WO2018198599A1 - Isolation method of droplets for analysis derived from cell, and cell analysis method - Google Patents

Isolation method of droplets for analysis derived from cell, and cell analysis method Download PDF

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Publication number
WO2018198599A1
WO2018198599A1 PCT/JP2018/010971 JP2018010971W WO2018198599A1 WO 2018198599 A1 WO2018198599 A1 WO 2018198599A1 JP 2018010971 W JP2018010971 W JP 2018010971W WO 2018198599 A1 WO2018198599 A1 WO 2018198599A1
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droplet
analysis
amplification
droplets
nucleic acid
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PCT/JP2018/010971
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French (fr)
Japanese (ja)
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伸也 砂永
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株式会社エンプラス
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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to a cell-derived analysis droplet isolation method and a cell analysis method.
  • sorting In the case where target cells are isolated from samples containing various cells and the target genome is analyzed, cell sorting has become common in recent years as a method for isolating target cells. In particular, in the method of forming droplets (droplets) containing cells by emulsification, sorting (sorting) of droplets containing target cells is performed using nucleic acid amplification.
  • a sample containing cells is emulsified to form droplets in the emulsion.
  • a short chain sequence derived from the target cell is amplified by a nucleic acid amplification method such as PCR, and the presence or absence of the amplification is detected using a probe or the like.
  • the droplet by which amplification was confirmed is fractionated as a droplet containing a target cell, and this is made into samples, such as a genome analysis (nonpatent literature 1).
  • the target genome of the target cell is further subjected to nucleic acid amplification, and the obtained target genome amplification product is subjected to next-generation sequencing (NGS) and assembled.
  • NGS next-generation sequencing
  • the target genome of the target cell can be analyzed (Non-patent Document 2).
  • Nucleic acid amplification in the cell sorting is aimed at confirming the presence or absence of cells in the droplets. Therefore, as described above, for example, a short region in the genome (for example, about 80 to 300 bp) is set and amplification is performed. Done.
  • the droplets collected by amplification detection contain a large amount of short-chain amplification products by cell sorting, and the amount is compared to the cell-derived genome contained in the droplets. Very many. For this reason, even if nucleic acid amplification of the target genome is further performed on the collected droplets for genome analysis, in addition to target amplification products derived from the target genome, samples to be analyzed are not subject to analysis. There is a problem that these short-chain amplification products are sequenced while a large amount of short-chain amplification products are not mixed, and the analysis result of the target genome of the target cell is affected.
  • an object of the present invention is to provide a method for isolating a droplet that can be subjected to analysis without affecting subsequent analysis, and an analysis method.
  • the present invention is a method for isolating a droplet for analysis, An emulsion formation step, a nucleic acid amplification step, a detection step, a first fractionation step, an amplification product removal step, and a second fractionation step
  • the emulsion forming step includes A step of contacting a sample containing cells with an emulsion-forming solvent to form a plurality of droplets containing cells in the sample in the emulsion-forming solvent;
  • the nucleic acid amplification step includes For the droplet, using a primer, a step of performing nucleic acid amplification of the nucleic acid derived from the cells, The primer has a complementary sequence to the nucleic acid and a first additional sequence
  • the detection step includes Detecting the presence or absence of the amplification product of the nucleic acid in the droplet,
  • the first sorting step includes A step of separating a droplet in which the amplification product of the nucleic acid is detected;
  • the present invention is a cell analysis method, Including a step of isolating a liquid-derived analysis droplet in a sample, and an analysis step,
  • the isolation step includes A step of isolating the cell-derived analysis droplet from a sample containing cells by the isolation method of the present invention,
  • the analysis step includes It is a step of analyzing the isolated analysis droplet.
  • nucleic acid amplification is performed in order to confirm the presence or absence of cells in the droplet for droplet collection, and the obtained amplification product can be removed from the droplet.
  • the amplification product for separating the droplets is removed from the droplet, for example, when genome analysis is performed using the analysis droplet, the amplification product as described above is used. Can be avoided, and genome analysis with higher accuracy becomes possible.
  • FIG. 1 is a schematic diagram showing an example of an amplification product removal step in the isolation method of the present invention.
  • the carrier in the solid-phased carrier is a magnetic carrier
  • the amplification product is bound from the droplet by a magnetic field in the first sorting step. Separate the solid support.
  • the magnetic carrier is a magnetic bead.
  • a droplet is formed in the presence of the amplification reagent containing the primer and the solid-phased carrier,
  • the nucleic acid amplification step using the amplification reagent contained in the droplet, the cell-derived nucleic acid is amplified,
  • the nucleic acid amplification product is removed using the solid-phased support contained in the droplet.
  • a droplet is formed in the absence of the amplification reagent containing the primer and the immobilized carrier, After the emulsion formation step, the amplification reagent and the solid-phase support are added to the droplets.
  • the detection reagent for detection of amplification coexists in the amplification reagent containing the primer, and the detection reagent contains an intercalator or a probe.
  • the analysis droplet isolation method of the present invention further includes, for example, an analysis step,
  • the analysis step is a step of analyzing a cell-derived genome contained in the droplet using the analysis droplet sorted in the second sorting step.
  • the emulsion forming solvent contains a water-insoluble solvent, and the plurality of liquid droplets are formed in the water-insoluble solvent.
  • the plurality of droplets are water-soluble droplets containing the sample.
  • the second sorting step is further performed in the flow path.
  • the cell analysis method of the present invention further includes, for example, an amplification step, and the amplification step is a step of amplifying the cell-derived genome with respect to the isolated analysis droplet, and the analysis step includes: Genomic analysis is performed on the amplification product of the genome contained in the analysis droplet.
  • the point is to remove the amplification products from the droplets prior to the separation of the analysis droplets, and other steps and conditions are not particularly limited.
  • isolation method and analysis method of the present invention will be described by way of examples.
  • the isolation method and analysis method of the present invention are not limited or restricted by the following embodiments. Moreover, the description of each embodiment can mutually be used.
  • the isolation method of the present invention is a method for isolating a droplet for analysis, An emulsion formation step, a nucleic acid amplification step, a detection step, a first fractionation step, an amplification product removal step, and a second fractionation step,
  • the emulsion forming step includes A step of contacting a sample containing cells with an emulsion-forming solvent to form a plurality of droplets containing cells in the sample in the emulsion-forming solvent;
  • the nucleic acid amplification step includes For the droplet, using a primer, a step of performing nucleic acid amplification of the nucleic acid derived from the cells, The primer has a complementary sequence to the nucleic acid and a first additional sequence,
  • the detection step includes Detecting the presence or absence of the amplification product of the nucleic acid in the droplet,
  • the first sorting step includes A step of separating a droplet in which the a
  • the emulsion forming step is a step of bringing a sample containing cells into contact with an emulsion forming solvent to form a plurality of droplets containing cells in the sample in the emulsion forming solvent.
  • the sample can be divided and divided into a plurality of droplets. Then, various cells in the sample are distributed to each of the droplets.
  • the type of the sample is not limited at all, and examples include samples that are considered to contain cells.
  • Examples of the sample include a biological sample, and specific examples include blood, lymph, cerebrospinal fluid, semen, urine, nasal discharge, nasal swab, and the like.
  • the living body is not particularly limited and includes, for example, humans; non-human mammals such as cows, pigs, sheep, mice, rats, rabbits and horses; birds; animals such as fish.
  • the emulsion-forming solvent is not particularly limited as long as it can form droplets containing the sample therein.
  • the emulsion-forming solvent is, for example, a water-insoluble solvent, and water-soluble droplets containing the sample can be formed in the water-insoluble solvent.
  • water-insoluble solvent examples include oil, mineral oil, chloroform, and aromatic compounds.
  • the water-insoluble solvent for example, one kind may be used alone, or two or more kinds may be used in combination.
  • a water-soluble solvent may be further present.
  • the water-soluble solvent include water, a buffer solution, and a water-soluble polymer solution.
  • the water-soluble solvent for example, one kind may be used alone, or two or more kinds may be used in combination.
  • the size of the droplets to be formed is not particularly limited, and the average volume has a lower limit of, for example, 4 pL or more and an upper limit of, for example, 10 nL or less.
  • the number of cells contained in the droplet is not particularly limited.
  • the number of cells contained in one droplet is, for example, 5 or less, 2 or less, and preferably 1 cell.
  • the method for forming droplets in the emulsion forming solvent is not particularly limited, and a plurality of droplets can be formed in the emulsion forming solvent by bringing the emulsion forming solvent into contact with the sample.
  • the sample may be brought into contact with the emulsion-forming solvent, or the emulsion-forming solvent may be brought into contact with the sample.
  • a known droplet manufacturing method can be applied as the droplet forming method.
  • an emulsification device having a microchannel can be used.
  • the device includes, as the microchannel, for example, a sample channel, an emulsion-forming solvent channel, a connecting portion thereof, and a lead-out channel derived from the connecting portion.
  • droplets are formed as follows. First, the emulsion forming solvent is introduced into the connecting portion from the emulsion forming solvent flow path, and then toward the connecting portion into which the emulsion forming solvent is introduced, from the sample flow path, The sample (eg, optionally containing the water-soluble solvent) is introduced.
  • the emulsion-forming solvent and the sample come into contact with each other to be emulsified, and from the connecting portion to the outlet channel, an emulsion (a state in which droplets are dispersed in the emulsion-forming solvent) Derived.
  • the sample and the water-soluble solvent may be introduced into the channel of the device as a mixed liquid mixed in advance as described above, or may be separately introduced into the channel. In the latter case, the sample channel and the reagent channel are provided upstream of the connecting portion, and the sample is introduced from the sample channel toward the connecting portion, and the reagent channel.
  • the water-soluble solvent and optionally the various reagents may be introduced.
  • the amplification step is a step of performing nucleic acid amplification of the cell-derived nucleic acid using a primer for the droplets formed in the emulsion formation step.
  • the type of nucleic acid to be amplified is not particularly limited, and can be appropriately determined according to, for example, the type of cell to be analyzed.
  • the nucleic acid amplification method is not particularly limited, and a known method such as a PCR method can be employed.
  • the amplification reagent used for the nucleic acid amplification is not particularly limited, and examples thereof include polymerase, dNTP and the like in addition to the primer.
  • the primer used in the amplification step has a complementary sequence to the nucleic acid and a first additional sequence as described above.
  • the complementary sequence can be appropriately determined according to the sequence of the nucleic acid to be amplified.
  • the complementary sequence only needs to be annealed as a primer to the nucleic acid to be amplified, for example, and the degree of complementarity (%) with respect to the nucleic acid is not particularly limited, and may be completely complementary (100%), for example. In the latter case, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the first additional sequence is not particularly limited.
  • the first additional sequence is a non-complementary sequence to the nucleic acid from the viewpoint of preventing the first additional sequence from being annealed and starting extension. It is preferable that For example, the first additional sequence is bound to the 5 'side of the complementary sequence so as not to prevent extension from the 3' end side of the complementary sequence.
