WO2016151719A1 - Nucleic acid reaction device - Google Patents

Nucleic acid reaction device Download PDF

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Publication number
WO2016151719A1
WO2016151719A1 PCT/JP2015/058675 JP2015058675W WO2016151719A1 WO 2016151719 A1 WO2016151719 A1 WO 2016151719A1 JP 2015058675 W JP2015058675 W JP 2015058675W WO 2016151719 A1 WO2016151719 A1 WO 2016151719A1
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nucleic acid
probe
sequence
acid reaction
cell
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PCT/JP2015/058675
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French (fr)
Japanese (ja)
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白井　正敬
友幸 坂井
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株式会社日立製作所
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Priority to PCT/JP2015/058675 priority Critical patent/WO2016151719A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • 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

Definitions

  • the present invention relates to a gene expression analysis method and a device therefor.
  • the present invention relates to a method for analyzing gene expression at the level of one cell and a device therefor.
  • Single cell analysis is a technology for detecting and quantifying biomolecules such as DNA, RNA, and proteins in cells with high accuracy for each single cell. There are the following two types of single cell analysis techniques.
  • the first technique does not include a means for isolating cells, and is a technique consisting only of means for treating biological materials in cells with high efficiency.
  • the first technique will be described using gene expression analysis at the single cell level as an example.
  • the cells are separated and dispensed one by one into a resin container (for example, a PCR tube or a well of a microtiter plate) one by one by an apparatus such as a cell sorter generally well known by those skilled in the art.
  • a resin container for example, a PCR tube or a well of a microtiter plate
  • an apparatus such as a cell sorter generally well known by those skilled in the art.
  • a tag sequence for identifying wells of a PCR tube or a microtiter plate is inserted, for example, at the end sequence connection (adapter ligation).
  • the sequence analysis data by the DNA sequencer is totaled and the gene expression analysis is performed.
  • the cells are subjected to chemical treatment such as trypsin treatment to separate the cells into pieces, and then to a cell sorter or the like. It is necessary to introduce. Therefore, information on the positional relationship between cells and gene analysis data for each cell cannot be associated with each other.
  • the reagent is dispensed into the resin container manually or by robot, the amount of the reaction solution containing the reagent is often on the order of ⁇ L due to evaporation and dispensing accuracy problems.
  • the second technique includes means for isolating cells, and means for isolating cells and means for treating biological materials in the cells with high efficiency are functionally connected.
  • a technique for treating cells captured in a reaction vessel in a nucleic acid reaction device in the same reaction vessel or an adjacent reaction vessel is known.
  • the second technique will be described using gene expression analysis at the single cell level as an example.
  • cells are captured on a nucleic acid reaction device and a nucleic acid reaction is performed on the same device.
  • Patent Document 1 describes a device in which a plurality of cell capture units for individually capturing individual cells in a reaction chamber in a flow cell device are arranged in an array to isolate cells to be measured. Has been.
  • a nucleic acid probe containing a poly-T sequence for capturing extracted mRNA and a cell recognition tag sequence having a known sequence and different for each captured cell position is included in the device. It is described that it is fixed. Further, it is described that a cDNA library in which different sequences are introduced for each cell is constructed by capturing mRNA extracted from cells with this nucleic acid probe and synthesizing corresponding cDNA.
  • Amplification products for sequencing by a large-scale nucleic acid sequence analyzer can be obtained by PCR amplification using this cDNA library as a template. From the obtained sequence data, gene expression analysis data is obtained by a DNA sequencer or the like as in the first technique. In this second technique, it is possible to correlate the information on the positional relationship of the cells with the gene analysis results, and because high-density parallel processing on the cell device is possible, the amount of reagent can be reduced, Reagent costs can be reduced.
  • the second technique it is possible to correlate the information on the positional relationship between the cells and the gene analysis results, and high-density parallel processing of cells on the device is possible.
  • the cost can be reduced and the reagent cost can be reduced, it has been difficult to efficiently perform the sequence analysis of the region other than the 3 ′ end.
  • sequence analysis of only the 3 'end for example, it is difficult to analyze mutations that characterize cancer cells, and sequence analysis of all regions of mRNA is required.
  • genome analysis is important in cancer diagnosis and research, and therefore, sequence analysis of an arbitrary region is required for genome sequences.
  • the resin reaction vessel in order to reduce the volume of the reaction reagent, the resin reaction vessel must be made smaller and arranged with high density.
  • the planar microchannel structure In order to introduce different DNA probes to a container arranged at high density, the planar microchannel structure requires a space for the channel wiring, which increases the area of the device and makes the device expensive. There was a problem of becoming.
  • the present inventor captured each cell in the nucleic acid reaction unit, and released the probe fixed in advance in the nucleic acid reaction unit after the cell capture, so that each nucleic acid reaction unit We have developed a device that can process samples without supplying a separate reagent for each.
  • the present invention includes the following aspects.
  • a nucleic acid reaction device comprising a substrate (101) including at least one nucleic acid reaction section (104), wherein the nucleic acid reaction section (104) is provided from a first opening that holds at least one cell.
  • a first probe comprising a capture sequence (203) comprising a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid extracted from a cell (1) (201), and (2) a tag sequence (204) comprising a unique base sequence for each nucleic acid reaction part, and a single-stranded nucleic acid captured by the first probe on the 3 ′ end side of the tag sequence
  • a second probe 211 containing a base sequence (208) complementary to a part of the base sequence of the nucleic acid reaction part (104) via the immobilization part.
  • the nucleic acid reaction device wherein the second probe or the fixing part of the second probe includes a cutting part (207).
  • the first probe (201) includes the common sequence (205) and the tag sequence (204) on the 5 ′ end side of the capture sequence (203) in this order from the 5 ′ end side.
  • (6) The nucleic acid reaction device according to any one of (1) to (5), wherein the nucleic acid reaction part has a porous structure.
  • (7) The nucleic acid reaction device according to (6), wherein the first probe (201) and / or the second probe (211) is fixed to the porous surface.
  • (8) The nucleic acid reaction device according to any one of (1) to (7), wherein the nucleic acid reaction unit has one or more carriers.
  • a flow cell device comprising the nucleic acid reaction device according to (11), a first channel in contact with the first opening, and a second channel in contact with the second opening.
  • a step of cleaving the cleaved portion (207) to release the second probe (211), the released second probe (211) is a part of the single-stranded RNA captured by the first probe
  • a nucleic acid comprising a base sequence complementary to a part of the single-stranded RNA captured by the first probe (201) using the hybridized step and the hybridized second probe (201) as a primer;
  • the method comprising.
  • a gene analysis method comprising a step of obtaining cDNA by the method according to (13), a step of amplifying the cDNA, and a step of sequencing the amplified nucleic acid.
  • FIG. 1 is a schematic view of an example of a nucleic acid reaction device according to the present invention.
  • FIG. 2-1 is a diagram showing a process of capturing and amplifying a nucleic acid to which a single-stranded nucleic acid is added and a tag sequence is added using the method of the present invention.
  • FIG. 2-2 is a diagram showing a process of capturing and amplifying a nucleic acid to which a single-stranded nucleic acid is added, a tag sequence is added, using the method of the present invention.
  • FIG. 2-3 is a diagram showing a process of capturing a single-stranded nucleic acid, synthesizing and amplifying a nucleic acid added with a tag sequence using the method of the present invention.
  • FIG. 2-4 is a diagram showing a process of capturing and amplifying a nucleic acid to which a single-stranded nucleic acid is added and a tag sequence is added using the method of the present invention.
  • FIG. 3 is a diagram showing an example of the nucleic acid reaction device of the present invention.
  • FIG. 4 is a diagram showing an example of a flow cell device of the present invention.
  • FIG. 5 is a diagram showing details of the restriction enzyme cleavage site.
  • FIG. 6 is a diagram showing an example of the nucleic acid reaction device of the present invention.
  • FIG. 7 is a diagram showing an example of the flow cell device of the present invention.
  • FIG. 8 is a diagram showing a second probe cleaved by a photoreaction.
  • FIG. 9 is a diagram showing details of the cleavage of the second probe by photoreaction.
  • FIG. 10 is a diagram showing an example of the flow cell device of the present invention.
  • nucleic acid reaction device The nucleic acid reaction device of the present invention comprises a substrate including at least one nucleic acid reaction part including one cell holding region. The configuration of the nucleic acid reaction device of the present invention will be described below.
  • substrate refers to a support comprising one or more nucleic acid reaction units.
  • the material of the substrate is not particularly limited as long as it is made of a material generally used in the technical field in gene expression analysis of DNA and RNA.
  • a metal made of an alloy such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, nickel, and stainless steel; silicon; glass, quartz glass, fused quartz, synthetic quartz, alumina, and photosensitive glass Glass materials (these materials are basically transparent); polyester resins, polystyrene, polyethylene resins, polypropylene resins, ABS resins (Acrylonitrile Butadiene Styrene resins), plastics such as nylon, acrylic resins and vinyl chloride resins (generally Non-transparent but can also be transparent to allow optical measurements); agarose, dextran, cellulose, polyvinyl alcohol, nitrocellulose, chitin, chitosan.
  • the substrate may be composed of two or more different materials.
  • the substrate skeleton is composed of the plastic or metal, and the sheet with pores is, for example, a film made of alumina, glass, and silicon; acrylamide gel, gelatin, Examples include a gel film made of modified polyethylene glycol, modified polyvinyl pyrrolidone, and hydrogel; or a cellulose acetate, nitrocellulose, or a mixed membrane thereof, and a membrane made of nylon membrane.
  • the substrate can be processed into a housing or the like as necessary. Further, it may be made of a material that is transparent, that is, capable of transmitting at least a part of light having a wavelength of 300 nm to 10000 nm. This makes it possible to optically analyze gene expression on the substrate.
  • the nucleic acid reaction part is two-dimensionally arranged on the substrate.
  • substrate is not ask
  • it is a flat substrate that can be manufactured by a manufacturing apparatus for semiconductor process.
  • Nucleic acid reaction part is an area
  • a tag sequence consisting of a unique base sequence for each nucleic acid reaction part is introduced at the end of the nucleic acid extracted from the cells held in the cell holding region described later. Do.
  • nucleic acid amplification may be performed in this region.
  • the number of nucleic acid reaction units per substrate is not particularly limited. There may be one or more. Usually, it may be in the range of 10 to 10 5 .
  • the nucleic acid reaction part includes one cell holding region, a first probe, and a second probe. The cell holding region, the first probe, and the second probe constituting the nucleic acid reaction part will be described below.
  • the “cell holding area” is composed of one or a plurality of first openings.
  • the “first opening” is a hole opened to the outside in the nucleic acid reaction part.
  • the number of holes in the first opening is not limited. Cells may be held by one hole, or cells may be held by a plurality of holes.
  • the shape of the first opening is not particularly limited. For example, a cylindrical shape, a substantially cylindrical shape, an elliptical columnar shape, a substantially elliptical columnar shape, a rectangular shape, a substantially rectangular shape, a cubic shape, a substantially cubic shape, a conical shape, a substantially conical shape, a pyramid shape, a substantially pyramid shape, and the like are applicable.
  • the opening diameter of the cell holding region is slightly smaller than the diameter of the cell so that one cell just fits.
  • the diameter may be in the range of 5 ⁇ m to 50 ⁇ m.
  • the depth of the cell holding region is preferably from 1 ⁇ m to a depth that allows just one cell to fit, for example, in the range of 5 to 100 ⁇ m.
  • the “cell holding region” may be composed of a plurality of first openings, but is preferably composed of one first opening. If the cell holding region comprising the first opening holds only one cell (in this specification, the cell holding region holds only one cell is also referred to as “isolation”), one cell at a time It is particularly preferable that the cell holding region holds only one cell because it can be processed by an individual nucleic acid reaction part.
  • the cell holding region may hold two or more cells, but in this case, since the cell holding region and the nucleic acid reaction part correspond spatially, The analysis result can be obtained in a form including the positional information of the tissue section in the two-dimensional plane.
  • the “first probe” is a probe composed of a nucleic acid.
  • the first probe is composed of DNA in principle, but is not limited thereto, and may include, for example, RNA or artificial nucleic acid.
  • the first probe includes a capture sequence (203) having a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid extracted from the cells held by the cell holding region. Further, the first probe further includes a common sequence and / or a tag sequence as necessary. Hereinafter, each sequence constituting the first probe will be specifically described.
