for example, TwisAmp (registered trademark) series (TwistDx) can be used.
  • Isothermal nucleic acid amplification is performed inside the gel capsule under the conditions of 37 ° C to 42 ° C using a primer / fluorescent probe designed according to the target sequence to be detected.
  • the gel capsule in which the amplified nucleic acid containing the target organism or the target gene is present is specificized using the fluorescent substance accumulated inside the gel capsule as an index. Can be detected.
  • Example 3 Secondary amplification
  • the secondary amplification step was further performed on the samples contained in the individual capsules prepared in Example 2. The procedure is shown below.
  • Example 2 Of those prepared in Example 2, two types of fluorescent droplets were fractionated under a microscope using a micropipette and individually collected in a PCR tube. After breaking the droplets by heating the collected individual droplets at 65 ° C. using a thermal cycler (S1000 thermal cycler, Bio-Rad), secondary amplification by the MDA method in the wells of each tube. Was done. This makes it possible to obtain a 1-cell amplified genomic library containing the sequence of the target gene.
  • S1000 thermal cycler Bio-Rad
  • a library was prepared using the Nextera XT DNA sample prep kit (Illumina, FC-131-1096) for eight samples containing genomic information derived from coral coexisting microorganisms, and Miseq (Illumina, SY-410-).
  • a 2 ⁇ 75 bp paired end read (3.99 Gb) was obtained by whole genome sequencing using 1003).
  • Assembly of sequence data using SPAdes (Bankevich A et al., J Comput Biol. 2012 May; 19 (5): 455-77. This was performed to prepare a Contig.
  • CheckM Parks et al., Genome Res. 2015.
  • Example 3-2 Analysis without secondary amplification
  • the following method is available as a procedure for skipping the secondary amplification step described in Example 3 and proceeding to the analysis.
  • droplets showing two types of fluorescence were collected under a microscope using a micropipette and individually collected in a PCR tube.
  • a thermal cycler S1000 thermal cycler, Bio-Rad
  • the Nextera XT DNA genome prep kit in the wells of each tube.
  • a library can be prepared using (Illumina, FC-131-1096) and a whole genome sequence can be performed using Miseq (Illumina, SY-410-1003).
  • DNA sequence library preparation reagent other than the Nextera XT DNA sample prep kit.
  • an adapter sequence is inserted by a transposase reaction as in the above kit, or a reaction such as fragmentation / ligation is performed to add the adapter sequence, and then a PCR reaction is sufficient to perform sequence analysis. Acquire the amount of DNA. After that, the analysis can be performed in the same manner as described in Example 3.
  • Example 4 When it is desired to selectively acquire data of cells having specific characteristics from various cells) A gel containing a polynucleotide when the purpose is to obtain genomic data of one or more specific microorganisms of interest to those skilled in the art among animal symbiotic microorganisms such as gut flora and marine / soil microorganisms. By confirming the presence or absence of the gene fragment of the target microorganism in advance for the capsule, it is possible to reduce unnecessary gene sequence data acquisition and the cost involved.
  • measurement targets include comparative analysis of microorganisms of the same strain (for example, analysis of subspecies in a group of microorganisms related to diseases, etc.) and microorganisms having a specific gene (for example, secondary metabolites and enzymes produced by microorganisms). It is assumed that the purpose is to search for or analyze bacteria, archaea, fungi, and other eukaryotic cells individually from among various species.
  • Example 5 When a large amount of host DNA or the like is mixed, the analysis sample is feces, saliva, sputum or skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning solution, tissue extract or blood, and the microorganisms contained in the sample are analyzed. Contains cells, intracellular small organs, and nucleic acids from many host animals in the sample. Some of these can also be encapsulated inside the gel capsule to perform polynucleotide amplification.
  • the intestinal flora and host animals present in the gastrointestinal tract have a symbiotic relationship in which the host provides an anaerobic environment for colonization of the gastrointestinal tract, while the intestinal flora affects the health of the host. It is known to have.
  • the major effects on the health status of the host include the production of nutrients, the defense against infectious diseases, and the development of the immune system.
  • inflammatory bowel disease is a disease caused by an abnormality of intestinal environmental factors such as intestinal bacteria in addition to a genetic predisposition.
  • Example 6 Example of performing encapsulation for the third time or more
  • Analysis samples include feces, saliva, sputum and skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning fluid, tissue extract and blood, or crushed fluid of plants, insects, and animals containing symbiotic microorganisms.
  • each gel capsule is encapsulated with host-derived cells, organelles, nucleic acids, and nucleic acids derived from symbiotic microorganisms. All of these can be encapsulated inside the gel capsule and polynucleotide amplification can be performed.
  • the target may be a combination of two or more microorganisms or host-derived substances.
  • This disclosure is useful in industries that use single cell analysis.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021219567A1 (fr) * 2020-04-27 2021-11-04 De Paulou Massat Sophie Microcompartiment pour la culture de cellules de cnidaires
WO2024202886A1 (ja) * 2023-03-29 2024-10-03 国立研究開発法人産業技術総合研究所 核酸検出方法並びにヒドロゲルカプセル及びその分散液

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002531056A (ja) * 1998-08-07 2002-09-24 セレイ， エルエルシー 遺伝子分析におけるゲルマイクロドロップ
JP2015528283A (ja) * 2012-08-14 2015-09-28 テンエックス・ジェノミクス・インコーポレイテッド マイクロカプセル組成物および方法
WO2017184707A1 (en) * 2016-04-19 2017-10-26 President And Fellows Of Harvard College Immobilization-based systems and methods for genetic analysis and other applications
WO2019216271A1 (ja) * 2018-05-07 2019-11-14 ｂｉｔＢｉｏｍｅ株式会社 シングルセル解析を行う方法およびそのための装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002531056A (ja) * 1998-08-07 2002-09-24 セレイ， エルエルシー 遺伝子分析におけるゲルマイクロドロップ
JP2015528283A (ja) * 2012-08-14 2015-09-28 テンエックス・ジェノミクス・インコーポレイテッド マイクロカプセル組成物および方法
WO2017184707A1 (en) * 2016-04-19 2017-10-26 President And Fellows Of Harvard College Immobilization-based systems and methods for genetic analysis and other applications
WO2019216271A1 (ja) * 2018-05-07 2019-11-14 ｂｉｔＢｉｏｍｅ株式会社 シングルセル解析を行う方法およびそのための装置

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021219567A1 (fr) * 2020-04-27 2021-11-04 De Paulou Massat Sophie Microcompartiment pour la culture de cellules de cnidaires
WO2024202886A1 (ja) * 2023-03-29 2024-10-03 国立研究開発法人産業技術総合研究所 核酸検出方法並びにヒドロゲルカプセル及びその分散液

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