  • the total length of the primer and the lengths of the complementary sequence and the first additional sequence are not particularly limited.
  • the total length of the primer is, for example, 20 to 25 mer
  • the length of the complementary sequence is, for example, 80 to 300 bp
  • the length of the first additional sequence is, for example, 10 to 20 bp.
  • the amplification reagent used in the amplification step may be previously contained in the droplet, for example, or may be added to the droplet during the amplification step, and the operation is easy. preferable.
  • a droplet is formed in the presence of the amplification reagent, and in the nucleic acid amplification step, amplification is performed using the amplification reagent contained in the droplet.
  • droplets may be formed in the emulsion formation step in the absence of the amplification reagent, and amplification may be performed by adding the amplification reagent in the nucleic acid amplification step.
  • the amplification step is preferably performed in the microchannel of the device as well.
  • the device has, for example, an amplification channel connected downstream of the outlet channel, and the amplification can be performed in the amplification channel.
  • the detection step is a step of detecting the presence or absence of the amplification product of the nucleic acid in the droplet as described above.
  • the method for detecting the presence or absence of the amplification is not particularly limited, and can be appropriately set depending on, for example, the amplification method, the type of the amplification reagent used, and the like.
  • the presence / absence of the amplification is detected for each droplet, for example.
  • the method for detecting the presence or absence of amplification is not particularly limited, and examples thereof include a general intercalator method and probe method.
  • a detection reagent such as an intercalator or a probe can be used according to the detection method.
  • the type of the probe is not particularly limited, and for example, a TaqMan (registered trademark) probe or the like can be used.
  • the detection reagent may coexist with the amplification reagent, for example.
  • the detection reagent used in the detection step may be previously contained in the droplet, for example, or may be added to the droplet during the detection step, and is easy to operate. preferable.
  • a droplet is formed in the presence of the amplification reagent and the detection reagent, and in the nucleic acid amplification step, the amplification reagent contained in the droplet is used. Amplification is performed, and in the detection step, the presence or absence of the amplification may be detected using the detection reagent contained in the droplet.
  • a droplet is formed in the absence of the amplification reagent and the detection reagent, and the amplification reagent and the detection reagent are added to the droplet. Then, amplification may be performed in the nucleic acid amplification step, and the presence or absence of amplification may be detected in the detection step.
  • the order of addition of the amplification reagent and the detection reagent is not particularly limited. For example, both may be added in the amplification step, or the amplification reagent is added in the amplification step, and the detection is performed. In the step, the detection reagent may be added.
  • the detection step is preferably performed in the microchannel of the device as well.
  • the device has a detection channel downstream of the amplification channel, and can perform the amplification in the detection channel.
  • the amplification step and the detection step may be performed sequentially, for example, but can be performed simultaneously. In the latter case, for example, the amplification step and the detection step may be performed in the presence of the amplification reagent and the detection reagent.
  • the device When using a device having the microchannel, the device has, for example, an amplification channel connected downstream of the outlet channel, and performs the amplification and the detection in the amplification channel. be able to.
  • the first sorting step is a step of sorting the droplet in which the amplification product of the nucleic acid is detected.
  • the first sorting step is similarly performed in the microchannel of the device.
  • the device has, for example, a pair of branched flow channels downstream of the detection flow channel, and one of the flow channels is a flow channel of the droplet in which amplification is detected (first sorting flow channel). ) And the other channel is a droplet channel in which amplification was not detected.
  • amplification is detected by detecting whether or not each droplet is amplified in the detection flow channel, and distributing each droplet in the flow channel according to the presence or absence of the amplification. Droplets can be collected.
  • the sorting method is not particularly limited, and for example, a conventionally known method in cell sorting can be adopted.
  • the amplification product removal step is a step of removing amplification products from the sorted droplets from the droplets using a solid-phased carrier in which the second additional sequence is solid-phased on the carrier.
  • the second additional sequence in the immobilized carrier is at least one of the first additional sequence and the complementary sequence to the first additional sequence in the primer.
  • the carrier in the solid phase carrier is not particularly limited, and is, for example, a magnetic carrier.
  • the shape of the carrier is not particularly limited, and examples thereof include beads.
  • Specific examples of the magnetic carrier include magnet beads, permanent magnets, electromagnets and the like.
  • the second additional sequence in the solid-phased carrier only needs to be able to bind to the amplification product of the primer, and may be, for example, the same sequence as the first additional sequence in the primer or a complementary sequence to the first additional sequence.
  • the solid-phase support may be, for example, a solid-phase support having the former sequence (the same sequence as the first addition sequence) or a solid-phase support having the latter sequence (complementary sequence to the first addition sequence). Alternatively, both the above may be used in combination.
  • the solid-phase support used in the amplification product removal step may be previously contained in the droplet, for example, or may be added to the droplet, and the former is preferable because the operation is easy. .
  • the former case for example, in the emulsion formation step, droplets are formed in the presence of the amplification reagent, the solid-phase support, and optionally the detection reagent, and in the nucleic acid amplification step, the droplets are formed.
  • Amplification is performed using the amplification reagent contained, and in the detection step, presence or absence of the amplification is detected using the detection reagent contained in the droplet, and in the amplification product removal step, the solid phase is detected.
  • the amplification product may be removed using a phased carrier.
  • a droplet is formed in the absence of the amplification reagent, the solid phase support, and optionally the detection reagent, and the amplification is performed on the droplet.
  • the order of addition of the amplification reagent, the immobilized carrier and the detection reagent is not particularly limited. For example, all may be added in the amplification step, or each may be added in each step. May be.
  • the amplification product removing step is preferably performed in the microchannel of the device as well.
  • the device has, for example, an amplification product removal flow path downstream of a flow path (first sorting flow path) of a droplet in which amplification is detected, of the pair of branched flow paths, The amplification product can be removed in the amplification product removal channel.
  • the method for removing the amplification product from the droplet in the amplification product removal channel using the solid-phased carrier is not particularly limited.
  • an example in which the solid-phased magnetic beads are used as the solid-phase support will be described with reference to FIG. 1.
  • the present invention is not limited to the following description.
  • FIG. 1 is a schematic view showing a process of removing an amplification product from the droplet in the amplification product removal channel that is a part of the micro channel, and is a plan view of the micro channel as viewed from above. It is.
  • an arrow X indicates a method of flowing a droplet.
  • an amplification product removal flow path 11 is connected to the micro flow path of the device downstream of the first sorting flow path 10.
  • the amplification product removal channel 11 includes a magnetic field generation region 11 a having a width wider than that of the first sorting channel 10 and a droplet recovery region 11 b having a width equivalent to that of the first sorting channel 10.
  • An external magnetic field is generated in the magnetic field generation region 11a.
  • the magnetic field can be generated, for example, by arranging a magnet outside the magnetic field generation region 11a.
  • a droplet 20 including an amplification product (not shown) and a solid-phased magnetic bead 30 is introduced as an emulsion from the first sorting channel 10 to the amplification product removal channel 11. . Since a magnetic field is generated in the magnetic field generation region 11 a of the amplification product removal channel 11, the solid-phased magnetic beads 30 included in the droplet 20 are pulled by the magnetic field and captured by the magnetic field. Excluded. Then, the droplet 20 ′ from which the solid-phased magnetic beads 30 have been removed flows into the droplet collection region 11 b of the amplification product removal channel 11.
  • the flow rate of the emulsion when introduced into the amplification product removal channel 11 is relatively slow, and the emulsion when passing through the amplification product removal channel 11 It is preferable to relatively slow the flow rate.
  • the flow rate before introduction into the amplification product removal channel 11 is 1 ⁇ m / min
  • the flow rate of the emulsion when introduced into the amplification product removal channel 11 is, for example, 0.5 to
  • the flow rate of the emulsion when passing through the amplification product removal flow path 11 is, for example, 0.1 to 1 ⁇ m / min.
  • the second sorting step is a step of sorting the droplets from which the amplification products have been removed by the amplification product removing step as analysis droplets.
  • the sorted analysis droplets can be applied to analysis such as genome analysis as described later.
  • the droplet is led out from the device and collected in another container (microtube or the like). Can be separated.
  • the present invention releases intracellular components such as nucleic acids from the cells contained in the droplets in the emulsion after the emulsion formation step, before the nucleic acid amplification step, or simultaneously with the nucleic acid amplification step.
  • the release of the intracellular component is not particularly limited, and for example, a general cell lysis reagent can be used.
  • the cell lysis reagent for example, may be added to the droplets in the emulsion after the emulsion formation step, or in the emulsion formation step, by contacting the sample and the emulsion formation solvent, You may make it contain in the droplet in the said emulsion formation solvent.
  • the reagent examples include a solution containing a surfactant, a salt, and a solvent.
  • the surfactant include anionic surfactants such as sodium deoxycholate and sodium lauryl sulfate.
  • the salt examples include sodium chloride.
  • the solvent include various buffers. Liquid. Specific examples of the composition of the reagent include, for example, 50 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.5 w / v% sodium deoxycholate, 0.1 w / v% sodium lauryl sulfate, and 1.0 w / An example is v% NIP-40.
  • the present invention may further include an analysis step.
  • the analysis step is, for example, a step of analyzing the analysis droplet. The analysis will be described later.
  • the analysis method of the present invention is a cell analysis method as described above, Including a step of isolating a liquid-derived analysis droplet in a sample, and an analysis step,
  • the isolation step includes A step of isolating the cell-derived analysis droplet from a sample containing cells by the isolation method of the present invention,
  • the analysis step includes It is a step of analyzing the isolated analysis droplet.
  • the point is that the droplet for analysis is isolated by the isolation method of the present invention, and other steps and conditions are not particularly limited.
  • the description of the method for isolating the analysis droplet of the present invention can be used for the isolation method of the isolation step.
  • the analysis droplet in the analysis method of the present invention has an amplification product obtained by nucleic acid amplification for confirming the presence or absence of cells in the droplet. For this reason, for example, when genome analysis is performed using the analysis droplet, it is possible to avoid the influence of the conventional amplification product and to obtain an analysis result with higher reliability.
  • the analysis method of the present invention may further include an amplification step, for example.
  • the amplification step is, for example, a step of amplifying the cell-derived genome with respect to the isolated analysis droplet.
  • the genome amplification product contained in the analysis droplet is Analysis can be performed.
  • the type of analysis is not particularly limited, and can be appropriately determined according to the purpose.
  • the analysis is genome analysis, for example, a known method can be mentioned, and specific examples include Fluorescence In Situ Hybridization (FISH), G-Band, Next-Generation Sequencing (NGS), and the like.
  • nucleic acid amplification is performed in order to confirm the presence or absence of cells in the droplet for droplet collection, and the obtained amplification product can be removed from the droplet.
  • the amplification product for separating the droplets is removed from the droplet, for example, when genome analysis is performed using the analysis droplet, the amplification product as described above is used. Can be avoided, and genome analysis with higher accuracy becomes possible.