  • the “capture sequence” is an essential sequence constituting the first probe, a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid extracted from a cell held in the cell holding region, or a random sequence And is configured to capture the extracted single-stranded nucleic acid.
  • the base sequence of the capture sequence is not particularly limited as long as it can hybridize with and capture the target single-stranded nucleic acid. Therefore, it can be designed appropriately in consideration of the type and sequence of the nucleic acid.
  • the target single-stranded nucleic acid includes messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, single-stranded DNA, and fragments thereof.
  • the length of the capture sequence may be any length as long as it can capture a single-stranded nucleic acid as a target (target) by hybridization.
  • the capture sequence is preferably a base sequence complementary to a sequence close to or near the 3 'end of the base sequence of the single-stranded nucleic acid.
  • an oligo (dT) sequence complementary to a poly A sequence that is a part of the mRNA sequence may be used as a capture sequence.
  • the polymerization degree of dT constituting the oligo (dT) sequence may be any polymerization degree that can capture the poly A sequence of mRNA by hybridization. For example, 8 to 40, preferably 8 to 30.
  • an oligo (dT) sequence is used as a capture sequence, it is preferable to add a random sequence of 2 bases to the 3 ′ end. This makes it possible to greatly reduce the amount of artifact when synthesizing cDNA. Examples of such a random sequence include a VN sequence (V is A, G, or C, and N is A, G, C, or T).
  • the target single-stranded nucleic acid is a single-stranded nucleic acid derived from microRNA or genomic DNA
  • a base sequence or random sequence complementary to a part of the base sequence of the single-stranded nucleic acid can be used.
  • the “tag sequence” is a sequence included in the first probe as necessary, and is an identification tag to be attached to the reaction product in the nucleic acid reaction part. Therefore, when there are a plurality of nucleic acid reaction units, the tag sequence includes a base sequence unique to each nucleic acid reaction unit.
  • the nucleic acid reaction part in the nucleic acid reaction device can identify the nucleic acid derived from the extracted nucleic acid. Since the nucleic acid reaction part corresponds to the cell holding region, the corresponding cell can be grasped from the information of the tag sequence.
  • the base sequence constituting the tag sequence differs in principle for each nucleic acid reaction part when a plurality of nucleic acid reaction parts exist on the substrate, but may be common to a plurality of nucleic acid reaction parts if necessary. For example, the case where a common tag sequence is used for every five nucleic acid reaction parts on one substrate corresponds.
  • the “common sequence” is a selected sequence that can function as a forward (Fw) primer sequence for amplifying a cleaved fragment in the nucleic acid amplification step of the gene analysis method using the nucleic acid reaction device of the present invention. . Therefore, in the first probe, as a rule, it is arranged on the 5 ′ end side.
  • the base length of the common sequence is not particularly limited as long as it is an appropriate length as a primer.
  • the length can be 8 to 60 bases, preferably 10 to 50 bases.
  • the base sequence of the common sequence is not particularly limited, but it is preferable to design the base sequence so as to have an appropriate Tm value as a primer sequence.
  • the Tm value may be 50 ° C. or higher, preferably 60 ° C. or higher.
  • the fixing site of the first probe is not particularly limited as long as the single-stranded nucleic acid can be captured, but the 5 'end is particularly preferably fixed.
  • the common sequence or tag sequence is preferably arranged on the 5 ′ end side of the capture sequence, and the 5 ′ end side of the capture sequence is 5 ′ end side. It is preferable that a common sequence and a tag sequence are included in order from the side.
  • nucleic acid probe The first probe and the second probe described later (in the present specification, these are often collectively referred to as “nucleic acid probe”) are fixed in the nucleic acid reaction part via a fixing part.
  • fixing the nucleic acid probe in the nucleic acid reaction part in advance, it is possible to obtain genetic information from nucleic acids derived from individual cells without damaging cells or tissues and without using robots. It becomes. In particular, since it does not damage cells or tissues, changes in gene expression due to the damage can be avoided.
  • the nucleic acid reaction part preferably has a porous structure, and the nucleic acid probe is preferably immobilized on a porous surface.
  • the “porous surface” includes not only the surface inside the pores but also the surface of the nucleic acid reaction part in the portion where no pores are present.
  • An example of the porous structure is a sheet with pores.
  • An example of indirectly fixing the nucleic acid probe to the nucleic acid reaction part is a case where the nucleic acid probe is fixed to the surface of a carrier held on the surface of the nucleic acid reaction part.
  • the “carrier” is an intervening substance that links the nucleic acid reaction part and the nucleic acid probe, fixes the nucleic acid probe to the surface thereof, and is itself dissociable on the inner surface of the nucleic acid reaction part as necessary. It is fixed.
  • the material of the carrier is not limited.
  • resin materials polystyrene, etc.
  • oxides glass, silica, etc.
  • metals iron, gold, platinum, silver, etc.
  • polymeric polysaccharide supports e.g., Sepharose or Sephadex
  • ceramics latex, and these Composed of a combination.
  • the shape of the carrier is not particularly limited, but spherical particles such as beads are preferable because they have a large binding surface area and high operability. Therefore, magnetic beads are suitable as a carrier.
  • the nucleic acid probe is fixed to the nucleic acid reaction part via the fixing part by any fixing method known in the art.
  • the immobilization method include biological bonding, covalent bonding, ionic bonding, or physical adsorption to the surface of the nucleic acid reaction part or the surface of the carrier. It is also possible to fix both probes to the surface of the nucleic acid reaction part and the carrier via a spacer sequence.
  • biological binding examples include binding via a junction molecule such as binding between biotin and avidin, streptavidin or neutravidin, binding between an antigen and an antibody.
  • a junction molecule such as binding between biotin and avidin, streptavidin or neutravidin
  • binding between an antigen and an antibody For example, it can be achieved by reacting a biotin-modified nucleic acid probe with the surface of a nucleic acid reaction part to which avidin, streptavidin or neutravidin is bound.
  • covalent bonding for example, it can be achieved by introducing a functional group into the nucleic acid probe, introducing a functional group reactive to the functional group to the surface of the nucleic acid reaction part, and reacting both.
  • an amino group is introduced into a nucleic acid probe, and a covalent bond is formed by introducing an active ester group, epoxy group, aldehyde group, carbodiimide group, isothiocyanate group or isocyanate group on the surface of the nucleic acid reaction part.
  • a mercapto group may be introduced into the nucleic acid probe, and an active ester group, maleimide group or disulfide group may be introduced into the surface inside the cell holding region.
  • Examples of the method for introducing the functional group into the surface inside the cell holding region or the surface of the carrier include a method of treating the surface of the nucleic acid reaction part with a silane coupling agent having a desired functional group.
  • Examples of coupling agents include ⁇ -aminopropyltriethoxysilane, N- ⁇ - (aminoethyl) - ⁇ -aminopropyltrimethoxysilane, N- ⁇ - (aminoethyl) - ⁇ -aminopropylmethyldimethoxysilane, etc.
  • plasma treatment may be mentioned.
  • Examples of physical adsorption include a method in which the surface of the nucleic acid reaction part is surface-treated with a polycation (polylysine, polyallylamine, polyethyleneimine, etc.) and electrostatically coupled using the charge of the nucleic acid probe.
  • the nucleic acid reaction part and the carrier are preferably pre-coated with a surface so that other substances (such as nucleic acids and proteins) do not adsorb.
  • the “second probe” is a probe composed of nucleic acid in the same manner as the first probe.
  • the second probe is also composed of DNA in principle, but is not limited thereto, and may include, for example, RNA or artificial nucleic acid.
  • the second probe is a tag sequence consisting of a unique base sequence for each nucleic acid reaction part, and a part of the base sequence of the single-stranded nucleic acid captured by the first probe on the 3 ′ end side of the tag sequence Contains a complementary nucleotide sequence. Further, the second probe includes a common sequence and / or a stem sense sequence and a stem antisense sequence as necessary. Hereinafter, each sequence constituting the second probe will be described.
  • a base sequence complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe is complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe. It comprises a basic nucleotide sequence or a random sequence and is configured to hybridize to a single-stranded nucleic acid captured by the first probe.
  • the base sequence of the sequence is not particularly limited as long as it can hybridize with the single-stranded nucleic acid captured by the target first probe. Therefore, it can be designed appropriately in consideration of the type and sequence of the nucleic acid.
  • the capture sequence may be any length as long as it can hybridize to a single-stranded nucleic acid serving as a target (target).
  • a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid captured by the first probe can hybridize to any region of the nucleic acid captured by the first probe. Therefore, a random sequence such as a random hexamer is preferable.
  • the tag sequence has the same configuration as the tag sequence described in the first probe, a specific description is omitted here. As described above, since the tag sequence is an identification tag indicating that it is derived from the same nucleic acid reaction part, the tag sequences of the first probe and the second probe arranged in one nucleic acid reaction part are in principle Consists of the same sequence.
  • a ⁇ common sequence '' is a selected sequence that can function as a forward (Fw) primer sequence for amplifying a cleaved fragment in the nucleic acid amplification step of the gene analysis method using the gene analysis system of the present invention.
  • the configuration is basically the same as the common sequence described in the first probe.
  • the second probe includes a consensus sequence
  • the consensus sequence is preferably included at the 5 'end.
  • the second probe in order from the 5 ′ end side, has a common sequence, a tag sequence consisting of a unique base sequence for each nucleic acid reaction part, and a base of the single-stranded nucleic acid captured by the first probe.
  • a base sequence complementary to a part of the sequence is included.
  • the “stem sense sequence and stem antisense sequence” are selection sequences unique to the second probe, and are composed of complementary base sequences.
  • the base length of each sequence is the same.
  • the length is not particularly limited as long as both sequences can form a stable stem structure.
  • the base length may be in the range of 3 to 7 bases.
  • the base sequences constituting each sequence are not particularly limited as long as they are complementary to each other.
  • the stem sense sequence and the stem antisense sequence may exist in a state where a pair is separated by an arbitrary base sequence arranged therebetween.
  • Arbitrary base sequences include tag sequences and / or common sequences.
  • the stem sense sequence and the stem antisense sequence can form a stem structure by base pairing with each other in the second probe.
  • the arbitrary sequence has a loop structure
  • the second probe forms a loop stem structure as a whole.
  • the second probe or the fixing part of the second probe includes a cutting part. By cleaving the cleaved portion, the second probe is liberated, and the liberated second probe can hybridize to the single-stranded nucleic acid captured by the first probe.
  • the cleavage part is not particularly limited as long as it can release the second probe from the nucleic acid reaction part at an arbitrary position, and a known part in this technical field may be used.
  • the cleaving part may be cleaved by a chemical reaction, light of a specific wavelength, and vibration (ultrasonic waves), and is preferably a chemical bond cleaved by a chemical reaction.
  • Examples of chemical reactions that can cleave the cleavage site include hydrolysis reactions, oxidation reactions, reduction reactions, and enzyme reactions.
  • the second probe includes a cleavage site
  • a restriction enzyme recognition site is included in the sequence of the second probe.
  • an enzyme capable of cleaving either a double-stranded DNA, a single-stranded DNA, or a DNA / RNA strand is used.
  • restriction enzymes capable of cleaving single-stranded DNA include AccI, AccII, AvaII, BspRI, CfoI, DdeI, EcoRI, HaeIII, HapII, HhaI, HinfI, MspI, MboI, MboII, MspI, Sau3AI, SfaI, and TthHB81. It is done.
  • a restriction enzyme capable of cleaving double-stranded DNA comprising the DNA and a DNA complementary strand obtained using the DNA as a template can be used. More preferably, it is a restriction enzyme (enzyme producing enzyme) capable of cleaving only one DNA strand of double-stranded DNA.
  • ultraviolet irradiation may be used as a method for cutting the cutting portion of the second probe.
  • vinylC-T as the cutting part array
  • the bond between the arrays can be cut by irradiating 312 nm ultraviolet rays (FIG. 8).
  • FIG. 8 For details of the reaction mechanism of this reaction, see Y. Yoshimura and K. Fujimoto, Organic Letters., Vol. 10, no. 5, pp. 3227-3230, 2008.
  • the fixing part of the second probe and the nucleic acid reaction part for example, an aspect in which the fixing part is a disulfide bond can be mentioned.
