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WO2018198599A1 - Procédé d'isolement de gouttelettes pour analyse dérivées d'une cellule, et procédé d'analyse de cellules - Google Patents

Procédé d'isolement de gouttelettes pour analyse dérivées d'une cellule, et procédé d'analyse de cellules Download PDF

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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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Japanese (ja)
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伸也 砂永
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Enplas Corp
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Enplas Corp
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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.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Selon l'invention, afin de fournir un procédé qui, sans affecter une analyse ultérieure, permet d'isoler des gouttelettes devant être fournies en vue d'une analyse, et de fournir un procédé d'analyse, le procédé d'isolement de gouttelettes pour analyse est caractérisé en ce qu'il comprend une étape de formation d'émulsion, une étape d'amplification d'acide nucléique, une étape de détection, une première étape de tri, une étape d'élimination de produit d'amplification, et une seconde étape de tri. À l'étape de formation d'émulsion, un échantillon qui contient des cellules et un solvant de formation d'émulsion sont mis en contact pour former de multiples gouttelettes qui contiennent les cellules dans l'échantillon dans le solvant de formation d'émulsion. À l'étape d'amplification d'acide nucléique, une amorce est utilisée dans les gouttelettes pour effectuer une amplification d'acide nucléique d'acides nucléiques dérivés des cellules. L'amorce présente une séquence complémentaire de celle des acides nucléiques, et une première séquence ajoutée. À l'étape de détection, la présence ou l'absence de produits d'amplification des acides nucléiques dans les gouttelettes est détectée. À la première étape de tri, les gouttelettes dans lesquelles les produits d'amplification des acides nucléiques ont été détectés sont triées. À l'étape d'élimination de produit d'amplification, les produits d'amplification dans les gouttelettes triées sont éliminés des gouttelettes au moyen d'un support immobilisé comprenant une seconde séquence ajoutée immobilisée sur un support, la seconde séquence ajoutée sur le support immobilisé étant la première séquence ajoutée dans l'amorce et/ou une séquence complémentaire de la première séquence ajoutée, les produits d'amplification dans les gouttelettes et les supports immobilisés sont mis en contact, les produits d'amplification sont liés aux supports immobilisés, et par séparation des supports immobilisés des gouttelettes, les produits d'amplification sont éliminés des gouttelettes. À la seconde étape de tri, les gouttelettes à partir desquelles les produits d'amplification ont été éliminés à l'étape d'élimination de produit d'amplification sont triées sous forme de gouttelettes pour analyse.
PCT/JP2018/010971 2017-04-26 2018-03-20 Procédé d'isolement de gouttelettes pour analyse dérivées d'une cellule, et procédé d'analyse de cellules Ceased WO2018198599A1 (fr)

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JP2017087236A JP2018183097A (ja) 2017-04-26 2017-04-26 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

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KR20210138017A (ko) * 2019-02-11 2021-11-18 울티마 제노믹스, 인크. 핵산 분석 방법

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JP2007535892A (ja) * 2003-01-29 2007-12-13 ４５４ コーポレーション ビーズエマルジョン核酸増幅

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