Abstract

In order to provide a method which, without affecting subsequent analysis, enables isolating droplets to be provided for analysis, and to provide an analysis method, this method for isolating droplets for analysis is characterized by involving an emulsion forming step, a nucleic acid amplification step, a detection step, a first sorting step, an amplification product removal step, and a second sorting step. In the emulsion forming step, a sample that contains cells and an emulsion forming solvent are brought into contact to form multiple droplets that contain the cells in the sample in the emulsion forming solvent. In the nucleic acid amplification step, a primer is used in the droplets to perform nucleic acid amplification of nucleic acids derived from the cells, and the primer has a sequence complementary to that of the nucleic acids, and a first added sequence. In the detection step, the presence or absence of amplification products of the nucleic acids in the droplets is detected. In the first sorting step, droplets in which amplification products of the nucleic acids have been detected are sorted. In the amplification product removal step, amplification products in the sorted droplets are removed from the droplets by means of an immobilized carrier comprising a second added sequence immobilized on a carrier, wherein the second added sequence on the immobilized carrier is the first added sequence in the primer and/or a sequence complementary to the first added sequence, the amplification products in the droplets and the immobilized carriers are brought into contact, the amplification products are bonded to the immobilized carriers, and by separating the immobilized carriers from the droplets, the amplification products are removed from the droplets. In the second sorting step, the droplets from which the amplification products have been removed in the amplification product removal step are sorted as droplets for analysis.

Description

細胞由来の解析用液滴の単離方法、および細胞の解析方法Method for isolating cell-derived droplet for analysis and cell analysis method
　本発明は、細胞由来の解析用液滴の単離方法、および細胞の解析方法に関する。 The present invention relates to a cell-derived analysis droplet isolation method and a cell analysis method.
　様々な細胞を含む試料から、ターゲット細胞を単離して、ターゲットゲノムについての解析を行う場合、ターゲット細胞の単離方法として、近年、セルソーティングが一般的になっている。中でも、エマルション化により細胞を含む液滴（ドロップレット）を形成する方法では、核酸増幅を利用して、ターゲット細胞を含む液滴の分取（ソーティング）が行われている。 In the case where target cells are isolated from samples containing various cells and the target genome is analyzed, cell sorting has become common in recent years as a method for isolating target cells. In particular, in the method of forming droplets (droplets) containing cells by emulsification, sorting (sorting) of droplets containing target cells is performed using nucleic acid amplification.
　セルソーティングの具体的な方法としては、例えば、まず、細胞を含む試料についてエマルション化を行い、前記エマルション中に液滴を形成する。そして、前記エマルション中の液滴について、ＰＣＲ等の核酸増幅法により、ターゲット細胞に由来する短鎖の配列を増幅させ、プローブ等を用いて、前記増幅の有無を検出する。そして、増幅が確認された液滴を、ターゲット細胞を含む液滴として分取し、これを、ゲノム解析等の試料とする（非特許文献１）。ゲノム解析においては、例えば、前記液滴について、さらに、前記ターゲット細胞のターゲットゲノムの核酸増幅を行い、得られたターゲットゲノムの増幅産物を次世代シーケンシング（ＮＧＳ）に供し、アッセンブリングすることで、前記ターゲット細胞のターゲットゲノムを解析できる（非特許文献２）。 As a specific method of cell sorting, for example, first, a sample containing cells is emulsified to form droplets in the emulsion. Then, for the droplets in the emulsion, a short chain sequence derived from the target cell is amplified by a nucleic acid amplification method such as PCR, and the presence or absence of the amplification is detected using a probe or the like. And the droplet by which amplification was confirmed is fractionated as a droplet containing a target cell, and this is made into samples, such as a genome analysis (nonpatent literature 1). In the genome analysis, for example, the target genome of the target cell is further subjected to nucleic acid amplification, and the obtained target genome amplification product is subjected to next-generation sequencing (NGS) and assembled. The target genome of the target cell can be analyzed (Non-patent Document 2).
　前記セルソーティングにおける核酸増幅は、前記液滴中の細胞の有無の確認を目的とするため、前述のように、例えば、ゲノムにおける短い領域（例えば、８０～３００ｂｐ程度）を設定して、増幅が行われる。しかしながら、増幅の検出により分取された液滴には、セルソーティングでの短鎖の増幅産物が大量に混入されており、その量は、液滴中に含まれる細胞由来のゲノムと比較して非常に多い。このため、ゲノム解析のために、前記分取した液滴について、さらに、ターゲットゲノムの核酸増幅を行っても、ゲノム解析に供する試料には、ターゲットゲノム由来の増幅産物の他に、解析対象ではない大量の短鎖の増幅産物が混入されたままで、これらの短鎖の増幅産物がシーケンシングされ、ターゲット細胞のターゲットゲノムの解析結果に影響を与えるという問題がある。 Nucleic acid amplification in the cell sorting is aimed at confirming the presence or absence of cells in the droplets. Therefore, as described above, for example, a short region in the genome (for example, about 80 to 300 bp) is set and amplification is performed. Done. However, the droplets collected by amplification detection contain a large amount of short-chain amplification products by cell sorting, and the amount is compared to the cell-derived genome contained in the droplets. Very many. For this reason, even if nucleic acid amplification of the target genome is further performed on the collected droplets for genome analysis, in addition to target amplification products derived from the target genome, samples to be analyzed are not subject to analysis. There is a problem that these short-chain amplification products are sequenced while a large amount of short-chain amplification products are not mixed, and the analysis result of the target genome of the target cell is affected.
　そこで、本発明は、後の解析に影響を与えることなく、解析に供することができる液滴を単離する方法、および解析方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a method for isolating a droplet that can be subjected to analysis without affecting subsequent analysis, and an analysis method.
　前記目的を達成するために、本発明は、解析用液滴の単離方法であり、
エマルション形成工程と、核酸増幅工程と、検出工程と、第１分取工程と、増幅産物除去工程と、第２分取工程とを含み、
前記エマルション形成工程は、
　　細胞を含む試料と、エマルション形成溶媒とを接触させて、前記エマルション形成溶媒中に、前記試料中の細胞を含む複数の液滴を形成する工程であり、
前記核酸増幅工程は、
　　前記液滴について、プライマーを用いて、前記細胞由来の核酸の核酸増幅を行う工程であり、
　　前記プライマーは、前記核酸に対する相補配列と第１付加配列とを有し、
前記検出工程は、
　　前記液滴における前記核酸の増幅産物の有無を検出する工程であり、
前記第１分取工程は、
　　前記核酸の増幅産物が検出された液滴を分取する工程であり、
前記増幅産物除去工程は、
　　前記分取した液滴における増幅産物を、担体に第２付加配列が固相化された固相化担体により、前記液滴から除去する工程であり、
　　前記固相化担体における第２付加配列は、前記プライマーにおける前記第１付加配列および前記第１付加配列に対する相補配列の少なくとも一方であり、
　　前記液滴中の前記増幅産物と前記固相化担体とを接触させて、前記固相化担体に前記増幅産物を結合させ、前記液滴から前記固相化担体を分離することで、前記液滴から前記増幅産物を除去し、
前記第２分取工程は、
　　前記増幅産物除去工程により前記増幅産物が除去された液滴を、解析用液滴として分取する工程であることを特徴とする。 In order to achieve the above object, the present invention is a method for isolating a droplet for analysis,
An emulsion formation step, a nucleic acid amplification step, a detection step, a first fractionation step, an amplification product removal step, and a second fractionation step,
The emulsion forming step includes
A step of contacting a sample containing cells with an emulsion-forming solvent to form a plurality of droplets containing cells in the sample in the emulsion-forming solvent;
The nucleic acid amplification step includes
For the droplet, using a primer, a step of performing nucleic acid amplification of the nucleic acid derived from the cells,
The primer has a complementary sequence to the nucleic acid and a first additional sequence,
The detection step includes
Detecting the presence or absence of the amplification product of the nucleic acid in the droplet,
The first sorting step includes
A step of separating a droplet in which the amplification product of the nucleic acid is detected;
The amplification product removal step includes:
A step of removing amplification products in the sorted droplets from the droplets by a solid-phased carrier in which a second additional sequence is immobilized on a carrier;
The second additional sequence in the solid phase support is at least one of the first additional sequence and the complementary sequence to the first additional sequence in the primer;
Contacting the amplification product in the droplet with the immobilized carrier, binding the amplification product to the immobilized carrier, and separating the immobilized carrier from the droplet; Removing the amplification product from the drop;
The second sorting step includes
It is a step of separating the droplet from which the amplification product has been removed by the amplification product removal step as an analysis droplet.
　本発明は、細胞の解析方法であり、
試料中の細胞由来の解析用液滴の単離工程と、解析工程とを含み、
前記単離工程は、
　細胞を含む試料から、前記本発明の単離方法により、前記細胞由来の解析用液滴を単離する工程であり、
前記解析工程は、
　前記単離された解析用液滴について、解析を行う工程であることを特徴とする。 The present invention is a cell analysis method,
Including a step of isolating a liquid-derived analysis droplet in a sample, and an analysis step,
The isolation step includes
A step of isolating the cell-derived analysis droplet from a sample containing cells by the isolation method of the present invention,
The analysis step includes
It is a step of analyzing the isolated analysis droplet.
　本発明によれば、液滴分取のために、液滴中の細胞の有無を確認すべく、核酸増幅を行うが、得られた増幅産物は、前記液滴から除去できる。このように、前記液滴から、前記液滴分取のための増幅産物は除去されているため、例えば、前記解析用液滴を用いてゲノム解析を行う場合、前述のような、前記増幅産物の影響を回避することができ、より精度に優れたゲノム解析が可能となる。 According to the present invention, nucleic acid amplification is performed in order to confirm the presence or absence of cells in the droplet for droplet collection, and the obtained amplification product can be removed from the droplet. Thus, since the amplification product for separating the droplets is removed from the droplet, for example, when genome analysis is performed using the analysis droplet, the amplification product as described above is used. Can be avoided, and genome analysis with higher accuracy becomes possible.
図１は、本発明の単離方法における増幅産物除去工程の一例を示す概略図である。FIG. 1 is a schematic diagram showing an example of an amplification product removal step in the isolation method of the present invention.
　本発明の解析用液滴の単離方法は、例えば、前記固相化担体における担体が、磁性担体であり、前記第１分取工程において、磁場により、前記液滴から前記増幅産物が結合した固相化担体を分離する。 In the method for isolating a droplet for analysis of the present invention, for example, the carrier in the solid-phased carrier is a magnetic carrier, and the amplification product is bound from the droplet by a magnetic field in the first sorting step. Separate the solid support.
　本発明の解析用液滴の単離方法は、例えば、前記磁性担体が、磁性ビーズである。 In the analysis droplet isolation method of the present invention, for example, the magnetic carrier is a magnetic bead.