  • the disulfide bond is cleaved by a reduction reaction with a reducing agent, and the second probe Can be liberated.
  • the nucleic acid reaction device of the present invention optionally includes a second opening.
  • the “second opening” is constituted by a hole opened to the outside at a position different from the first opening.
  • the size, shape, and number of “second openings” are not limited.
  • the invention relates to a flow cell device.
  • the flow cell device of the present invention comprises a substrate including the nucleic acid reaction device and a flow path. Since the configuration of the substrate is the same as that of the nucleic acid reaction device, description thereof is omitted here.
  • the flow cell device of the present invention comprises a nucleic acid reaction device of the present invention having only a first opening and a substrate including a first flow channel in contact with the first opening. In this case, both inflow and outflow of the solution to the nucleic acid reaction unit are performed via the first opening with respect to the first channel.
  • the flow cell device of the present invention comprises a nucleic acid reaction device of the present invention having a first opening and a second opening, a first flow path in contact with the first opening, and a second
  • the substrate includes a second flow path in contact with the opening.
  • the sample can be processed while passing the solution through the two openings.
  • the first flow path is a flow path for inflow and the second flow path is a flow path for outflow, so that the sample can be processed continuously with the flow of the solution in one direction. Is possible.
  • the flow cell device of the present invention can process a large number of samples in parallel.
  • the flow cell device of the present invention may include liquid feeding means such as a liquid feeding pump in order to arbitrarily send a solution.
  • the present invention provides a method for obtaining a nucleic acid to which the tag sequence is added from a single-stranded nucleic acid derived from a cell using the nucleic acid reaction device of the present invention, and optionally, the nucleic acid reaction of the present invention.
  • a step of flowing a plurality of cells on the substrate of the device and holding each cell in the cell holding region extracting a single-stranded nucleic acid from the cells held in the cell holding region
  • a step of allowing the first probe to capture a strand nucleic acid by hybridization with the capture sequence (single-stranded nucleic acid capturing step), a step of cleaving the cleavage portion of the second probe, and releasing the second probe (Second probe releasing step), the step of hybridizing the released second probe to a part of the single-stranded nucleus A captured by the first probe (hybridizing step of the second probe) )
  • hive A nucleic acid to which the tag sequence is added by synthesizing a nucleic acid containing a base sequence complementary to a part of a single-stranded nucleic acid captured by the first probe, using the soybean second probe as a primer A method (nucleic acid synthesis step).
  • the “cell holding step” is a step that can optionally be included in the method of the present invention, and is a step of flowing a plurality of cells on the substrate of the nucleic acid reaction device of the present invention and holding the cells in the cell holding region.
  • the sample used for analysis in the present invention is not particularly limited as long as it is a biological sample to be analyzed for gene expression, and any sample such as a cell sample, a tissue sample, a liquid sample, or the like can be used. Specific examples include a sample composed of one cell, a sample containing a plurality of cells, a tissue slice sample, a sample in which a plurality of individual cells are two-dimensionally held and arranged in an array.
  • the living organism from which the sample is derived is not particularly limited, and vertebrates (for example, mammals, birds, reptiles, fish, amphibians, etc.), invertebrates (for example, insects, nematodes, crustaceans, etc.), protozoa
  • vertebrates for example, mammals, birds, reptiles, fish, amphibians, etc.
  • invertebrates for example, insects, nematodes, crustaceans, etc.
  • protozoa A sample derived from any living body such as an organism, a plant, a fungus, a bacterium, or a virus can be used.
  • the “single-stranded nucleic acid capturing step” is a step of extracting nucleic acid from the cells held in the cell holding region and capturing the obtained single-stranded nucleic acid with the first probe in the cell holding region.
  • the single-stranded nucleic acid extracted from the cells held in the cell holding region is captured by hybridization with the first probe fixed to the nucleic acid reaction part immediately below.
  • the target single-stranded nucleic acid to be captured is not limited, but is a messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, and one Examples include double-stranded DNA, and fragments thereof, particularly mRNA.
  • Extraction of nucleic acids from cells may be performed by methods known in the art. For example, using a proteolytic enzyme such as Proteinase K, chaotropic salts such as guanidine thiocyanate and guanidine hydrochloride, surfactants such as Tween and SDS, or commercially available reagents for cell lysis, nucleic acids contained therein, That is, DNA and RNA can be eluted.
  • a proteolytic enzyme such as Proteinase K
  • chaotropic salts such as guanidine thiocyanate and guanidine hydrochloride
  • surfactants such as Tween and SDS
  • the “second probe releasing step” is a step of releasing the second probe by cleaving the second probe or the cleavage part included in the fixing part of the second probe.
  • the structure of the cut portion is as described in the item “Second probe” above.
  • the cutting part can be cut using a method known to those skilled in the art, such as chemical reaction, light of a specific wavelength, and vibration (ultrasonic waves), depending on the structure of the cutting part.
  • the second probe hybridization step the second probe released by cleaving the cleavage site is complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe in the probe. Is a step of hybridizing with a part of the base sequence of the single-stranded nucleic acid captured by the first probe in the same nucleic acid reaction part.
  • the “base sequence complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe” is a random sequence such as a random hexamer
  • the first probe It becomes possible to analyze an arbitrary region of the captured nucleic acid.
  • the “second probe releasing step” and the “second probe hybridizing step” are preferably performed simultaneously.
  • Nucleic acid synthesis step means that the hybridized second probe is used as a primer, the single-stranded nucleic acid captured by the first probe is used as a template, and its complementary strand is complemented by reverse transcriptase or DNA polymerase. It is a step of synthesizing a chain.
  • the complementary strand can be synthesized by a method known in the art.
  • the nucleic acid is RNA such as mRNA
  • cDNA can be synthesized by a reverse transcription reaction using reverse transcriptase.
  • the nucleic acid is DNA
  • cDNA can be synthesized by a replication reaction using DNA polymerase.
  • the second probe By simultaneously performing the “second probe releasing step”, the “second probe hybridizing step”, and the “nucleic acid synthesis step”, the second probe efficiently hybridizes to the mRNA as a primer. Since these functions, it is preferable to perform these steps simultaneously.
  • the method of the present invention optionally includes a step of amplifying the nucleic acid.
  • the amplification step can be performed by a method known to those skilled in the art. For example, for a nucleic acid synthesized by the “nucleic acid synthesis step”, a polyA sequence is added to the 3 ′ end by terminal transferase, followed by a “nucleic acid synthesis step” using a primer containing a poly T sequence complementary to the poly A sequence. The complementary strand of the nucleic acid synthesized by is synthesized.
  • the primer includes a common primer sequence corresponding to the common sequence that can be included in the second probe described above (for example, a common reverse primer sequence having a sequence different from the common forward primer sequence that can be included in the second probe). )
  • a common primer sequence corresponding to the common sequence that can be included in the second probe described above (for example, a common reverse primer sequence having a sequence different from the common forward primer sequence that can be included in the second probe).
  • PCR polymerase chain reaction
  • NASBA Nucleic Acid-Sequence-Based Amplification
  • LAMP Loop-Mediated Isothermal Thermal Amplification
  • RCA rolling A circle amplification
  • the gene sequence of the amplification product obtained in this amplification step can also be analyzed by any method known in the art. Gene expression analysis is also possible by performing sequence analysis. For example, in one embodiment, the presence or absence of expression of the gene to be analyzed, the expression level, etc. can be analyzed by determining the sequence of the amplification product. In another embodiment, a labeled probe having a base sequence complementary to the gene-specific sequence is used to hybridize the probe to cDNA or the obtained amplification product, and the analysis target is based on the label. Can be detected (eg, optically detected). Those skilled in the art can appropriately design a probe used for such detection.
  • the label used can also be any label known in the art, such as fluorescent labels (Cy3, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), etc.), chemiluminescent labels (luciferin). Etc.), enzyme labels (peroxidase, ⁇ -galactosidase, alkaline phosphatase, etc.), and radioactive labels (tritium, iodine 125, etc.).
  • fluorescent labels Cy3, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), etc.
  • chemiluminescent labels luciferin
  • Etc. enzyme labels
  • peroxidase, ⁇ -galactosidase, alkaline phosphatase, etc. enzyme labels
  • radioactive labels tritium, iodine 125, etc.
  • the nucleic acid amplification reaction is performed using a probe having a base sequence complementary to the gene-specific sequence, and the presence or absence of amplification is detected based on chemiluminescence or fluorescence, thereby analyzing the nucleic acid.
  • the expression of the gene of interest can be analyzed.
  • the correlation between a specific position in a cell or tissue and gene expression is obtained by associating the result of gene analysis in the nucleic acid obtained as described above with the two-dimensional position information of the sample (cell, tissue, etc.). It is also possible to obtain data.
  • the two-dimensional position information of such a sample is, for example, a microscopic image of a cell sample or a tissue section sample, a fluorescent image or a chemiluminescent image obtained by other labeling methods, etc.
  • FIG. 1 shows a schematic diagram of an example of a nucleic acid reaction device formed on a substrate.
  • Fig. 1 (a) shows a top view of the nucleic acid reaction device
  • Fig. 1 (b) shows a cross-sectional view at A-A '.
  • a plurality of cell holding regions (103) are formed in an array on the substrate (101), and a nucleic acid reaction part (104) made of a porous material is formed directly below the cell capturing region in a one-to-one correspondence with the cell capturing region.
  • the cell (102) is held in the cell holding region, and the nucleic acid in the cell is captured in the nucleic acid reaction part for each individual cell by crushing with the cell crushing solution while holding the cell.
  • the cell holding region has a diameter of 5 ⁇ m to 10 ⁇ m, the same size as that of one cell, and the cell-containing suspension is aspirated from the opposite side of the cell region as viewed from the nucleic acid reaction part.
  • One cell can be isolated and captured in one cell retention region.
  • a plurality of cells and tissue sections may be arranged in the cell holding region of the substrate.
  • the plurality of cell holding regions and the nucleic acid reaction part are arranged separately in a plane, but the plurality of cell holding regions and the nucleic acid reaction part may be arranged adjacent to each other.
  • Such a configuration is particularly effective for analysis of tissue sections.
  • FIG. A first probe (201) and a second probe (211) are fixed to the nucleic acid reaction part.
  • the first probe is chemically and firmly fixed to the inner wall (202) of the nucleic acid reaction part composed of a porous material in the device.
  • the first probe includes a capture sequence (203) for capturing a single-stranded nucleic acid to be sequence-analyzed, and a tag sequence (for specifying the position of the nucleic acid reaction part in the nucleic acid reaction device, if necessary) 204) and a common sequence (205) for nucleic acid amplification.
  • the second probe (211) is also firmly fixed to the inner wall made of a porous material, but at least a part of the sequence including the tag sequence (204) is designed to be cut and released. Yes.
  • the second probe (211) includes a base sequence (208) complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe, 207), and, if necessary, a common sequence for nucleic acid amplification (205). This second probe (211) is cleaved and released at a predetermined position by chemical treatment such as enzyme, so that the nucleic acid captured by the first probe is hybridized at multiple positions with the complementary sequence (208).
  • nucleic acid extraction is performed.
  • the cell or tissue to be analyzed is spread on the nucleic acid reaction device, and the solution is aspirated from the opposite side of the cell holding region as seen from the nucleic acid reaction part. Aspirate, fix and hold.
  • the nucleic acid to be analyzed is captured.
  • the solution is sucked from the opposite side of the cell holding region as viewed from the nucleic acid reaction part, thereby supplying a solution for crushing the cells in the same manner as in the cell aspiration.
  • the nucleic acid in the cell passes through the nucleic acid reaction part (104).
  • the capture sequence (203) in the first probe captures the nucleic acid extracted from the cell by hybridization. can do. Since the nucleic acid extracted from the cells passes through the nucleic acid reaction part together with the cell disruption solution, generally, nucleic acid extraction and nucleic acid capture are performed simultaneously.
  • the capture sequence may be a continuous sequence of T (poly T sequence) as described above.
  • T poly T sequence
  • the following reaction process is described as an example of analyzing mRNA.
  • the captured mRNA is indicated by (209), and (210) is a continuous sequence of mRNA A (polyA) that hybridizes with the capture sequence at the 3 ′ end of the first probe.