　本発明の解析用液滴の単離方法は、例えば、前記エマルション形成工程において、前記プライマーを含む増幅用試薬および前記固相化担体の存在下、液滴を形成し、
前記核酸増幅工程において、前記液滴に含まれる前記増幅用試薬を用いて、前記細胞由来の核酸の増幅を行い、
前記第２分取工程において、前記液滴に含まれる前記固相化担体を用いて、前記核酸増幅物の除去を行う。 In the method for isolating a droplet for analysis of the present invention, for example, in the emulsion formation step, a droplet is formed in the presence of the amplification reagent containing the primer and the solid-phased carrier,
In the nucleic acid amplification step, using the amplification reagent contained in the droplet, the cell-derived nucleic acid is amplified,
In the second sorting step, the nucleic acid amplification product is removed using the solid-phased support contained in the droplet.
　本発明の解析用液滴の単離方法は、例えば、前記エマルション形成工程において、前記プライマーを含む増幅用試薬および前記固相化担体の非存在下、液滴を形成し、
前記エマルション形成工程後、前記液滴に、前記増幅用試薬および前記固相化担体を添加する。 In the method for isolating a droplet for analysis of the present invention, for example, in the emulsion formation step, a droplet is formed in the absence of the amplification reagent containing the primer and the immobilized carrier,
After the emulsion formation step, the amplification reagent and the solid-phase support are added to the droplets.
　本発明の解析用液滴の単離方法は、例えば、前記プライマーを含む増幅用試薬に、前記増幅の検出のための検出試薬を共存させ、前記検出試薬が、インターカレータまたはプローブを含む。 In the method for isolating analysis droplets of the present invention, for example, the detection reagent for detection of amplification coexists in the amplification reagent containing the primer, and the detection reagent contains an intercalator or a probe.
　本発明の解析用液滴の単離方法は、例えば、さらに、解析工程を含み、
前記解析工程は、前記第２分取工程で分取した前記解析用液滴を用いて、前記液滴に含まれる細胞由来のゲノムを解析する工程である。 The analysis droplet isolation method of the present invention further includes, for example, an analysis step,
The analysis step is a step of analyzing a cell-derived genome contained in the droplet using the analysis droplet sorted in the second sorting step.
　本発明の解析用液滴の単離方法は、例えば、前記エマルション形成工程において、前記エマルション形成溶媒が、非水溶性溶媒を含み、前記非水溶性溶媒中に、前記複数の液滴が形成され、前記複数の液滴は、前記試料を含む水溶性の液滴である。 In the method for isolating liquid droplets for analysis according to the present invention, for example, in the emulsion forming step, the emulsion forming solvent contains a water-insoluble solvent, and the plurality of liquid droplets are formed in the water-insoluble solvent. The plurality of droplets are water-soluble droplets containing the sample.
　本発明の解析用液滴の単離方法は、例えば、前記流路内で、さらに、前記第２分取工程を行う。 In the method for isolating analysis droplets of the present invention, for example, the second sorting step is further performed in the flow path.
　本発明の細胞の解析方法は、例えば、さらに、増幅工程を含み、前記増幅工程は、前記単離した解析用液滴について、前記細胞由来のゲノムを増幅させる工程であり、前記解析工程は、前記解析用液滴に含まれる前記ゲノムの増幅産物について、ゲノム解析を行う。 The cell analysis method of the present invention further includes, for example, an amplification step, and the amplification step is a step of amplifying the cell-derived genome with respect to the isolated analysis droplet, and the analysis step includes: Genomic analysis is performed on the amplification product of the genome contained in the analysis droplet.
　本発明の単離方法および解析方法は、前記解析用液滴の分取に先だって、前記液滴から増幅産物を除去することがポイントであり、その他の工程や条件は、特に制限されない。 In the isolation method and analysis method of the present invention, the point is to remove the amplification products from the droplets prior to the separation of the analysis droplets, and other steps and conditions are not particularly limited.
　以下、本発明の単離方法および解析方法の実施形態について、例をあげて説明する。本発明の単離方法および解析方法は、下記の実施形態によって何ら限定および制限されない。また、各実施形態の記載は、それぞれ互いに援用できる。 Hereinafter, embodiments of the isolation method and analysis method of the present invention will be described by way of examples. The isolation method and analysis method of the present invention are not limited or restricted by the following embodiments. Moreover, the description of each embodiment can mutually be used.
（解析用液滴の単離方法）
　本発明の単離方法は、前述のように、解析用液滴の単離方法であり、
エマルション形成工程と、核酸増幅工程と、検出工程と、第１分取工程と、増幅産物除去工程と、第２分取工程とを含み、
前記エマルション形成工程は、
　　細胞を含む試料と、エマルション形成溶媒とを接触させて、前記エマルション形成溶媒中に、前記試料中の細胞を含む複数の液滴を形成する工程であり、
前記核酸増幅工程は、
　　前記液滴について、プライマーを用いて、前記細胞由来の核酸の核酸増幅を行う工程であり、
　　前記プライマーは、前記核酸に対する相補配列と第１付加配列とを有し、
前記検出工程は、
　　前記液滴における前記核酸の増幅産物の有無を検出する工程であり、
前記第１分取工程は、
　　前記核酸の増幅産物が検出された液滴を分取する工程であり、
前記増幅産物除去工程は、
　　前記分取した液滴における増幅産物を、担体に第２付加配列が固相化された固相化担体により、前記液滴から除去する工程であり、
　　前記固相化担体における第２付加配列は、前記プライマーにおける前記第１付加配列および前記第１付加配列に対する相補配列の少なくとも一方であり、
　　前記液滴中の前記増幅産物と前記固相化担体とを接触させて、前記固相化担体に前記増幅産物を結合させ、前記液滴から前記固相化担体を分離することで、前記液滴から前記増幅産物を除去し、
前記第２分取工程は、
　　前記増幅産物除去工程により前記増幅産物が除去された液滴を、解析用液滴として分取する工程であることを特徴とする。 (Method for isolating droplets for analysis)
As described above, the isolation method of the present invention is a method for isolating a droplet for analysis,
An emulsion formation step, a nucleic acid amplification step, a detection step, a first fractionation step, an amplification product removal step, and a second fractionation step,
The emulsion forming step includes
A step of contacting a sample containing cells with an emulsion-forming solvent to form a plurality of droplets containing cells in the sample in the emulsion-forming solvent;
The nucleic acid amplification step includes
For the droplet, using a primer, a step of performing nucleic acid amplification of the nucleic acid derived from the cells,
The primer has a complementary sequence to the nucleic acid and a first additional sequence,
The detection step includes
Detecting the presence or absence of the amplification product of the nucleic acid in the droplet,
The first sorting step includes
A step of separating a droplet in which the amplification product of the nucleic acid is detected;
The amplification product removal step includes:
A step of removing amplification products in the sorted droplets from the droplets by a solid-phased carrier in which a second additional sequence is immobilized on a carrier;
The second additional sequence in the solid phase support is at least one of the first additional sequence and the complementary sequence to the first additional sequence in the primer;
Contacting the amplification product in the droplet with the immobilized carrier, binding the amplification product to the immobilized carrier, and separating the immobilized carrier from the droplet; Removing the amplification product from the drop;
The second sorting step includes
It is a step of separating the droplet from which the amplification product has been removed by the amplification product removal step as an analysis droplet.
　前記エマルション形成工程は、前述のように、細胞を含む試料と、エマルション形成溶媒とを接触させて、前記エマルション形成溶媒中に、前記試料中の細胞を含む複数の液滴を形成する工程である。前記エマルション形成溶媒中における前記液滴の形成によって、前記試料を、複数の液滴に分割して区画することができる。そして、前記液滴のそれぞれに、前記試料中の様々な細胞が、分配されることになる。 As described above, the emulsion forming step is a step of bringing a sample containing cells into contact with an emulsion forming solvent to form a plurality of droplets containing cells in the sample in the emulsion forming solvent. . By forming the droplets in the emulsion-forming solvent, the sample can be divided and divided into a plurality of droplets. Then, various cells in the sample are distributed to each of the droplets.
　前記試料の種類は、何ら制限されず、細胞が存在すると考えられる試料があげられる。前記試料は、例えば、生体試料があげられ、具体例としては、血液、リンパ液、脳脊髄液、精液、尿、鼻水、鼻腔ぬぐい液（スワブ）等である。前記生体は、特に制限されず、例えば、ヒト；ウシ、ブタ、ヒツジ、マウス、ラット、ウサギおよびウマ等の非ヒト哺乳類；鳥類；魚類等の動物の生体があげられる。 The type of the sample is not limited at all, and examples include samples that are considered to contain cells. Examples of the sample include a biological sample, and specific examples include blood, lymph, cerebrospinal fluid, semen, urine, nasal discharge, nasal swab, and the like. The living body is not particularly limited and includes, for example, humans; non-human mammals such as cows, pigs, sheep, mice, rats, rabbits and horses; birds; animals such as fish.
　前記エマルション形成溶媒は、特に制限されず、その中に、前記試料を含む液滴を形成できるものであればよい。前記エマルション形成溶媒は、例えば、非水溶性溶媒であり、前記非水溶性溶媒中に、前記試料を含む水溶性の液滴が形成できる。 The emulsion-forming solvent is not particularly limited as long as it can form droplets containing the sample therein. The emulsion-forming solvent is, for example, a water-insoluble solvent, and water-soluble droplets containing the sample can be formed in the water-insoluble solvent.
　前記非水溶性溶媒は、例えば、油、ミネラルオイル、クロロホルム、芳香族化合物等があげられる。前記非水溶性溶媒は、例えば、一種類を単独で使用してもよいし、二種類以上を併用してもよい。 Examples of the water-insoluble solvent include oil, mineral oil, chloroform, and aromatic compounds. As the water-insoluble solvent, for example, one kind may be used alone, or two or more kinds may be used in combination.
　前記液滴の形成においては、例えば、さらに、水溶性溶媒を共存させてもよい。前記水溶性溶媒は、例えば、水、緩衝液、水溶性高分子溶液等があげられる。前記水溶性溶媒は、例えば、一種類を単独で使用してもよいし、二種類以上を併用してもよい。 In the formation of the droplets, for example, a water-soluble solvent may be further present. Examples of the water-soluble solvent include water, a buffer solution, and a water-soluble polymer solution. As the water-soluble solvent, for example, one kind may be used alone, or two or more kinds may be used in combination.
　前記液滴の形成においては、例えば、後述する増幅工程、検出工程、第１分取工程において使用する各種試薬を共存させてもよい。これにより、前記各種試薬を含有する液滴が形成されるため、例えば、後述する各工程において、新たに前記各種試薬を添加することなく、各工程を実施できる。前記試薬に関しては、後述する。 In the formation of the droplets, for example, various reagents used in the amplification process, the detection process, and the first sorting process described later may coexist. Thereby, since the droplet containing the said various reagents is formed, in each process mentioned later, each process can be implemented, without adding the said various reagents newly, for example. The reagent will be described later.
　前記形成する液滴の大きさは、特に制限されず、その平均体積は、下限が、例えば、４ｐＬ以上であり、上限が、例えば、１０ｎＬ以下である。 The size of the droplets to be formed is not particularly limited, and the average volume has a lower limit of, for example, 4 pL or more and an upper limit of, for example, 10 nL or less.
　前記液滴に含まれる細胞の個数は、特に制限されない。前記液滴１個に含まれる細胞数は、例えば、５個以下、２個以下、好ましくは１個である。 The number of cells contained in the droplet is not particularly limited. The number of cells contained in one droplet is, for example, 5 or less, 2 or less, and preferably 1 cell.
　前記エマルション形成溶媒中における液滴の形成方法は、特に制限されず、前記エマルション形成溶媒と、前記試料とを接触させることで、前記エマルション形成溶媒中に、複数の液滴を形成できる。前記接触は、例えば、前記エマルション形成溶媒に、前記試料を接触させてもよいし、前記試料に、前記エマルション形成溶媒を接触させてもよい。前記液滴の形成方法は、例えば、公知のドロップレット作製方法が適用できる。 The method for forming droplets in the emulsion forming solvent is not particularly limited, and a plurality of droplets can be formed in the emulsion forming solvent by bringing the emulsion forming solvent into contact with the sample. In the contact, for example, the sample may be brought into contact with the emulsion-forming solvent, or the emulsion-forming solvent may be brought into contact with the sample. For example, a known droplet manufacturing method can be applied as the droplet forming method.
　前記液滴の形成には、例えば、マイクロ流路を有するエマルション化デバイスを使用できる。前記デバイスは、前記マイクロ流路として、例えば、サンプル用流路と、エマルション形成溶媒用流路と、これらの連結部と、前記連結部から導出される導出流路とを含む。このデバイスを用いた場合、例えば、以下のように液滴が形成される。まず、前記連結部には、前記エマルション形成溶媒用流路から、前記エマルション形成溶媒が導入され、つぎに、前記エマルション形成溶媒が導入された前記連結部に向かって、前記サンプル用流路から、前記試料（例えば、任意で前記水溶性溶媒を含む）が導入される。そして、前記連結部において、前記エマルション形成溶媒と前記試料とが接触し、エマルション化され、前記連結部から前記導出流路に、エマルション（前記エマルション形成溶媒中に液滴が分散された状態）として導出される。なお、前記試料と前記水溶性溶媒とは、例えば、前述のように、予め混合した混合液として、前記デバイスの流路に導入してもよいし、別々に流路に導入してもよい。後者の場合、前記連結部の上流において、前記サンプル用流路と試薬用流路とを有し、前記連結部に向かって、前記サンプル用流路から前記試料が導入され、前記試薬用流路から前記水溶性溶媒や任意で前記各種試薬等が導入されてもよい。 For the formation of the droplets, for example, an emulsification device having a microchannel can be used. The device includes, as the microchannel, for example, a sample channel, an emulsion-forming solvent channel, a connecting portion thereof, and a lead-out channel derived from the connecting portion. When this device is used, for example, droplets are formed as follows. First, the emulsion forming solvent is introduced into the connecting portion from the emulsion forming solvent flow path, and then toward the connecting portion into which the emulsion forming solvent is introduced, from the sample flow path, The sample (eg, optionally containing the water-soluble solvent) is introduced. Then, in the connecting portion, the emulsion-forming solvent and the sample come into contact with each other to be emulsified, and from the connecting portion to the outlet channel, an emulsion (a state in which droplets are dispersed in the emulsion-forming solvent) Derived. Note that the sample and the water-soluble solvent may be introduced into the channel of the device as a mixed liquid mixed in advance as described above, or may be separately introduced into the channel. In the latter case, the sample channel and the reagent channel are provided upstream of the connecting portion, and the sample is introduced from the sample channel toward the connecting portion, and the reagent channel. The water-soluble solvent and optionally the various reagents may be introduced.
　前記増幅工程は、前述のように、前記エマルション形成工程で形成された液滴について、プライマーを用いて、前記細胞由来の核酸の核酸増幅を行う工程である。 As described above, the amplification step is a step of performing nucleic acid amplification of the cell-derived nucleic acid using a primer for the droplets formed in the emulsion formation step.
　前記増幅工程において、増幅させる核酸の種類は、特に制限されず、例えば、解析対象の細胞の種類に応じて適宜決定できる。 In the amplification step, the type of nucleic acid to be amplified is not particularly limited, and can be appropriately determined according to, for example, the type of cell to be analyzed.
　前記増幅工程において、前記核酸増幅の方法は、特に制限されず、ＰＣＲ法等の公知の手法が採用できる。前記核酸増幅に使用する増幅用試薬は、特に制限されず、プライマーの他に、例えば、ポリメラーゼ、ｄＮＴＰ等があげられる。 In the amplification step, the nucleic acid amplification method is not particularly limited, and a known method such as a PCR method can be employed. The amplification reagent used for the nucleic acid amplification is not particularly limited, and examples thereof include polymerase, dNTP and the like in addition to the primer.
　前記増幅工程に使用するプライマーは、前述のように、核酸に対する相補配列と第１付加配列とを有する。前記相補配列は、前述のように、増幅させる核酸の配列に応じて、適宜決定できる。前記相補配列は、例えば、増幅させる核酸に対して、プライマーとしてアニーリングできればよく、その前記核酸に対する相補性の程度（％）は、特に制限されず、例えば、完全相補（１００％）でもよく、部分相補でもよく、後者の場合は、例えば、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上である。 The primer used in the amplification step has a complementary sequence to the nucleic acid and a first additional sequence as described above. As described above, the complementary sequence can be appropriately determined according to the sequence of the nucleic acid to be amplified. The complementary sequence only needs to be annealed as a primer to the nucleic acid to be amplified, for example, and the degree of complementarity (%) with respect to the nucleic acid is not particularly limited, and may be completely complementary (100%), for example. In the latter case, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
　前記第１付加配列は、特に制限されず、例えば、第１付加配列が、前記増幅対象である核酸にアニーリングして伸長が開始されることを防止する点から、前記核酸に対して非相補配列であることが好ましい。第１付加配列は、例えば、前記相補配列の３’末端側からの伸長を妨げないため、前記相補配列の５’側に結合している。 The first additional sequence is not particularly limited. For example, the first additional sequence is a non-complementary sequence to the nucleic acid from the viewpoint of preventing the first additional sequence from being annealed and starting extension. It is preferable that For example, the first additional sequence is bound to the 5 'side of the complementary sequence so as not to prevent extension from the 3' end side of the complementary sequence.
　前記プライマーの全長、前記相補配列と前記第１付加配列の各長さは、特に制限されない。前記プライマーの全長は、例えば、２０～２５ｍｅｒであり、前記相補配列の長さは、例えば、８０～３００ｂｐであり、前記第１付加配列の長さは、例えば、１０～２０ｂｐである。 The total length of the primer and the lengths of the complementary sequence and the first additional sequence are not particularly limited. The total length of the primer is, for example, 20 to 25 mer, the length of the complementary sequence is, for example, 80 to 300 bp, and the length of the first additional sequence is, for example, 10 to 20 bp.
　前記増幅工程で使用する前記増幅用試薬は、例えば、予め前記液滴に含有させてもよいし、前記増幅工程時に前記液滴に添加してもよく、操作が容易であることから、前者が好ましい。前者の場合、例えば、前記エマルション形成工程において、前記増幅用試薬の存在下、液滴を形成し、前記核酸増幅工程において、前記液滴に含まれる前記増幅用試薬を用いて、増幅を行えばよい。また、後者の場合、例えば、前記エマルション形成工程において、前記増幅用試薬の非存在下、液滴を形成し、前記核酸増幅工程において、前記増幅用試薬を添加し、増幅を行えばよい。 The amplification reagent used in the amplification step may be previously contained in the droplet, for example, or may be added to the droplet during the amplification step, and the operation is easy. preferable. In the former case, for example, in the emulsion formation step, a droplet is formed in the presence of the amplification reagent, and in the nucleic acid amplification step, amplification is performed using the amplification reagent contained in the droplet. Good. In the latter case, for example, droplets may be formed in the emulsion formation step in the absence of the amplification reagent, and amplification may be performed by adding the amplification reagent in the nucleic acid amplification step.
　前述のように、前記エマルション形成工程は、マイクロ流路を有するデバイスを用いて行えることから、前記増幅工程も、同様に、前記デバイスのマイクロ流路内で行うことが好ましい。この場合、前記デバイスは、例えば、前記導出流路の下流に連結された増幅流路を有し、前記増幅流路内で前記増幅を行うことができる。 As described above, since the emulsion forming step can be performed using a device having a microchannel, the amplification step is preferably performed in the microchannel of the device as well. In this case, the device has, for example, an amplification channel connected downstream of the outlet channel, and the amplification can be performed in the amplification channel.
　前記検出工程は、前述のように、前記液滴における前記核酸の増幅産物の有無を検出する工程である。前記増幅の有無を検出する方法は、特に制限されず、例えば、前記増幅方法、使用した前記増幅用試薬の種類等に応じて、適宜設定できる。前記検出工程において、前記増幅の有無の検出は、例えば、前記液滴ごとに行う。 The detection step is a step of detecting the presence or absence of the amplification product of the nucleic acid in the droplet as described above. The method for detecting the presence or absence of the amplification is not particularly limited, and can be appropriately set depending on, for example, the amplification method, the type of the amplification reagent used, and the like. In the detection step, the presence / absence of the amplification is detected for each droplet, for example.
　前記増幅の有無を検出する方法は、特に制限されず、例えば、一般的な、インターカレータ法、プローブ法等があげられる。前記検出には、例えば、前記検出方法に応じて、インターカレータ、プローブ等の検出用試薬が使用できる。前記プローブの種類は、特に制限されず、例えば、ＴａｑＭａｎ（登録商標）プローブ等が使用できる。前記検出用試薬は、例えば、前記増幅用試薬と共存させてもよい。 The method for detecting the presence or absence of amplification is not particularly limited, and examples thereof include a general intercalator method and probe method. For the detection, for example, a detection reagent such as an intercalator or a probe can be used according to the detection method. The type of the probe is not particularly limited, and for example, a TaqMan (registered trademark) probe or the like can be used. The detection reagent may coexist with the amplification reagent, for example.