  • Array mRNA A
  • a complementary strand of the captured nucleic acid is synthesized.
  • the captured nucleic acid is mRNA, this corresponds to the synthesis of 1st cDNA.
  • a solution containing a restriction enzyme was prepared in the same manner as the cell disruption solution. Pass through the nucleic acid reaction part. By the action of this restriction enzyme, the nucleic acid is cleaved at the cleavage site (207) corresponding to the restriction enzyme recognition sequence (206). The second probe is released by the cleavage, and the released second probe hybridizes to nearby mRNA (within the same nucleic acid reaction site). It is preferable to use a short random sequence of about 6 mer as the base sequence (208) complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe for hybridization.
  • the second probe can hybridize to an arbitrary sequence region of mRNA.
  • the second probe includes a common sequence (205), a restriction enzyme recognition sequence (206), and a stem sense sequence and a stem antisense sequence as necessary.
  • the stem sense sequence and the stem antisense sequence are formed by the second probe released as shown in FIG. 2 (c) in order to avoid hybridization of the sequence other than the random sequence to the nucleic acid captured by the first probe.
  • the first probe (201) and the second probe (211) function as primers for this synthesis reaction.
  • the second probe (211) may include a random sequence that hybridizes to multiple positions of the mRNA, in which case 1st cDNA is synthesized from the multiple positions.
  • the tag sequence (204) is introduced into the 5 ′ end of all synthesized 1st cDNAs.
  • the 1st cDNA with the tag sequence (212 ((when the first probe serves as a primer) or (213) ((when the second probe serves as a primer)
  • the sequence of the sequencer performing the sequence analysis is analyzed. It is preferable to fix the second probe so that the quantity ratio corresponding to the length (the ratio of the second probe number to the number of molecules to be sequenced) is approximately 50 times the number of mRNA molecules. It is preferable to synthesize two probes and synthesize 1st cDNA having an average of 200 bp, but the quantity ratio can be changed according to the read base length of the sequencer for sequence analysis.
  • the second probe efficiently hybridizes to mRNA and functions as a primer by simultaneously releasing the second probe and synthesizing the 1st cDNA, these steps are preferably performed simultaneously.
  • mRNA is decomposed as shown in FIG. 2 (d).
  • An RNase enzyme for mRNA treatment is introduced into the nucleic acid reaction part by the same method as in the previous step.
  • a sequence addition reaction for hybridizing a reverse primer for nucleic acid amplification is performed.
  • a solution containing a terminal transferase enzyme was introduced into the nucleic acid reaction part by the same method as in the previous step, and a continuous A sequence was placed at the 3 'end of the 1st cDNA synthesized in Fig. 2 (c). (214) is added.
  • This continuous sequence may be other bases instead of A.
  • the DNA polymerase, as well as continuous T sequence is a nucleotide sequence complementary to the contiguous A sequence (215) and consensus sequence (216) Into the nucleic acid reaction part.
  • PCR amplification is performed using the reverse-direction common primer (217 (complementary strand of 205)) in FIG. 2 (f) and the forward-direction common primer (216) in FIG. 2 (g).
  • the reaction is completed by introducing the common primer (216) necessary for PCR amplification, the common primer (217) in the reverse direction, the PCR enzyme, and the substrate into the nucleic acid reaction part as in the previous step.
  • the obtained double-stranded PCR products (218 and 219) shown in FIG. 2 (g) are sequences that can be sequence-analyzed (more precisely, sequences that can be pre-processed for sequence analysis (such as emulsion PCR)). ) Called sequencing library.
  • Example 1 shows a structure of a substrate using magnetic beads and a manufacturing method thereof.
  • a schematic diagram of the substrate is shown in FIG.
  • the chip (303) made of PDMS (polydimethylsiloxane) that forms the nucleic acid reaction part (104) and the cell holding region (103) was produced by injection molding.
  • a substrate made of a resin (polycarbonate, cyclic polyolefin, and polypropylene) manufactured by nanoimprint technology or injection molding, a commercially available nylon mesh, or a track etch membrane may be used.
  • Thermal adhesion can also be used for adhesion of the pore array sheet.
  • the cell holding region (103) is a through-hole having a diameter of 10 ⁇ m and arranged in an array at intervals of 125 ⁇ m.
  • the size of the chip (303) side is square 1.125 mm, a cell holding area therein the (103) arranged two 10.
  • the diameter of the through hole is as wide as 75 ⁇ m, and magnetic beads are packed in this part to form the nucleic acid reaction part (104).
  • a pore array sheet (301) was placed under the chip (303) in which the through holes were arranged in an array.
  • the pore diameter of this pore array sheet (301) was 200 nm. Since the diameter of the magnetic beads is 1 ⁇ m and the diameter of the pores is smaller than the diameter of the magnetic beads, the magnetic beads do not flow out of the pores.
  • the packing of the magnetic beads is performed individually for each nucleic acid reaction unit using an ink jet printer head.
  • the magnetic beads nucleic acid is fixed comprising a different tag sequence for each nucleic acid reaction unit, 5 ⁇ 10 9 cells / mL each nucleic acid reaction suspension containing the number density of 2 nL of each portion (104) was filled.
  • the inner wall of the pores in the pore array sheet (301) is a hydrophilic surface, so that only moisture in the bead solution is absorbed by capillary action. Only the beads remain in the nucleic acid reaction part (104).
  • pore array sheet (301) various materials such as a monolith sheet made of porous glass, a capillary plate obtained by bundling capillaries and sliced, a nylon membrane or a gel thin film can be used.
  • alumina is used as an anode.
  • a pore array sheet obtained by oxidation was used.
  • Those skilled in the art can easily produce the pore sheet by anodic oxidation, but those having a pore diameter of 20 nm to 200 nm and a diameter of 25 mm are commercially available.
  • a sheet (Anodisc, GE Healthcare) having a pore diameter of 200 nm is used, and the pores in the sheet serve as a flow path (302) that connects the nucleic acid reaction part and the lower region.
  • the method for immobilizing the DNA probe on the magnetic beads is as follows. That is, in a separate reaction tube for each tag sequence, a magnetic bead immobilized with streptavidin and a DNA probe solution modified with a biotin group at the 5 'end in Tris buffer (pH 7.4) containing 1.5M NaCl for 10 minutes. By mixing while rotating, the DNA probe was immobilized on the magnetic beads via the biotin-streptavidin reaction.
  • the produced sheet can be used repeatedly, and by using the sheet repeatedly, it is possible to perform highly accurate expression distribution measurement for a necessary type of gene.
  • Example 2 This example shows an example of analyzing an arbitrary region of mRNA using a nucleic acid reaction device including magnetic beads in the nucleic acid reaction part.
  • FIG. 3 shows a cross-sectional view of an example of the nucleic acid reaction device of the present invention. This device was fabricated using a semiconductor process.
  • FIG. 3 (a) is a vertical sectional view of an example of the nucleic acid device of the present invention. The solution flows from the upper side to the lower side in the figure.
  • (103) is a cell holding region
  • (104) is a nucleic acid reaction part.
  • the cell holding region (103) and the nucleic acid reaction part (104) are formed by forming a through hole on a resin chip (303).
  • the through hole has a small diameter in the cell holding region (103) and a large diameter in the nucleic acid reaction part (104).
  • the number of molecules in the reaction part can be increased, and the capture efficiency of the extracted nucleic acid can be improved.
  • the diameter of the holes constituting the flow path (302) in the pore array sheet (301) is smaller than the diameter of the magnetic beads as described above. .
  • FIG. 3 (b) is a cross-sectional view corresponding to the A-A 'cross section of FIG. 3 (a)
  • FIG. 3 (c) is a cross-sectional view corresponding to the B-B' cross section of FIG. 3 (a).
  • the cells 102 contained in the solution are isolated and held by capturing them in the cell holding region (103) in the nucleic acid reaction device.
  • a tag sequence (204) that is different for each nucleic acid reaction part is fixed to the surface of the bead included in the nucleic acid reaction part immediately below the cell holding region, that is, in the B-B 'cross section. This makes it possible to introduce a different tag sequence for each cell by the following reaction process.
  • a flow cell device including a plurality of nucleic acid reaction devices and capable of supplying cell solutions and reagents was used.
  • a schematic diagram of this flow cell device is shown in FIG.
  • the flow cell device (401) includes a plurality of reaction chambers (402), and each reaction chamber (402) includes one nucleic acid reaction device (105).
  • This nucleic acid reaction device is the same as that shown in FIG. 3, and includes a plurality of nucleic acid reaction units (104) including one cell holding region (103).
  • Cells (102) flow from the upper inlet (404) toward the upper outlet (405) in a common channel (403) on the flow cell device.
  • a negative pressure is applied to the lower outlet (407), the cells are drawn to the cell holding region via the suction channel (406), and the cells are captured through an opening smaller than the cell diameter.
  • the solution in the channel (403) is caused by the negative pressure applied through the suction channel (406). Flows into the suction channel (406).
  • the diameter of the beads By setting the diameter of the beads to 1 ⁇ m, the voids are on the order of sub ⁇ m, and macromolecules such as enzymes can also pass through.
  • 1000 cells were washed with 500 ⁇ L of 1 ⁇ PBS so as not to be damaged, suspended in 10 ⁇ L of 1 ⁇ PBS buffer cooled to 4 ° C., introduced from the upper inlet (404) of FIG. ) From the top outlet (405) to fill with this solution.
  • the nucleic acid reaction device (105) is filled with the PBS buffer containing the cells.
  • a negative pressure of 1.0 atm is applied to the lower outlet (407) so that the solution flows through the cell holding region (103) toward the suction channel (406), and the solution is sucked.
  • the cell trapping region (103) When the cell 102 moves along the flow of the solution and reaches the cell holding region (103), the cell trapping region (103) has a smaller opening diameter than the cell 102, so the cell is trapped in the cell holding region. The Since the trapped cells act as plugs for the solution flow, the solution flow goes to the cell holding region (103) where the cells have not yet been trapped. Therefore, the remaining cells move to the cell holding region (103) where the cells are not yet captured and are captured.
  • a Lysis buffer e.g., a surfactant such as Tween 20
  • Tween 20 a surfactant such as Tween 20
  • the channel (302) below the nucleic acid reaction part is a channel composed of a porous material having a diameter of 0.2 ⁇ m, and in order to suppress pressure loss, the Lysis buffer is transferred from the reaction chamber (402) to the cell holding region. The solution may continue to flow slowly through the (103) toward the suction channel (406) for about 5 minutes.
  • the cell 102 is crushed by the cell lysate (Lysis buffer), and the mRNA (209) is released to the outside of the cell, but the mRNA (209) does not diffuse to the periphery, and the cell holding region (103 ) To reach the nucleic acid reaction part (104).
  • an electric field may be applied in the direction of the flow, and the nucleic acid (mRNA (209)) in the cell may be moved to the nucleic acid reaction part (104) by electrophoresis.
  • the mRNA (209) reaches the nucleic acid reaction part (104)
  • it is captured by the first probe immobilized on the beads contained in the nucleic acid reaction part (104) by hybridization with the capture sequence.
  • the nucleic acid reaction is performed.
  • a tag sequence (204) having a different sequence for each position is introduced into the first probe (201) and the second probe (211).
  • the first probe and the second probe contain a common sequence (205) for PCR amplification on the 5 ′ end side.
  • the first probe of (201) is, for example, from the 5 ′ end, a 30 base PCR amplification common primer (205), a 7 base tag sequence (204), an 18 base poly T (dT ) Sequence + 2 base VN sequence (203).
  • V represents the base of A, C, or G.
  • FIG. 5 shows details of the restriction enzyme cleavage site (207) and restriction enzyme recognition sequence (206) used in this example.
  • Y in the recognition sequence (206) of the restriction enzyme is T or C
  • R represents A or G
  • N represents any base of A, C, G, or T.
  • N with a line on the top represents a base complementary to N.
  • (501) is a nick, and by placing a nick at this position, a single-stranded portion (502) of a 6-base random sequence can be exposed (by changing the position of this nick
  • the length of the random sequence that hybridizes to mRNA can be freely designed between 5 and 12 bases).
  • the second probe before being subjected to the action of the restriction enzyme is immobilized on the surface of the bead (503).
  • streptavidin was immobilized on the bead surface, biotin was modified at the 5 'end and 3' end of the DNA, and the probe was immobilized by biotin-streptavidin reaction.