　前記検出工程で使用する前記検出用試薬は、例えば、予め前記液滴に含有させてもよいし、前記検出工程時に前記液滴に添加してもよく、操作が容易であることから、前者が好ましい。前者の場合、例えば、前記エマルション形成工程において、前記増幅用試薬および前記検出用試薬の存在下、液滴を形成し、前記核酸増幅工程において、前記液滴に含まれる前記増幅用試薬を用いて、増幅を行い、前記検出工程において、前記液滴に含まれる前記検出用試薬を用いて、前記増幅の有無を検出すればよい。また、後者の場合、例えば、前記エマルション形成工程において、前記増幅用試薬および前記検出用試薬の非存在下、液滴を形成し、前記液滴に、前記増幅用試薬および前記検出用試薬を添加し、前記核酸増幅工程において、増幅を行い、前記検出工程において、増幅の有無を検出すればよい。前記増幅用試薬と前記検出用試薬の添加の順序は、特に制限されず、例えば、前記増幅工程において、両者を添加してもよいし、前記増幅工程において前記増幅用試薬を添加し、前記検出工程において、前記検出用試薬を添加してもよい。 The detection reagent used in the detection step may be previously contained in the droplet, for example, or may be added to the droplet during the detection step, and is easy to operate. preferable. In the former case, for example, in the emulsion formation step, a droplet is formed in the presence of the amplification reagent and the detection reagent, and in the nucleic acid amplification step, the amplification reagent contained in the droplet is used. Amplification is performed, and in the detection step, the presence or absence of the amplification may be detected using the detection reagent contained in the droplet. In the latter case, for example, in the emulsion forming step, a droplet is formed in the absence of the amplification reagent and the detection reagent, and the amplification reagent and the detection reagent are added to the droplet. Then, amplification may be performed in the nucleic acid amplification step, and the presence or absence of amplification may be detected in the detection step. The order of addition of the amplification reagent and the detection reagent is not particularly limited. For example, both may be added in the amplification step, or the amplification reagent is added in the amplification step, and the detection is performed. In the step, the detection reagent may be added.
　前述のように、前記エマルション形成工程および前記増幅工程は、マイクロ流路を有するデバイスを用いて行えることから、前記検出工程も、同様に、前記デバイスのマイクロ流路内で行うことが好ましい。この場合、前記デバイスは、例えば、前記増幅流路の下流に検出流路を有し、前記検出流路内で前記増幅を行うことができる。 As described above, since the emulsion formation step and the amplification step can be performed using a device having a microchannel, the detection step is preferably performed in the microchannel of the device as well. In this case, for example, the device has a detection channel downstream of the amplification channel, and can perform the amplification in the detection channel.
　本発明において、前記増幅工程と前記検出工程は、例えば、順番に行ってもよいが、同時に行うこともできる。後者の場合、例えば、前記増幅用試薬と前記検出用試薬の共存下で、前記増幅工程と前記検出工程とを行えばよい。また、前記マイクロ流路を有するデバイスを用いる場合、前記デバイスは、例えば、前記導出流路の下流に連結された増幅流路を有し、前記増幅流路内で前記増幅と前記検出とを行うことができる。 In the present invention, the amplification step and the detection step may be performed sequentially, for example, but can be performed simultaneously. In the latter case, for example, the amplification step and the detection step may be performed in the presence of the amplification reagent and the detection reagent. When using a device having the microchannel, the device has, for example, an amplification channel connected downstream of the outlet channel, and performs the amplification and the detection in the amplification channel. be able to.
　前記第１分取工程は、前述のように、前記核酸の増幅産物が検出された液滴を分取する工程である。 As described above, the first sorting step is a step of sorting the droplet in which the amplification product of the nucleic acid is detected.
　前述のように、前述の各工程は、マイクロ流路を有するデバイスを用いて行えることから、前記第１分取工程も、同様に、前記デバイスのマイクロ流路内で行うことが好ましい。この場合、前記デバイスは、例えば、前記検出流路の下流に、分岐する一対の流路を有し、一方の流路が、増幅が検出された液滴の流路（第１分取流路）であり、他方の流路が、増幅が検出されなかった液滴の流路である。前記デバイスによれば、例えば、前記検出流路において、各液滴の増幅の有無を検出し、前記増幅の有無に応じて、各液滴を前記流路の分配することで、増幅が検出された液滴を分取することができる。前記分取の方法は、特に制限されず、例えば、セルソーティングにおける従来公知の方法を採用できる。 As described above, since each of the above-described steps can be performed using a device having a microchannel, it is preferable that the first sorting step is similarly performed in the microchannel of the device. In this case, the device has, for example, a pair of branched flow channels downstream of the detection flow channel, and one of the flow channels is a flow channel of the droplet in which amplification is detected (first sorting flow channel). ) And the other channel is a droplet channel in which amplification was not detected. According to the device, for example, amplification is detected by detecting whether or not each droplet is amplified in the detection flow channel, and distributing each droplet in the flow channel according to the presence or absence of the amplification. Droplets can be collected. The sorting method is not particularly limited, and for example, a conventionally known method in cell sorting can be adopted.
　前記増幅産物除去工程は、前述のように、前記分取した液滴における増幅産物を、担体に第２付加配列が固相化された固相化担体により、前記液滴から除去する工程である。ここで、前記固相化担体における第２付加配列は、前記プライマーにおける前記第１付加配列および前記第１付加配列に対する相補配列の少なくとも一方である。このため、前記液滴中の前記増幅産物と前記固相化担体とを接触させることで、前記固相化担体に前記増幅産物を結合させ、さらに、前記液滴から前記固相化担体を分離することで、前記液滴から前記増幅産物を除去することができる。 As described above, the amplification product removal step is a step of removing amplification products from the sorted droplets from the droplets using a solid-phased carrier in which the second additional sequence is solid-phased on the carrier. . Here, the second additional sequence in the immobilized carrier is at least one of the first additional sequence and the complementary sequence to the first additional sequence in the primer. For this reason, the amplification product in the droplet is brought into contact with the immobilized carrier, thereby binding the amplified product to the immobilized carrier, and further separating the immobilized carrier from the droplet. Thus, the amplification product can be removed from the droplet.
　前記固相化担体における担体は、特に制限されず、例えば、磁性担体である。前記担体の形状は、特に制限されず、例えば、ビーズがあげられる。前記磁性担体の具体例としては、マグネットビーズ、永久磁石、電磁石等があげられる。前記固相化担体が固相化磁性担体の場合、例えば、磁場により、前記液滴から前記増幅産物が結合した固相化担体を分離することで、前記液滴から前記増幅産物を除去できる。 The carrier in the solid phase carrier is not particularly limited, and is, for example, a magnetic carrier. The shape of the carrier is not particularly limited, and examples thereof include beads. Specific examples of the magnetic carrier include magnet beads, permanent magnets, electromagnets and the like. When the solid-phased carrier is a solid-phased magnetic carrier, for example, the amplification product can be removed from the droplet by separating the solid-phased carrier to which the amplification product is bound from the droplet by a magnetic field.
　前記固相化担体における第２付加配列は、前記プライマーによる増幅産物に結合できればよく、例えば、前記プライマーにおける前記第１付加配列と同じ配列でもよいし、前記第１付加配列に対する相補配列でもよい。前記固相化担体は、例えば、前者の配列（第１付加配列と同じ配列）を有する固相化担体でもよいし、後者の配列（第１付加配列に対する相補配列）を有する固相化担体でもよいし、前記両者を併用してもよい。 The second additional sequence in the solid-phased carrier only needs to be able to bind to the amplification product of the primer, and may be, for example, the same sequence as the first additional sequence in the primer or a complementary sequence to the first additional sequence. The solid-phase support may be, for example, a solid-phase support having the former sequence (the same sequence as the first addition sequence) or a solid-phase support having the latter sequence (complementary sequence to the first addition sequence). Alternatively, both the above may be used in combination.
　前記増幅産物除去工程で使用する前記固相化担体は、例えば、予め前記液滴に含有させてもよいし、前記液滴に添加してもよく、操作が容易であることから、前者が好ましい。前者の場合、例えば、前記エマルション形成工程において、前記増幅用試薬、前記固相化担体、および任意で前記検出用試薬の存在下、液滴を形成し、前記核酸増幅工程において、前記液滴に含まれる前記増幅用試薬を用いて、増幅を行い、前記検出工程において、前記液滴に含まれる前記検出用試薬を用いて、前記増幅の有無を検出し、前記増幅産物除去工程において、前記固相化担体を用いて、前記増幅産物を除去すればよい。また、後者の場合、例えば、前記エマルション形成工程において、前記増幅用試薬、前記固相化担体、および任意で前記検出用試薬の非存在下、液滴を形成し、前記液滴に、前記増幅用試薬、前記固相化担体、および任意で前記検出用試薬を添加し、前記核酸増幅工程において、増幅を行い、前記検出工程において、増幅の有無を検出し、前記増幅産物除去工程において、前記増幅産物を除去すればよい。前記増幅用試薬、前記固相化担体および前記検出用試薬の添加の順序は、特に制限されず、例えば、前記増幅工程において、全てを添加してもよいし、各工程において、それぞれを添加してもよい。 The solid-phase support used in the amplification product removal step may be previously contained in the droplet, for example, or may be added to the droplet, and the former is preferable because the operation is easy. . In the former case, for example, in the emulsion formation step, droplets are formed in the presence of the amplification reagent, the solid-phase support, and optionally the detection reagent, and in the nucleic acid amplification step, the droplets are formed. Amplification is performed using the amplification reagent contained, and in the detection step, presence or absence of the amplification is detected using the detection reagent contained in the droplet, and in the amplification product removal step, the solid phase is detected. The amplification product may be removed using a phased carrier. In the latter case, for example, in the emulsion formation step, a droplet is formed in the absence of the amplification reagent, the solid phase support, and optionally the detection reagent, and the amplification is performed on the droplet. Reagent, the solid-phased support, and optionally the detection reagent, and performing amplification in the nucleic acid amplification step, detecting the presence or absence of amplification in the detection step, and in the amplification product removal step, What is necessary is just to remove an amplification product. The order of addition of the amplification reagent, the immobilized carrier and the detection reagent is not particularly limited. For example, all may be added in the amplification step, or each may be added in each step. May be.
　前述のように、前述の工程は、マイクロ流路を有するデバイスを用いて行えることから、前記増幅産物除去工程も、同様に、前記デバイスのマイクロ流路内で行うことが好ましい。この場合、前記デバイスは、例えば、前記分岐した一対の流路のうち、増幅が検出された液滴の流路（第１分取流路）の下流に増幅産物除去流路を有し、前記増幅産物除去流路内で前記増幅産物の除去を行うことができる。 As described above, since the above-described steps can be performed using a device having a microchannel, the amplification product removing step is preferably performed in the microchannel of the device as well. In this case, the device has, for example, an amplification product removal flow path downstream of a flow path (first sorting flow path) of a droplet in which amplification is detected, of the pair of branched flow paths, The amplification product can be removed in the amplification product removal channel.
　前記固相化担体を用いて、前記増幅産物除去流路内で、前記液滴から前記増幅産物を除去する方法は、特に制限されない。具体例として、図１を用いて、前記固相化担体として前記固相化磁性ビーズを使用する例をあげて説明するが、本発明は、以下の説明には何ら制限されない。 The method for removing the amplification product from the droplet in the amplification product removal channel using the solid-phased carrier is not particularly limited. As a specific example, an example in which the solid-phased magnetic beads are used as the solid-phase support will be described with reference to FIG. 1. However, the present invention is not limited to the following description.
　図１は、前記マイクロ流路の一部である前記増幅産物除去流路内で、前記液滴から増幅産物を除去する工程を示す概略図であり、前記マイクロ流路を上から見た平面図である。図１において、矢印Xは、液滴の流れる方法を示す。図１に示すように、前記デバイスのマイクロ流路は、第１分取流路１０の下流に、増幅産物除去流路１１が連結されている。増幅産物除去流路１１は、第１分取流路１０の幅よりも広い幅の磁場発生領域１１ａと、第１分取流路１０と同等の幅の液滴回収領域１１ｂとを有する。磁場発生領域１１ａには、外部からの磁場が発生している。前記磁場は、例えば、磁場発生領域１１ａの外部に磁石を配置することで発生させることができる。 FIG. 1 is a schematic view showing a process of removing an amplification product from the droplet in the amplification product removal channel that is a part of the micro channel, and is a plan view of the micro channel as viewed from above. It is. In FIG. 1, an arrow X indicates a method of flowing a droplet. As shown in FIG. 1, an amplification product removal flow path 11 is connected to the micro flow path of the device downstream of the first sorting flow path 10. The amplification product removal channel 11 includes a magnetic field generation region 11 a having a width wider than that of the first sorting channel 10 and a droplet recovery region 11 b having a width equivalent to that of the first sorting channel 10. An external magnetic field is generated in the magnetic field generation region 11a. The magnetic field can be generated, for example, by arranging a magnet outside the magnetic field generation region 11a.
　図１に示すように、増幅産物（図示せず）と固相化磁性ビーズ３０とを含む液滴２０は、エマルションとして、第１分取流路１０から増幅産物除去流路１１に導入される。増幅産物除去流路１１の磁場発生領域１１aには、磁場が発生しているため、液滴２０に含まれる固相化磁性ビーズ３０は、磁場に引っ張られ、磁場により捕捉され、液滴２０から除かれる。そして、固相化磁性ビーズ３０が除去された液滴２０’は、増幅産物除去流路１１の液滴回収領域１１ｂに流れていく。 As shown in FIG. 1, a droplet 20 including an amplification product (not shown) and a solid-phased magnetic bead 30 is introduced as an emulsion from the first sorting channel 10 to the amplification product removal channel 11. . Since a magnetic field is generated in the magnetic field generation region 11 a of the amplification product removal channel 11, the solid-phased magnetic beads 30 included in the droplet 20 are pulled by the magnetic field and captured by the magnetic field. Excluded. Then, the droplet 20 ′ from which the solid-phased magnetic beads 30 have been removed flows into the droplet collection region 11 b of the amplification product removal channel 11.
　磁場によって固相化磁性ビーズ３０を効果的に捕捉するには、増幅産物除去流路１１に導入する際のエマルションの流速を相対的に遅くし、増幅産物除去流路１１を通過する際のエマルションの流速を相対的にさらに遅くすることが好ましい。流速は、例えば、増幅産物除去流路１１に導入する前の流速を、例えば、１μｍ／ｍｉｎとした場合、増幅産物除去流路１１に導入する際のエマルションの流速は、例えば、０．５～１μｍ／ｍｉｎであり、増幅産物除去流路１１を通過する際のエマルションの流速は、例えば、０．１～１μｍ／ｍｉｎである。 In order to effectively capture the solid-phased magnetic beads 30 by a magnetic field, the flow rate of the emulsion when introduced into the amplification product removal channel 11 is relatively slow, and the emulsion when passing through the amplification product removal channel 11 It is preferable to relatively slow the flow rate. For example, when the flow rate before introduction into the amplification product removal channel 11 is 1 μm / min, the flow rate of the emulsion when introduced into the amplification product removal channel 11 is, for example, 0.5 to The flow rate of the emulsion when passing through the amplification product removal flow path 11 is, for example, 0.1 to 1 μm / min.
　前記第２分取工程は、前述のように、前記増幅産物除去工程により前記増幅産物が除去された液滴を、解析用液滴として分取する工程である。前記分取した解析用液滴は、例えば、後述するように、ゲノム解析等の解析に適用することができる。 As described above, the second sorting step is a step of sorting the droplets from which the amplification products have been removed by the amplification product removing step as analysis droplets. The sorted analysis droplets can be applied to analysis such as genome analysis as described later.
　前述のように、前記デバイスを用いて前記増幅産物除去工程までを行った場合には、例えば、前記デバイスから、前記液滴を導出させ、別の容器（マイクロチューブ等）に回収することで単離できる。 As described above, when the device is used up to the amplification product removal step, for example, the droplet is led out from the device and collected in another container (microtube or the like). Can be separated.
　本発明は、例えば、前記エマルション形成工程の後、前記核酸増幅工程の前、または前記核酸増幅工程と同時に、前記エマルション内の前記液滴に含まれる細胞について、核酸等の細胞内成分を放出する処理を行う。前記細胞内成分の放出は、特に制限されず、例えば、一般的な細胞溶解試薬を用いることができる。前記細胞溶解試薬は、例えば、前記エマルション形成工程の後、前記エマルション内の前記液滴に添加してもよいし、前記エマルション形成工程において、前記試料と前記エマルション形成溶媒とに接触させることで、前記エマルション形成溶媒中の液滴に含有させてもよい。前記試薬としては、例えば、界面活性剤と、塩と、溶媒とを含む溶液があげられる。前記界面活性剤としては、例えば、デオキシコール酸ナトリウム、ラウリル硫酸ナトリウム等の陰イオン界面活性剤等があげられ、前記塩としては、例えば、塩化ナトリウム等があげられ、前記溶媒としては、各種緩衝液があげられる。前記試薬の組成の具体例としては、例えば、５０ｍｍｏｌ／Ｌ　Ｔｒｉｓ－ＨＣｌ、１５０ｍｍｏｌ／Ｌ　ＮａＣｌ、０．５ｗ／ｖ％デオキシコール酸ナトリウム、０．１ｗ／ｖ％ラウリル硫酸ナトリウム、および１．０ｗ／ｖ％　ＮＩＰ－４０等が例示できる。前記細胞溶解試薬を前記液滴に含有させることによって、例えば、前記液滴中で、前記細胞から、核酸等の細胞内成分を放出させることができる。前記液滴中で前記細胞から前記細胞内成分を放出させる際、その処理条件は、特に制限されない。 The present invention, for example, releases intracellular components such as nucleic acids from the cells contained in the droplets in the emulsion after the emulsion formation step, before the nucleic acid amplification step, or simultaneously with the nucleic acid amplification step. Process. The release of the intracellular component is not particularly limited, and for example, a general cell lysis reagent can be used. The cell lysis reagent, for example, may be added to the droplets in the emulsion after the emulsion formation step, or in the emulsion formation step, by contacting the sample and the emulsion formation solvent, You may make it contain in the droplet in the said emulsion formation solvent. Examples of the reagent include a solution containing a surfactant, a salt, and a solvent. Examples of the surfactant include anionic surfactants such as sodium deoxycholate and sodium lauryl sulfate. Examples of the salt include sodium chloride. Examples of the solvent include various buffers. Liquid. Specific examples of the composition of the reagent include, for example, 50 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.5 w / v% sodium deoxycholate, 0.1 w / v% sodium lauryl sulfate, and 1.0 w / An example is v% NIP-40. By including the cell lysis reagent in the droplet, for example, intracellular components such as nucleic acids can be released from the cell in the droplet. When the intracellular components are released from the cells in the droplets, the treatment conditions are not particularly limited.
　本発明は、さらに、解析工程を含んでもよい。前記解析工程は、例えば、前記解析用液滴について解析を行う工程である。前記解析については、後述する。 The present invention may further include an analysis step. The analysis step is, for example, a step of analyzing the analysis droplet. The analysis will be described later.
（細胞由来の解析用液滴の解析方法）
　本発明の解析方法は、前述のように、細胞の解析方法であり、
試料中の細胞由来の解析用液滴の単離工程と、解析工程とを含み、
前記単離工程は、
　細胞を含む試料から、前記本発明の単離方法により、前記細胞由来の解析用液滴を単離する工程であり、
前記解析工程は、
　前記単離された解析用液滴について、解析を行う工程であることを特徴とする。 (Method for analyzing cell-derived droplets for analysis)
The analysis method of the present invention is a cell analysis method as described above,
Including a step of isolating a liquid-derived analysis droplet in a sample, and an analysis step,
The isolation step includes
A step of isolating the cell-derived analysis droplet from a sample containing cells by the isolation method of the present invention,
The analysis step includes
It is a step of analyzing the isolated analysis droplet.
　本発明の解析方法は、本発明の単離方法によって解析用液滴を単離することがポイントであり、その他の工程や条件は、特に制限されない。本発明の解析方法において、前記単離工程の単離方法は、前記本発明の解析用液滴の単離方法の記載を援用できる。 In the analysis method of the present invention, the point is that the droplet for analysis is isolated by the isolation method of the present invention, and other steps and conditions are not particularly limited. In the analysis method of the present invention, the description of the method for isolating the analysis droplet of the present invention can be used for the isolation method of the isolation step.
　本発明の解析方法における前記解析用液滴は、前述のように、前記液滴中の細胞の有無を確認するための核酸増幅により得られた増幅産物が除去されている。このため、前記解析用液滴を用いて、例えば、ゲノム解析を行う場合、従来のような増幅産物による影響を回避することができ、より信頼性に優れる解析結果を得ることができる。 As described above, the analysis droplet in the analysis method of the present invention has an amplification product obtained by nucleic acid amplification for confirming the presence or absence of cells in the droplet. For this reason, for example, when genome analysis is performed using the analysis droplet, it is possible to avoid the influence of the conventional amplification product and to obtain an analysis result with higher reliability.
　本発明の解析方法は、例えば、さらに、増幅工程を含んでもよい。前記増幅工程は、例えば、前記単離した解析用液滴について、前記細胞由来のゲノムを増幅させる工程であり、前記解析工程では、前記解析用液滴に含まれる前記ゲノムの増幅産物について、ゲノム解析を行うことができる。 The analysis method of the present invention may further include an amplification step, for example. The amplification step is, for example, a step of amplifying the cell-derived genome with respect to the isolated analysis droplet. In the analysis step, the genome amplification product contained in the analysis droplet is Analysis can be performed.