  • the immobilization method other methods available to the technician may be used.
  • the entire flow cell device (401) was kept at 85 ° C. for 1.5 minutes to inactivate the reverse transcriptase.
  • the RNA is decomposed by repeating the process of discharging from the lower outlet (407) and removing the buffer in the reaction chamber from the upper outlet five times, and the remaining and decomposed products in the nucleic acid reaction part are removed. Removed and washed.
  • a cDNA library array was constructed in which a tag sequence consisting of a unique base sequence was inserted for each cell holding region.
  • FIG. 2 (d) shows the added poly A sequence (214).
  • RNase-free sterilized water 22.8 uL, 10 x PCR Buffer II 3 uL, 25 mM MgCl 2 1.8 uL, 100 mM ATP 0.9 uL, and terminal transferase (2 U / ⁇ L) 1.5 uL were mixed, and the nucleic acid reaction part (404) from the upper inlet (404) was mixed as described above. 104).
  • the reaction was allowed to proceed by raising the temperature to 37 ° C and holding for 15 minutes.
  • 2nd DNA which is the complementary strand of 1st cDNA Synthesize a chain.
  • a primer containing a common forward sequence for PCR amplification (216) and a poly-T sequence (dT30 + VN) (215) 2nd DNA which is the complementary strand of 1st cDNA Synthesize a chain.
  • the mixed solution was introduced into the nucleic acid reaction part (104) from the upper inlet (404) as in the previous step.
  • the nucleic acid secondary structure was solved by holding at 95 ° C. for 3 minutes, and then the primer annealing portion was hybridized at 44 ° C. for 2 minutes using the 1st cDNA strand as a template. Further, by raising the temperature to 72 ° C. for 6 minutes, a complementary strand extension reaction was performed to synthesize a 2nd DNA strand.
  • the arrow in FIG. 2 (e) indicates the direction of synthesis of the 2nd DNA strand.
  • PCR amplification was performed using common primers as shown in FIG. 2 (f) and FIG. 2 (g).
  • the reagent prepared above was introduced from the upper inlet (404) as in the previous step, and the reagent was introduced into the nucleic acid reaction part by aspiration from (407). Subsequently, the entire flow cell device is kept at 94 ° C. for 30 seconds, and the three-stage process of 94 ° C. for 30 seconds ⁇ 55 ° C. for 30 seconds ⁇ 68 ° C. for 30 seconds is repeated for 40 cycles.
  • the PCR amplification process was performed after cooling. This reaction is a common reaction, and PCR amplification was performed under the same reagent conditions for all nucleic acid reaction devices, so that the amplification efficiency was uniform among the nucleic acid reaction devices.
  • the PCR amplification product solution accumulated in the solution was recovered.
  • the product was purified using PCR Purification Kit (QIAGEN).
  • the obtained PCR products (218) and (219) are sequences that can be sequence-analyzed (more precisely, sequences that can be pre-processed for sequence analysis (such as emulsion PCR)) and are called sequencing libraries.
  • the gene expression level can be obtained for each tag sequence by sequence analysis of this sequencing library. That is, simultaneous analysis can be performed on the number of cells equal to or less than the number of types of tag sequences introduced simultaneously into the flow cell device.
  • a method for analyzing mRNA using the device of the present invention has been shown.
  • a proteolytic enzyme, a restriction enzyme for fragmenting to an appropriate length and By adding a mixed solution of terminal transferase for adding poly A to the cell disruption solution, the same method as in the analysis of mRNA can be applied.
  • a method generally known to those skilled in the art may be applied as a method for preparing a restriction enzyme mixed solution from which a fragment of an appropriate length can be obtained.
  • Example 3 This example is an example showing a nucleic acid reaction device using a pore array sheet to which a DNA probe is fixed without using beads. In this example, a photoreaction was used for releasing the second probe.
  • FIG. 6 The structure of the nucleic acid reaction device is shown in FIG. This device was fabricated using a semiconductor process. It is the same as the device of Example 2 that a plurality of nucleic acid reaction parts (104) including one cell holding region (103) are included.
  • the different patterns of the nucleic acid reaction part (104) in FIG. 6 (c) indicate that different tag sequences are fixed in each nucleic acid reaction part.
  • 6 (a) shows a cross-sectional view perpendicular to the substrate
  • FIG. 6 (b) shows a cross-sectional view along AA ′ in FIG. 6 (a)
  • FIG. 6 (c) shows a cross-sectional view along BB ′ in FIG. 6 (a). It was.
  • the through holes constituting the nucleic acid reaction part (104) were formed at intervals of 0.5 ⁇ m in a diameter of 0.3 ⁇ m in a SiO 2 film having a thickness of 5 ⁇ m. Thereafter, the silicon substrate was removed by wet etching. The first DNA probe was immobilized on the inner wall using a silane coupling agent.
  • the diameter of the cell holding region (103) is 10 ⁇ m, and this trapping part was prepared by periodically removing polyimide from the polyimide resin film (601) having a thickness of 10 ⁇ m by lithography and plasma etching to form an opening. .
  • the upper surface of the nucleic acid reaction part can be used as a cell holding region without the (601) uneven structure.
  • the function of isolating one cell in one cell holding region is lost, but the cells are adhered to each other as in a tissue section, for example, and are extracted from cells at any approximate position. It is an effective device configuration when it is desired to know the sequence of the nucleic acid.
  • borate buffer pH 8.5
  • 30 mM sodium citrate buffer 2 x SSC, pH 7.0
  • 10mTris containing 0.1% Tween 20 which is 100 times the internal volume is introduced and discharged to complete the washing.
  • 10 ⁇ M 5 'end biotin-modified DNA probe (first probe and second probe) in the same volume as the internal volume, 1 mM NaCl, and 10 mM TrisHCl solution containing 0.1% Tween20 Injection is performed for each individual nucleic acid reaction unit using an inkjet device.
  • the probe solution to be injected into one nucleic acid reaction part is injected so as to fill the region by dividing 40 pL into 250 times. After injecting the solution, react for 30 minutes, and then introduce and drain the 10 times volume of 2X SSC buffer heated to 60 ° C again.
  • the reaction part was washed by passing through the part to complete the immobilization reaction.
  • the structure of the flow cell device is shown in FIG.
  • the device includes a plurality of nucleic acid reaction devices (105) shown in FIG. 6 and is capable of introducing cells and reaction solutions. Since such functions and basic configurations are the same as those in the second embodiment, a description thereof will be omitted. Since the structure of the second probe and the release method are different, this point will be described below.
  • FIG. 9 A schematic diagram of the second probe fixed to the inner wall of the pore array sheet of the nucleic acid reaction part is shown in FIG.
  • the DNA is cleaved at the cleaving part (207).
  • the bond between C and T to which a vinyl group is added is broken.
  • the arrangement of the cut portions is shown in FIG. 8 as vinylC-T. Details of the photoreaction at the time of the photoreaction of this portion are shown in FIG.
  • the right side of FIG. 9 (a) is the DNA structure before cleavage, and the left side of FIG. 9 (a) is the structure after cleavage.
  • FIG. 9 (b) shows this change in chemical formula.
  • (901) is a vinyl group
  • (902) is a pyrimidine moiety of thymine (T)
  • (903) is a pyrimidine moiety of cytosine (C).
  • (904) represents the chemical formula of the portion corresponding to (901)-(903) before cutting.
  • sequencing, quantification, and identification of biomolecules can be performed on a large number of cultured cells, a large number of immune cells, (in blood) cancer cells, and the like, and to what extent the cell group is in what state. It is possible to measure whether there are as many of them in the living body. This makes it possible to measure early diagnosis of cancer and the like and heterogeneity of iPS cells.

Abstract

For analyzing the sequence for an arbitrary gene region, it is required to introduce different probes into individual reaction vessels, respectively. However, the supply of regents into individual reaction vessels has a problem that the structure of a device is complicated and therefore the cost for the analysis is increased. In the present invention, individual cells are captured in a cell hold region, probes that are respectively immobilized onto nucleic acid reaction sections in advance are released after the capture of the cells. In this manner, the supply of individual reagents to the nucleic acid reaction sections can be eliminated and it becomes possible to analyze many cells by employing an inexpensive device configuration.

Description

核酸反応デバイスNucleic acid reaction device
　本発明は遺伝子発現解析方法及びそのためのデバイスに関する。特に、本発明は1細胞レベルでの遺伝子発現解析方法及びそのためのデバイスに関する。 The present invention relates to a gene expression analysis method and a device therefor. In particular, the present invention relates to a method for analyzing gene expression at the level of one cell and a device therefor.
　単一細胞解析とは、単一細胞ごとに細胞中のDNA、RNA、及びタンパク質などの生体分子を高精度に検出、定量する技術である。単一細胞解析技術には、大きく以下の2種類の技術が存在する。 Single cell analysis is a technology for detecting and quantifying biomolecules such as DNA, RNA, and proteins in cells with high accuracy for each single cell. There are the following two types of single cell analysis techniques.
　第1の技術は、細胞を単離する手段を含まず、細胞中の生体物質を高効率に処理する手段のみからなる技術である。まず、第1の技術を、単一細胞レベルの遺伝子発現解析を例として説明する。 The first technique does not include a means for isolating cells, and is a technique consisting only of means for treating biological materials in cells with high efficiency. First, the first technique will be described using gene expression analysis at the single cell level as an example.
　当業者によって一般的によく知られているセルソーターなどの装置によって、細胞を一つずつ樹脂製容器(例えば、PCRチューブ又はマイクロタイタープレートのウェル)などに分離して分注する。この樹脂製容器に適切な試薬を加えることによって、単一細胞中の微量なmRNAを抽出し、逆転写によってその相補的な塩基配列を有するcDNAを合成し、核酸をPCR増幅した後、シーケンシングに適切な塩基長への断片化と末端配列(アダプター)の接続(ライゲーション)を行う(非特許文献1)。この方法では、細胞が樹脂製容器に分注された後、例えば末端配列接続(アダプターライゲーション)時に、PCRチューブやマイクロタイタープレートのウェルを識別するためのタグ配列の挿入が行われる。続いて、DNAシーケンサーによる配列解析データを集計し、遺伝子発現解析を行う。この技術においては、組織切片など、空間的な細胞間の位置関係が重要な場合であっても、細胞に対してトリプシン処理などの化学処理を行って、細胞をばらばらに分離した後にセルソーターなどに導入する必要がある。したがって、細胞の位置関係の情報と細胞ごとの遺伝子解析データを対応付けることはできない。また、樹脂製容器にマニュアル又はロボットで試薬を分注するため、蒸発や分注精度の問題で、試薬を含む反応溶液量はμLオーダーとなることが多い。 The cells are separated and dispensed one by one into a resin container (for example, a PCR tube or a well of a microtiter plate) one by one by an apparatus such as a cell sorter generally well known by those skilled in the art. By adding appropriate reagents to this resin container, a small amount of mRNA in a single cell is extracted, cDNA having its complementary base sequence is synthesized by reverse transcription, nucleic acid is PCR amplified, and sequencing is performed. Then, fragmentation to an appropriate base length and terminal sequence (adapter) connection (ligation) are performed (Non-patent Document 1). In this method, after cells are dispensed into a resin container, a tag sequence for identifying wells of a PCR tube or a microtiter plate is inserted, for example, at the end sequence connection (adapter ligation). Subsequently, the sequence analysis data by the DNA sequencer is totaled and the gene expression analysis is performed. In this technology, even if the positional relationship between spatial cells such as tissue sections is important, the cells are subjected to chemical treatment such as trypsin treatment to separate the cells into pieces, and then to a cell sorter or the like. It is necessary to introduce. Therefore, information on the positional relationship between cells and gene analysis data for each cell cannot be associated with each other. In addition, since the reagent is dispensed into the resin container manually or by robot, the amount of the reaction solution containing the reagent is often on the order of μL due to evaporation and dispensing accuracy problems.