　前記解析工程において、解析の種類は、特に制限されず、目的に応じて適宜決定できる。前記解析がゲノム解析の場合、例えば、公知の方法があげられ、具体例としては、Ｆｌｕｏｒｅｓｃｅｎｃｅ　Ｉｎ　Ｓｉｔｕ　Ｈｙｂｒｉｄｉｚａｔｉｏｎ（ＦＩＳＨ）、Ｇ－Ｂａｎｄ、Ｎｅｘｔ―Ｇｅｎｅｒａｔｉｏｎ　Ｓｅｑｕｅｎｃｉｎｇ（ＮＧＳ）等である。 In the analysis step, the type of analysis is not particularly limited, and can be appropriately determined according to the purpose. When the analysis is genome analysis, for example, a known method can be mentioned, and specific examples include Fluorescence In Situ Hybridization (FISH), G-Band, Next-Generation Sequencing (NGS), and the like.
　以上、実施形態および実施例を参照して、本発明を説明したが、本発明は、上記発明のスコープ内で当業者が理解し得る様々な変更をすることができる。また、本明細書で引用する特許文献および学術文献等の文献に記載の内容は、全て引用により本明細書に取り込むものとする。 The present invention has been described above with reference to the exemplary embodiments and examples. However, the present invention can be modified in various ways that can be understood by those skilled in the art within the scope of the above invention. In addition, the contents described in documents such as patent documents and academic documents cited in the present specification are all incorporated herein by reference.
　この出願は、２０１７年４月２６日に出願された日本出願特願２０１７－０８７２３６を基礎とする優先権を主張し、その開示のすべてをここに取り込む。 This application claims priority based on Japanese Patent Application No. 2017-087236 filed on Apr. 26, 2017, the entire disclosure of which is incorporated herein.
　本発明によれば、液滴分取のために、液滴中の細胞の有無を確認すべく、核酸増幅を行うが、得られた増幅産物は、前記液滴から除去できる。このように、前記液滴から、前記液滴分取のための増幅産物は除去されているため、例えば、前記解析用液滴を用いてゲノム解析を行う場合、前述のような、前記増幅産物の影響を回避することができ、より精度に優れたゲノム解析が可能となる。 According to the present invention, nucleic acid amplification is performed in order to confirm the presence or absence of cells in the droplet for droplet collection, and the obtained amplification product can be removed from the droplet. Thus, since the amplification product for separating the droplets is removed from the droplet, for example, when genome analysis is performed using the analysis droplet, the amplification product as described above is used. Can be avoided, and genome analysis with higher accuracy becomes possible.
１０　　第１分取流路
１１　　増幅産物除去流路
２０　　液滴
３０　　固相化磁性ビーズ

 
  10 First Sorting Channel 11 Amplification Product Removal Channel 20 Droplet 30 Solid Phase Magnetic Beads


Claims (12)

  1. エマルション形成工程と、核酸増幅工程と、検出工程と、第１分取工程と、増幅産物除去工程と、第２分取工程とを含み、
    前記エマルション形成工程は、
    　　細胞を含む試料と、エマルション形成溶媒とを接触させて、前記エマルション形成溶媒中に、前記試料中の細胞を含む複数の液滴を形成する工程であり、
    前記核酸増幅工程は、
    　　前記液滴について、プライマーを用いて、前記細胞由来の核酸の核酸増幅を行う工程であり、
    　　前記プライマーは、前記核酸に対する相補配列と第１付加配列とを有し、
    前記検出工程は、
    　　前記液滴における前記核酸の増幅産物の有無を検出する工程であり、
    前記第１分取工程は、
    　　前記核酸の増幅産物が検出された液滴を分取する工程であり、
    前記増幅産物除去工程は、
    　　前記分取した液滴における増幅産物を、担体に第２付加配列が固相化された固相化担体により、前記液滴から除去する工程であり、
    　　前記固相化担体における第２付加配列は、前記プライマーにおける前記第１付加配列および前記第１付加配列に対する相補配列の少なくとも一方であり、
    　　前記液滴中の前記増幅産物と前記固相化担体とを接触させて、前記固相化担体に前記増幅産物を結合させ、前記液滴から前記固相化担体を分離することで、前記液滴から前記増幅産物を除去し、
    前記第２分取工程は、
    　　前記増幅産物除去工程により前記増幅産物が除去された液滴を、解析用液滴として分取する工程であることを特徴とする解析用液滴の単離方法。     An emulsion formation step, a nucleic acid amplification step, a detection step, a first fractionation step, an amplification product removal step, and a second fractionation step,
    The emulsion forming step includes
    A step of contacting a sample containing cells with an emulsion-forming solvent to form a plurality of droplets containing cells in the sample in the emulsion-forming solvent;
    The nucleic acid amplification step includes
    For the droplet, using a primer, a step of performing nucleic acid amplification of the nucleic acid derived from the cells,
    The primer has a complementary sequence to the nucleic acid and a first additional sequence,
    The detection step includes
    Detecting the presence or absence of the amplification product of the nucleic acid in the droplet,
    The first sorting step includes
    A step of separating a droplet in which the amplification product of the nucleic acid is detected;
    The amplification product removal step includes:
    A step of removing amplification products in the sorted droplets from the droplets by a solid-phased carrier in which a second additional sequence is immobilized on a carrier;
    The second additional sequence in the solid phase support is at least one of the first additional sequence and the complementary sequence to the first additional sequence in the primer;
    Contacting the amplification product in the droplet with the immobilized carrier, binding the amplification product to the immobilized carrier, and separating the immobilized carrier from the droplet; Removing the amplification product from the drop;
    The second sorting step includes
    A method for isolating an analysis droplet, wherein the droplet from which the amplification product has been removed in the amplification product removal step is a step of separating the droplet as an analysis droplet.
  2. 前記固相化担体における担体が、磁性担体であり、
    前記第１分取工程において、磁場により、前記液滴から前記増幅産物が結合した固相化担体を分離する、請求項１記載の解析用液滴の単離方法。     The carrier in the solid-phased carrier is a magnetic carrier,
    The method for isolating a droplet for analysis according to claim 1, wherein, in the first sorting step, the solid-phase support to which the amplification product is bound is separated from the droplet by a magnetic field.
  3. 前記磁性担体が、磁性ビーズである、請求項２記載の解析用液滴の単離方法。 The method for isolating a droplet for analysis according to claim 2, wherein the magnetic carrier is a magnetic bead.
  4. 前記エマルション形成工程において、前記プライマーを含む増幅用試薬および前記固相化担体の存在下、液滴を形成し、
    前記核酸増幅工程において、前記液滴に含まれる前記増幅用試薬を用いて、前記細胞由来の核酸の増幅を行い、
    前記第２分取工程において、前記液滴に含まれる前記固相化担体を用いて、前記核酸増幅物の除去を行う、請求項１から３のいずれか一項に記載の解析用液滴の単離方法。     In the emulsion formation step, droplets are formed in the presence of the amplification reagent including the primer and the solid-phased carrier,
    In the nucleic acid amplification step, using the amplification reagent contained in the droplet, the cell-derived nucleic acid is amplified,
    4. The analysis droplet according to claim 1, wherein in the second sorting step, the nucleic acid amplification product is removed using the solid-phased support contained in the droplet. Isolation method.
  5. 前記エマルション形成工程において、前記プライマーを含む増幅用試薬および前記固相化担体の非存在下、液滴を形成し、
    前記エマルション形成工程後、前記液滴に、前記増幅用試薬および前記固相化担体を添加する、請求項１から３のいずれか一項に記載の解析用液滴の単離方法。     In the emulsion formation step, droplets are formed in the absence of the amplification reagent containing the primer and the solid phase support,
    The method for isolating the analysis droplet according to any one of claims 1 to 3, wherein the amplification reagent and the solid-phase support are added to the droplet after the emulsion formation step.
  6. 前記プライマーを含む増幅用試薬に、前記増幅の検出のための検出試薬を共存させ、
    前記検出試薬が、インターカレータまたはプローブを含む、請求項４または５記載の解析用液滴の単離方法。     The amplification reagent containing the primer coexists with a detection reagent for detection of the amplification,
    The method for isolating a droplet for analysis according to claim 4 or 5, wherein the detection reagent contains an intercalator or a probe.
  7. さらに、解析工程を含み、
    前記解析工程は、前記第２分取工程で分取した前記解析用液滴を用いて、前記液滴に含まれる細胞由来のゲノムを解析する工程である、請求項１から６のいずれか一項に記載の解析用液滴の単離方法。     In addition, the analysis process includes
    The analysis step is a step of analyzing a cell-derived genome contained in the droplet using the analysis droplet sorted in the second sorting step. The method for isolating the analysis droplet according to Item.
  8. 前記エマルション形成工程において、
    　前記エマルション形成溶媒は、非水溶性溶媒を含み、
    　前記非水溶性溶媒中に、前記複数の液滴が形成され、
    　前記複数の液滴は、前記試料を含む水溶性の液滴である、請求項１から７のいずれか一項に記載の解析用液滴の単離方法。     In the emulsion formation step,
    The emulsion forming solvent includes a water-insoluble solvent,
    The plurality of droplets are formed in the water-insoluble solvent,
    8. The method of claim 1, wherein the plurality of droplets are water-soluble droplets containing the sample.
  9. 流路を有するデバイスを使用し、
    前記流路内で、前記エマルション形成工程、前記増幅工程、前記検出工程、前記第１分取工程、および前記増幅産物除去工程を行う、請求項１から８のいずれか一項に記載の解析用液滴の単離方法。     Using a device with a flow path,
    The analysis according to any one of claims 1 to 8, wherein the emulsion formation step, the amplification step, the detection step, the first fractionation step, and the amplification product removal step are performed in the flow path. Droplet isolation method.
  10. 前記流路内で、さらに、前記第２分取工程を行う、請求項９記載の解析用液滴の単離方法。 The method for isolating a droplet for analysis according to claim 9, wherein the second sorting step is further performed in the flow path.
  11. 試料中の細胞由来の解析用液滴の単離工程と、解析工程とを含み、
    前記単離工程は、
    　細胞を含む試料から、請求項１から１０のいずれか一項に記載の単離方法により、前記細胞由来の解析用液滴を単離する工程であり、
    前記解析工程は、
    　前記単離された解析用液滴について、解析を行う工程であることを特徴とする細胞の解析方法。     Including a step of isolating a liquid-derived analysis droplet in a sample, and an analysis step,
    The isolation step includes
    It is a step of isolating the cell-derived analysis droplet from a sample containing cells by the isolation method according to any one of claims 1 to 10.
    The analysis step includes
    A method for analyzing a cell, which is a step of analyzing the isolated analysis droplet.
  12. さらに、増幅工程を含み、
    前記増幅工程は、前記単離した解析用液滴について、前記細胞由来のゲノムを増幅させる工程であり、
    前記解析工程は、前記解析用液滴に含まれる前記ゲノムの増幅産物について、ゲノム解析を行う、請求項１１記載の細胞の解析方法。

     
          And further includes an amplification step,
    The amplification step is a step of amplifying the cell-derived genome with respect to the isolated analysis droplet,
    The cell analysis method according to claim 11, wherein in the analysis step, genome analysis is performed on the amplification product of the genome contained in the analysis droplet.


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