　これに対し第2の技術は、細胞を単離する手段を含み、細胞を単離する手段と細胞中の生体物質を高効率に処理する手段が機能的に接続されている。例えば、核酸反応デバイス中で反応槽中に捕捉した細胞を同じ反応槽又は隣接する反応槽で処理する技術が知られている。第2の技術を、単一細胞レベルの遺伝子発現解析を例として説明する。 On the other hand, the second technique includes means for isolating cells, and means for isolating cells and means for treating biological materials in the cells with high efficiency are functionally connected. For example, a technique for treating cells captured in a reaction vessel in a nucleic acid reaction device in the same reaction vessel or an adjacent reaction vessel is known. The second technique will be described using gene expression analysis at the single cell level as an example.
　この技術では、核酸反応デバイス上で細胞を捕捉し、同じデバイス上で核酸反応を行う。 In this technology, cells are captured on a nucleic acid reaction device and a nucleic acid reaction is performed on the same device.
　特許文献1には、計測対象の細胞を単離するために、フローセルデバイス中の反応室内に一つ一つの細胞を個別に捕捉するための細胞捕捉部がアレイ状に複数配置されたデバイスが記載されている。特許文献１には、抽出されたmRNAを捕捉するためのポリT配列と、既知の配列を有しかつ捕捉された細胞の位置ごとに異なる細胞認識用タグ配列とを含む核酸プローブがデバイス中に固定されていることが記載されている。また、この核酸プローブで細胞から抽出されたmRNAを捕捉し、対応するcDNAを合成することで、細胞ごとに異なる配列が導入されたcDNAライブラリーが構築されることが記載されている。このcDNAライブラリーをテンプレートとしてPCR増幅することによって大規模核酸配列解析装置によるシーケンシングのための増幅産物が得られる。得られた配列データから上記第１の技術と同様にDNAシーケンサーなどにより遺伝子発現解析データを得る。この第2の技術では、細胞の位置関係の情報と遺伝子解析結果を対応付けることが可能であり、細胞のデバイス上での高密度並列処理が可能であるため、試薬量を低減することができ、試薬コストが低減できる。 Patent Document 1 describes a device in which a plurality of cell capture units for individually capturing individual cells in a reaction chamber in a flow cell device are arranged in an array to isolate cells to be measured. Has been. In Patent Document 1, a nucleic acid probe containing a poly-T sequence for capturing extracted mRNA and a cell recognition tag sequence having a known sequence and different for each captured cell position is included in the device. It is described that it is fixed. Further, it is described that a cDNA library in which different sequences are introduced for each cell is constructed by capturing mRNA extracted from cells with this nucleic acid probe and synthesizing corresponding cDNA. Amplification products for sequencing by a large-scale nucleic acid sequence analyzer can be obtained by PCR amplification using this cDNA library as a template. From the obtained sequence data, gene expression analysis data is obtained by a DNA sequencer or the like as in the first technique. In this second technique, it is possible to correlate the information on the positional relationship of the cells with the gene analysis results, and because high-density parallel processing on the cell device is possible, the amount of reagent can be reduced, Reagent costs can be reduced.
公開特許公報　WO2014/020657Published patent publication WO2014 / 020657
　上記第１の技術を用いた場合には、細胞の空間的な位置関係と細胞ごとの遺伝子解析データを対応付けることができないだけでなく、１細胞ごとにμLオーダーの試薬が必要なため、解析対象となる細胞の数に比例してシーケンシングのための試薬が増加し、コストが高くなることが問題であった。 When the first technique is used, not only the spatial positional relationship between cells and the gene analysis data for each cell cannot be associated, but also a reagent on the order of μL is required for each cell. The problem is that the number of reagents for sequencing increases in proportion to the number of cells to become, and the cost increases.
　一方、第2の技術を用いた場合には、細胞の位置関係の情報と遺伝子解析結果を対応付けることが可能であり、デバイス上での細胞の高密度並列処理が可能であるため、試薬量を低減することができ、試薬コストが低減できるが、3’末端以外の領域の配列解析を効率的に行うことが困難であった。しかし、3’末端のみの配列解析では、例えばがん細胞を特徴づける変異の解析を行うことは困難であり、mRNAのすべての領域の配列解析が求められている。また、例えばがんの診断や研究ではゲノム解析も重要となるため、ゲノム配列についても任意の領域の配列解析が求められている。 On the other hand, when the second technique is used, it is possible to correlate the information on the positional relationship between the cells and the gene analysis results, and high-density parallel processing of cells on the device is possible. Although the cost can be reduced and the reagent cost can be reduced, it has been difficult to efficiently perform the sequence analysis of the region other than the 3 ′ end. However, in the sequence analysis of only the 3 'end, for example, it is difficult to analyze mutations that characterize cancer cells, and sequence analysis of all regions of mRNA is required. Further, for example, genome analysis is important in cancer diagnosis and research, and therefore, sequence analysis of an arbitrary region is required for genome sequences.
　任意領域の配列解析を行うためには、断片化した配列に対してタグ配列を1細胞ごとに導入するか、ランダム配列によって、mRNAの任意の領域からcDNAを合成する必要がある。いずれの場合もデバイスの位置ごとに異なるタグ配列を挿入する必要がある。 In order to perform sequence analysis of an arbitrary region, it is necessary to introduce a tag sequence for each cell into a fragmented sequence or to synthesize cDNA from an arbitrary region of mRNA by a random sequence. In either case, it is necessary to insert a different tag sequence for each position of the device.
　この場合、反応試薬のボリュームを低減するためには樹脂製反応容器を小さくし、高密度に配置しなくてはならない。高密度に配置した容器に対して、異なるDNAプローブを導入するためには、プレーナ型のマイクロ流路構造では、流路配線のスペースをとる必要があるためデバイスの面積が大きくなり、デバイスが高価になってしまうという問題があった。 In this case, in order to reduce the volume of the reaction reagent, the resin reaction vessel must be made smaller and arranged with high density. In order to introduce different DNA probes to a container arranged at high density, the planar microchannel structure requires a space for the channel wiring, which increases the area of the device and makes the device expensive. There was a problem of becoming.
　上記課題を解決するために鋭意検討した結果、本発明者は、個々の細胞を核酸反応部で捕捉し、核酸反応部に事前に固定したプローブを細胞捕捉後に遊離させることで、各核酸反応部ごとに個別の試薬を供給することなくサンプルを処理できるデバイスを開発した。 As a result of earnest studies to solve the above problems, the present inventor captured each cell in the nucleic acid reaction unit, and released the probe fixed in advance in the nucleic acid reaction unit after the cell capture, so that each nucleic acid reaction unit We have developed a device that can process samples without supplying a separate reagent for each.
　すなわち、本発明は以下の態様を包含する。 That is, the present invention includes the following aspects.
（１）核酸反応部(104)を少なくとも1つ含む基板(101)からなる核酸反応デバイスであって、前記核酸反応部(104)は、少なくとも1つの細胞を保持する、第1の開口部からなる1つの細胞保持領域(103)を含み、かつ(1)細胞から抽出される一本鎖核酸の塩基配列の一部に相補的な塩基配列からなる捕捉配列(203)を含む第1のプローブ(201)、並びに、(2)核酸反応部ごとに固有の塩基配列からなるタグ配列(204)、及び該タグ配列の3'末端側に、前記第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列(208)を含む第2のプローブ(211)を含み、前記第1のプローブ及び第2のプローブは固定部を介して前記核酸反応部(104)に固定されており、前記第2のプローブ、又は第2のプローブの前記固定部が切断部(207)を含む、前記核酸反応デバイス。
（２）前記細胞保持領域(104)が、1つの細胞のみを保持する、（１）に記載の核酸反応デバイス。
（３）前記第2のプローブ(211)が、前記タグ配列の5’末端側に共通配列(205)を含む、（１）又は（２）に記載の核酸反応デバイス。
（４）前記第1のプローブ(201)の5'末端が核酸反応部に固定されている、（１）～（３）のいずれかに記載の核酸反応デバイス。
（５）前記第1のプローブ(201)が、前記捕捉配列(203)の5'末端側に、5'末端側から順に、前記共通配列(205)、及び前記タグ配列(204)を含む、（４）に記載の核酸反応デバイス。
（６）前記核酸反応部が、多孔質構造である、（１）～（５）のいずれかに記載の核酸反応デバイス。
（７）前記第1のプローブ(201)及び／又は第2のプローブ(211)が、前記多孔質の表面に固定されている、（６）に記載の核酸反応デバイス。
（８）前記核酸反応部が、1以上の担体を有する、（１）～（７）のいずれかに記載の核酸反応デバイス。
（９）前記第1のプローブ(201)及び／又は第2のプローブ(211)が、担体の表面に固定されている、（８）に記載の核酸反応デバイス。
（１０）前記核酸反応部(104)が基板上に複数配置されている、（１）～（９）のいずれかに記載の核酸反応デバイス。
（１１）前記第1の開口部とは異なる第2の開口部を有する（１）～（１０）のいずれかに記載の核酸反応デバイス。
（１２）（１１）に記載の核酸反応デバイス、前記第1の開口部に接する第1の流路、及び前記第2の開口部に接する第2の流路を含む、フローセルデバイス。
（１３）（１１）に記載の核酸反応デバイスを用いて、細胞由来の一本鎖RNAから、前記タグ配列(204)が付加されたcDNAを得る方法であって、前記細胞保持領域に保持された細胞から一本鎖RNAを抽出し、抽出された一本鎖RNAを、前記捕捉配列とのハイブリダイゼーションにより前記第1のプローブ(201)に捕捉させる工程、前記第2のプローブ(211)の切断部(207)を切断し、第2のプローブ(211)を遊離させる工程、遊離した前記第2のプローブ(211)を、前記第1のプローブによって捕捉された一本鎖RNAの一部にハイブリダイズさせる工程、及びハイブリダイズした前記第2のプローブ(201)をプライマーとして、前記第1のプローブ(201)によって捕捉された一本鎖RNAの一部に相補的な塩基配列を含む核酸を合成することにより、前記タグ配列が付加されたcDNAを得る工程、を含む前記方法。
（１４）（１３）に記載の方法によりcDNAを得る工程、前記cDNAを増幅する工程、及び増幅された核酸の配列決定を行う工程を含む、遺伝子解析方法。 (1) A nucleic acid reaction device comprising a substrate (101) including at least one nucleic acid reaction section (104), wherein the nucleic acid reaction section (104) is provided from a first opening that holds at least one cell. A first probe comprising a capture sequence (203) comprising a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid extracted from a cell (1) (201), and (2) a tag sequence (204) comprising a unique base sequence for each nucleic acid reaction part, and a single-stranded nucleic acid captured by the first probe on the 3 ′ end side of the tag sequence A second probe (211) containing a base sequence (208) complementary to a part of the base sequence of the nucleic acid reaction part (104) via the immobilization part. The nucleic acid reaction device, wherein the second probe or the fixing part of the second probe includes a cutting part (207).
(2) The nucleic acid reaction device according to (1), wherein the cell holding region (104) holds only one cell.
(3) The nucleic acid reaction device according to (1) or (2), wherein the second probe (211) includes a common sequence (205) on the 5 ′ end side of the tag sequence.
(4) The nucleic acid reaction device according to any one of (1) to (3), wherein the 5 ′ end of the first probe (201) is fixed to a nucleic acid reaction part.
(5) The first probe (201) includes the common sequence (205) and the tag sequence (204) on the 5 ′ end side of the capture sequence (203) in this order from the 5 ′ end side. The nucleic acid reaction device according to (4).
(6) The nucleic acid reaction device according to any one of (1) to (5), wherein the nucleic acid reaction part has a porous structure.
(7) The nucleic acid reaction device according to (6), wherein the first probe (201) and / or the second probe (211) is fixed to the porous surface.
(8) The nucleic acid reaction device according to any one of (1) to (7), wherein the nucleic acid reaction unit has one or more carriers.
(9) The nucleic acid reaction device according to (8), wherein the first probe (201) and / or the second probe (211) is fixed on the surface of a carrier.
(10) The nucleic acid reaction device according to any one of (1) to (9), wherein a plurality of the nucleic acid reaction units (104) are arranged on a substrate.
(11) The nucleic acid reaction device according to any one of (1) to (10), which has a second opening different from the first opening.
(12) A flow cell device comprising the nucleic acid reaction device according to (11), a first channel in contact with the first opening, and a second channel in contact with the second opening.
(13) A method for obtaining a cDNA to which the tag sequence (204) is added from a single-stranded RNA derived from a cell using the nucleic acid reaction device according to (11), which is retained in the cell retention region. Extracting single-stranded RNA from the obtained cells, and allowing the extracted single-stranded RNA to be captured by the first probe (201) by hybridization with the capture sequence, the second probe (211) A step of cleaving the cleaved portion (207) to release the second probe (211), the released second probe (211) is a part of the single-stranded RNA captured by the first probe A nucleic acid comprising a base sequence complementary to a part of the single-stranded RNA captured by the first probe (201) using the hybridized step and the hybridized second probe (201) as a primer; A step of obtaining a cDNA to which the tag sequence is added by synthesis. The method comprising.
(14) A gene analysis method comprising a step of obtaining cDNA by the method according to (13), a step of amplifying the cDNA, and a step of sequencing the amplified nucleic acid.
　任意の遺伝子領域の配列解析を行うためには、個々の微小反応槽への異なるプローブの導入が必要であったが、試薬又はデバイス等のコストが高くなるという問題があった。個別の反応槽への試薬供給を不要とすることによって、簡単なデバイス構成で多数の種類のサンプルの解析を安価に実行することができる。 In order to perform sequence analysis of an arbitrary gene region, it was necessary to introduce a different probe into each microreaction tank, but there was a problem that the cost of a reagent or a device was increased. By eliminating the need to supply reagents to individual reaction vessels, analysis of many types of samples can be performed at low cost with a simple device configuration.
図１は、本発明に関する核酸反応デバイスの一例の模式図である。FIG. 1 is a schematic view of an example of a nucleic acid reaction device according to the present invention. 図２－１は、本発明の方法を用いて、一本鎖核酸を捕捉し、タグ配列を付加した核酸を合成し、増幅する工程を示す図である。FIG. 2-1 is a diagram showing a process of capturing and amplifying a nucleic acid to which a single-stranded nucleic acid is added and a tag sequence is added using the method of the present invention. 図２－２は、本発明の方法を用いて、一本鎖核酸を捕捉し、タグ配列を付加した核酸を合成し、増幅する工程を示す図である。FIG. 2-2 is a diagram showing a process of capturing and amplifying a nucleic acid to which a single-stranded nucleic acid is added, a tag sequence is added, using the method of the present invention. 図２－３は、本発明の方法を用いて、一本鎖核酸を捕捉し、タグ配列を付加した核酸を合成し、増幅する工程を示す図である。FIG. 2-3 is a diagram showing a process of capturing a single-stranded nucleic acid, synthesizing and amplifying a nucleic acid added with a tag sequence using the method of the present invention. 図２－４は、本発明の方法を用いて、一本鎖核酸を捕捉し、タグ配列を付加した核酸を合成し、増幅する工程を示す図である。FIG. 2-4 is a diagram showing a process of capturing and amplifying a nucleic acid to which a single-stranded nucleic acid is added and a tag sequence is added using the method of the present invention. 図３は、本発明の核酸反応デバイスの一例を示す図である。FIG. 3 is a diagram showing an example of the nucleic acid reaction device of the present invention. 図４は、本発明のフローセルデバイスの一例を示す図である。FIG. 4 is a diagram showing an example of a flow cell device of the present invention. 図５は、制限酵素の切断部の詳細を示す図である。FIG. 5 is a diagram showing details of the restriction enzyme cleavage site. 図６は、本発明の核酸反応デバイスの一例を示す図である。FIG. 6 is a diagram showing an example of the nucleic acid reaction device of the present invention. 図７は、本発明のフローセルデバイスの一例を示す図である。FIG. 7 is a diagram showing an example of the flow cell device of the present invention. 図８は、光反応によって切断される第2のプローブを示す図である。FIG. 8 is a diagram showing a second probe cleaved by a photoreaction. 図９は、光反応による第2のプローブの切断の詳細を示す図である。FIG. 9 is a diagram showing details of the cleavage of the second probe by photoreaction. 図１０は、本発明のフローセルデバイスの一例を示す図である。FIG. 10 is a diagram showing an example of the flow cell device of the present invention.
１．核酸反応デバイス
　本発明の核酸反応デバイスは、1つの細胞保持領域を含む核酸反応部を少なくとも1つ含む基板からなる。本発明の核酸反応デバイスの構成について、以下説明する。 1. Nucleic acid reaction device The nucleic acid reaction device of the present invention comprises a substrate including at least one nucleic acid reaction part including one cell holding region. The configuration of the nucleic acid reaction device of the present invention will be described below.
１－１．基板
　本発明の遺伝子解析システムにおいて「基板」とは、核酸反応部を1個又は複数個含む支持体をいう。 1-1. Substrate In the gene analysis system of the present invention, “substrate” refers to a support comprising one or more nucleic acid reaction units.
　基板の素材は、DNA及びRNAの遺伝子発現解析において当技術分野で一般的に使用されている材料で作製されたものであれば特に限定されるものではない。例えば、金、銀、銅、アルミニウム、タングステン、モリブデン、クロム、白金、チタン、ニッケルなどやステンレスなどの合金からなる金属；シリコン；ガラス、石英ガラス、溶融石英、合成石英、アルミナ及び感光性ガラスなどのガラス材料(これらの材料は基本的に透明である)；ポリエステル樹脂、ポリスチレン、ポリエチレン樹脂、ポリプロピレン樹脂、ABS樹脂(Acrylonitrile Butadiene Styrene樹脂)、ナイロン、アクリル樹脂及び塩化ビニル樹脂などのプラスチック(一般には透明でないが光学計測を可能とするために透明とすることもできる)；アガロース、デキストラン、セルロース、ポリビニルアルコール、ニトロセルロース、キチン、キトサンが挙げられる。 The material of the substrate is not particularly limited as long as it is made of a material generally used in the technical field in gene expression analysis of DNA and RNA. For example, a metal made of an alloy such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, nickel, and stainless steel; silicon; glass, quartz glass, fused quartz, synthetic quartz, alumina, and photosensitive glass Glass materials (these materials are basically transparent); polyester resins, polystyrene, polyethylene resins, polypropylene resins, ABS resins (Acrylonitrile Butadiene Styrene resins), plastics such as nylon, acrylic resins and vinyl chloride resins (generally Non-transparent but can also be transparent to allow optical measurements); agarose, dextran, cellulose, polyvinyl alcohol, nitrocellulose, chitin, chitosan.
　基板は、異なる二以上の素材から構成されていてもよい。例えば、基板底部に細孔付きシートを有する基板の場合、基板骨格が前記プラスチックや金属などで構成され、細孔付きシートが、例えば、アルミナ、ガラス、及びシリコン製のフィルム；アクリルアミドゲル、ゼラチン、修飾ポリエチレングリコール、修飾ポリビニルピロリドン、及びハイドロゲル製のゲル薄膜；又はセルロースアセテート、ニトロセルロース又はこれらの混合メンブレン、及びナイロンメンブレン製のメンブレンで構成される場合などが該当する。 The substrate may be composed of two or more different materials. For example, in the case of a substrate having a sheet with pores at the bottom of the substrate, the substrate skeleton is composed of the plastic or metal, and the sheet with pores is, for example, a film made of alumina, glass, and silicon; acrylamide gel, gelatin, Examples include a gel film made of modified polyethylene glycol, modified polyvinyl pyrrolidone, and hydrogel; or a cellulose acetate, nitrocellulose, or a mixed membrane thereof, and a membrane made of nylon membrane.
　基板は、必要に応じてハウジングなどの加工を行うことができる。また、波長300nm～10000nmの光うち少なくとも一部の波長の光に対して透明な、すなわち透過可能な材料で作製してもよい。これにより、遺伝子発現の解析を基板上で光学的に行うことが可能となる。 The substrate can be processed into a housing or the like as necessary. Further, it may be made of a material that is transparent, that is, capable of transmitting at least a part of light having a wavelength of 300 nm to 10000 nm. This makes it possible to optically analyze gene expression on the substrate.
　基板上には、核酸反応部が二次元的に配置されていることが好ましい。基板の形状は問わないが、例えば平板、円筒形、及び球形であっても良い。好ましくは半導体プロセス用製造装置により製造可能な平面基板である。 It is preferable that the nucleic acid reaction part is two-dimensionally arranged on the substrate. Although the shape of a board | substrate is not ask | required, a flat plate, a cylindrical shape, and a spherical shape may be sufficient, for example. Preferably, it is a flat substrate that can be manufactured by a manufacturing apparatus for semiconductor process.
１－２．核酸反応部
　「核酸反応部」とは、細胞中の解析対象である核酸に対して酵素反応処理を行う領域である。核酸反応部では、位置情報込みで核酸を増幅するために、後述する細胞保持領域で保持された細胞から抽出した核酸の末端に、核酸反応部ごとに固有の塩基配列からなるタグ配列の導入を行う。また、この領域において核酸増幅を行ってもよい。一基板あたりの核酸反応部の数は特に限定はしない。1個であっても、また複数個であってもよい。通常は、10～105個の範囲にあればよい。核酸反応部は、1つの細胞保持領域、第1のプローブ、及び第2のプロ－ブを含む。核酸反応部を構成する細胞保持領域、第1のプローブ、及び第2のプロ－ブについて、以下説明する。 1-2. Nucleic acid reaction part "Nucleic acid reaction part" is an area | region which performs an enzyme reaction process with respect to the nucleic acid which is the analysis object in a cell. In the nucleic acid reaction part, in order to amplify the nucleic acid with positional information, a tag sequence consisting of a unique base sequence for each nucleic acid reaction part is introduced at the end of the nucleic acid extracted from the cells held in the cell holding region described later. Do. In addition, nucleic acid amplification may be performed in this region. The number of nucleic acid reaction units per substrate is not particularly limited. There may be one or more. Usually, it may be in the range of 10 to 10 5 . The nucleic acid reaction part includes one cell holding region, a first probe, and a second probe. The cell holding region, the first probe, and the second probe constituting the nucleic acid reaction part will be described below.
１－３．細胞保持領域
　本明細書において「細胞保持領域」は、1又は複数の第1の開口部からなる。「第1の開口部」は、核酸反応部において外界に開いた孔で構成される。第1の開口部の孔の個数は限定されない。一つの孔により細胞を保持してもよいし、複数の孔により細胞を保持してもよい。また、第1の開口部の形状は特に限定しない。例えば、円柱形、略円柱形、楕円柱形、略楕円柱形、直方形、略直方形、立方形、略立方形、円錐形、略円錐形、角錐形、略角錐形などが該当する。本発明の核酸反応デバイスにおいて、細胞保持領域の開口径は、細胞の直径よりもやや小さい大きさから細胞一つがちょうど収まる程度の大きさであることが好ましい。例えば、直径5μm～50μmの範囲にあればよい。細胞保持領域の深さは、1μmから細胞一つがちょうど収まる程度の深さ、例えば、5～100μmの範囲にあることが好ましい。 1-3. Cell Holding Area In the present specification, the “cell holding area” is composed of one or a plurality of first openings. The “first opening” is a hole opened to the outside in the nucleic acid reaction part. The number of holes in the first opening is not limited. Cells may be held by one hole, or cells may be held by a plurality of holes. Further, the shape of the first opening is not particularly limited. For example, a cylindrical shape, a substantially cylindrical shape, an elliptical columnar shape, a substantially elliptical columnar shape, a rectangular shape, a substantially rectangular shape, a cubic shape, a substantially cubic shape, a conical shape, a substantially conical shape, a pyramid shape, a substantially pyramid shape, and the like are applicable. In the nucleic acid reaction device of the present invention, it is preferable that the opening diameter of the cell holding region is slightly smaller than the diameter of the cell so that one cell just fits. For example, the diameter may be in the range of 5 μm to 50 μm. The depth of the cell holding region is preferably from 1 μm to a depth that allows just one cell to fit, for example, in the range of 5 to 100 μm.
　「細胞保持領域」は、複数の第1の開口部からなってもよいが、好ましくは一つの第1の開口部からなる。第1の開口部からなる細胞保持領域が一つの細胞のみを保持すれば(本明細書では、細胞保持領域が一つの細胞のみを保持することを「単離」とも称する)、一つの細胞ごとに個別の核酸反応部で処理できるため、細胞保持領域は一つの細胞のみを保持することが特に好ましい。 The “cell holding region” may be composed of a plurality of first openings, but is preferably composed of one first opening. If the cell holding region comprising the first opening holds only one cell (in this specification, the cell holding region holds only one cell is also referred to as “isolation”), one cell at a time It is particularly preferable that the cell holding region holds only one cell because it can be processed by an individual nucleic acid reaction part.
　多数の細胞からなる接着系細胞を処理する場合には、組織中の細胞を浮遊化して、細胞保持領域で単離することによって単一細胞解析が可能となる。また、組織切片などサンプルを対象とする場合は、細胞保持領域が二つ以上の細胞を保持してもよいが、この場合、細胞保持領域と核酸反応部が空間的に対応しているため、組織切片の2次元平面内の位置情報を含む形で解析結果を得ることができる。 When processing adherent cells consisting of a large number of cells, single cells can be analyzed by suspending the cells in the tissue and isolating them in the cell holding region. In addition, when targeting a sample such as a tissue section, the cell holding region may hold two or more cells, but in this case, since the cell holding region and the nucleic acid reaction part correspond spatially, The analysis result can be obtained in a form including the positional information of the tissue section in the two-dimensional plane.
１－４．第1のプローブ
　本発明の核酸反応デバイスにおいて、「第1のプローブ」とは、核酸で構成されたプローブである。第1のプローブは、原則としてDNAで構成されるが、それに限定するものではなく、例えば、RNAや人工核酸を含んでいてもよい。 1-4. First Probe In the nucleic acid reaction device of the present invention, the “first probe” is a probe composed of a nucleic acid. The first probe is composed of DNA in principle, but is not limited thereto, and may include, for example, RNA or artificial nucleic acid.
　第1のプローブは、前記細胞保持領域によって保持された細胞から抽出される一本鎖核酸の塩基配列の一部に相補的な塩基配列からなる捕捉配列(203)を含む。また、第1のプローブは、必要に応じて共通配列、及び／又はタグ配列をさらに含む。以下、第１のプローブを構成する各配列について具体的に説明をする。 The first probe includes a capture sequence (203) having a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid extracted from the cells held by the cell holding region. Further, the first probe further includes a common sequence and / or a tag sequence as necessary. Hereinafter, each sequence constituting the first probe will be specifically described.
　「捕捉配列」とは、第１のプローブを構成する必須の配列で、細胞保持領域に保持された細胞から抽出された一本鎖核酸の塩基配列の一部に相補的な塩基配列、又はランダムな配列を含み、抽出された一本鎖核酸を捕捉するように構成されている。捕捉配列の塩基配列は、標的である前記一本鎖核酸とハイブリダイズして、それを捕捉することができれば特に限定されない。それ故、核酸の種類及び配列を考慮して適宜設計することができる。本発明において、標的となる一本鎖核酸としては、メッセンジャーRNA(mRNA)、非コードRNA(ncRNA)、microRNA、及び一本鎖DNA、並びにそれらの断片が挙げられる。捕捉配列の長さは、ハイブリダイゼーションによってターゲット(標的)となる一本鎖核酸を捕捉しうる長さであればよい。捕捉配列は、一本鎖核酸の塩基配列の3’末端側又はそれに近い配列に相補的な塩基配列であることが好ましい。 The “capture sequence” is an essential sequence constituting the first probe, a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid extracted from a cell held in the cell holding region, or a random sequence And is configured to capture the extracted single-stranded nucleic acid. The base sequence of the capture sequence is not particularly limited as long as it can hybridize with and capture the target single-stranded nucleic acid. Therefore, it can be designed appropriately in consideration of the type and sequence of the nucleic acid. In the present invention, the target single-stranded nucleic acid includes messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, single-stranded DNA, and fragments thereof. The length of the capture sequence may be any length as long as it can capture a single-stranded nucleic acid as a target (target) by hybridization. The capture sequence is preferably a base sequence complementary to a sequence close to or near the 3 'end of the base sequence of the single-stranded nucleic acid.
　例えば、標的となる一本鎖核酸がmRNAの場合には、捕捉配列としてmRNAの配列の一部であるポリA配列に相補的なオリゴ(dT)配列を用いればよい。オリゴ(dT)配列を構成するdTの重合度は、mRNAのポリA配列をハイブリダイゼーションによって捕捉しうる重合度であればよい。例えば、8～40個、好ましくは8～30個である。また捕捉配列としてオリゴ(dT)配列を用いる場合には、その3'末端に2塩基のランダム配列を付加することが好ましい。それによりcDNAを合成する際のアーティファクトの量を大幅に低減させることが可能になる。そのようなランダム配列として、例えば、VN配列(VはA、G又はCであり、NはA、G、C又はTである)が挙げられる。 For example, when the target single-stranded nucleic acid is mRNA, an oligo (dT) sequence complementary to a poly A sequence that is a part of the mRNA sequence may be used as a capture sequence. The polymerization degree of dT constituting the oligo (dT) sequence may be any polymerization degree that can capture the poly A sequence of mRNA by hybridization. For example, 8 to 40, preferably 8 to 30. When an oligo (dT) sequence is used as a capture sequence, it is preferable to add a random sequence of 2 bases to the 3 ′ end. This makes it possible to greatly reduce the amount of artifact when synthesizing cDNA. Examples of such a random sequence include a VN sequence (V is A, G, or C, and N is A, G, C, or T).
　また、標的となる一本鎖核酸がmicroRNAやゲノムDNA由来の一本鎖核酸である場合には、一本鎖核酸の塩基配列の一部に相補的な塩基配列又はランダム配列を用いることができる。 In addition, when the target single-stranded nucleic acid is a single-stranded nucleic acid derived from microRNA or genomic DNA, a base sequence or random sequence complementary to a part of the base sequence of the single-stranded nucleic acid can be used. .
　「タグ配列」とは、必要に応じて第1のプローブに含まれる配列であって、核酸反応部内の反応産物に付すべき識別タグである。したがって、核酸反応部が複数個存在する場合には、タグ配列は、各核酸反応部に固有の塩基配列を含む。タグ配列は、任意の長さの既知の塩基配列から構成される。例えば、タグ配列が5塩基長からなる場合には、45(＝1024)種類の異なる核酸反応部に固有の塩基配列からなるタグ配列を付与することができる。同様に、例えば、タグ配列が10塩基長からなる場合には、410(＝1048576)種類の異なる核酸反応部に固有の塩基配列からなるタグ配列を付与することができる。したがって、タグ配列の長さは、遺伝子解析システム上の核酸反応部の位置及び／又は数に応じて、それらを識別できるように適宜決定すればよい。具体的には、5～30塩基とすることが好ましい。 The “tag sequence” is a sequence included in the first probe as necessary, and is an identification tag to be attached to the reaction product in the nucleic acid reaction part. Therefore, when there are a plurality of nucleic acid reaction units, the tag sequence includes a base sequence unique to each nucleic acid reaction unit. The tag sequence is composed of a known base sequence having an arbitrary length. For example, when the tag sequence has a length of 5 bases, tag sequences consisting of unique base sequences can be assigned to 4 5 (= 1024) different nucleic acid reaction sites. Similarly, for example, when the tag sequence has a length of 10 bases, 4 10 (= 1048576) types of different nucleic acid reaction units can be given tag sequences consisting of unique base sequences. Therefore, the length of the tag sequence may be determined as appropriate according to the position and / or number of nucleic acid reaction sites on the gene analysis system. Specifically, it is preferably 5 to 30 bases.
　タグ配列を解析することによって、核酸反応デバイス中のどの核酸反応部で抽出された核酸に由来する核酸であるかを識別することができる。核酸反応部は細胞保持領域と対応しているので、タグ配列の情報から、対応する細胞を把握することができる。 By analyzing the tag sequence, the nucleic acid reaction part in the nucleic acid reaction device can identify the nucleic acid derived from the extracted nucleic acid. Since the nucleic acid reaction part corresponds to the cell holding region, the corresponding cell can be grasped from the information of the tag sequence.
　タグ配列を構成する塩基配列は、核酸反応部が基板上に複数個存在する場合、原則として核酸反応部ごとに異なるが、必要であれば複数の核酸反応部に共通していてもよい。例えば、一基板上において、5つの核酸反応部ごとに共通のタグ配列を使用する場合が該当する。 The base sequence constituting the tag sequence differs in principle for each nucleic acid reaction part when a plurality of nucleic acid reaction parts exist on the substrate, but may be common to a plurality of nucleic acid reaction parts if necessary. For example, the case where a common tag sequence is used for every five nucleic acid reaction parts on one substrate corresponds.
　「共通配列」は、選択配列であって、本発明の核酸反応デバイスを用いた遺伝子解析方法の核酸増幅工程において、切断断片を増幅する際のフォワード(Fw)プライマー配列として機能し得る配列である。したがって、第1のプローブでは、原則として5’末端側に配置される。共通配列の塩基長は、プライマーとして適切な長さあれば特に限定されない。例えば、8～60塩基長、好ましくは10～50塩基長とすることができる。共通配列の塩基配列も特に制限はしないが、プライマー配列として適当なTm値となるような配列となるように設計することが好ましい。通常は、Tm値が50℃以上、好ましくは60℃以上となるようにすればよい。 The “common sequence” is a selected sequence that can function as a forward (Fw) primer sequence for amplifying a cleaved fragment in the nucleic acid amplification step of the gene analysis method using the nucleic acid reaction device of the present invention. . Therefore, in the first probe, as a rule, it is arranged on the 5 ′ end side. The base length of the common sequence is not particularly limited as long as it is an appropriate length as a primer. For example, the length can be 8 to 60 bases, preferably 10 to 50 bases. The base sequence of the common sequence is not particularly limited, but it is preferable to design the base sequence so as to have an appropriate Tm value as a primer sequence. Usually, the Tm value may be 50 ° C. or higher, preferably 60 ° C. or higher.
　第1のプローブが捕捉配列のみを含む場合、一本鎖核酸を捕捉できる限り、第1のプローブの固定部位は特に限定しないが、特に5’末端が固定されていることが好ましい。また、第1のプローブが共通配列又はタグ配列を含む場合、共通配列又はタグ配列は、捕捉配列の5’末端側に配置されるのが好ましく、捕捉配列の5'末端側に、5'末端側から順に、共通配列、及びタグ配列を含むことが好ましい。 When the first probe contains only the capture sequence, the fixing site of the first probe is not particularly limited as long as the single-stranded nucleic acid can be captured, but the 5 'end is particularly preferably fixed. When the first probe includes a common sequence or tag sequence, the common sequence or tag sequence is preferably arranged on the 5 ′ end side of the capture sequence, and the 5 ′ end side of the capture sequence is 5 ′ end side. It is preferable that a common sequence and a tag sequence are included in order from the side.
　第1のプローブ及び後述する第2のプローブ(本明細書では、しばしばこれらをまとめて「核酸プローブ」と称する。)は、核酸反応部内に固定部を介して固定される。予め核酸プローブを核酸反応部内に固定しておくことにより、細胞又は組織にダメージを及ぼすことなく、またロボットなどを使用することなく、個々の細胞に由来する核酸からの遺伝子情報を得ることが可能となる。特に、細胞又は組織にダメージを及ぼすことがないため、そのダメージに起因する遺伝子発現の変化を回避することができる。 The first probe and the second probe described later (in the present specification, these are often collectively referred to as “nucleic acid probe”) are fixed in the nucleic acid reaction part via a fixing part. By fixing the nucleic acid probe in the nucleic acid reaction part in advance, it is possible to obtain genetic information from nucleic acids derived from individual cells without damaging cells or tissues and without using robots. It becomes. In particular, since it does not damage cells or tissues, changes in gene expression due to the damage can be avoided.
　核酸プローブを核酸反応部内に直接的に固定する例として、核酸反応部内部の表面に固定する場合が挙げられる。固定する位置は問わない。例えば、核酸反応部の底面、壁面、若しくはその組み合わせ又は全面のいずれであってもよい。核酸反応部は多孔質構造であることが好ましく、核酸プローブは多孔質の表面に固定されていることが好ましい。「多孔質の表面」には、孔内部の表面のみならず、孔の存在しない部分における核酸反応部表面も含まれる。多孔質構造の例として細孔付きシートが挙げられる。 As an example of directly immobilizing a nucleic acid probe in a nucleic acid reaction part, there is a case where it is immobilized on the surface inside the nucleic acid reaction part. The position to fix is not ask | required. For example, any of the bottom surface, the wall surface, a combination thereof, or the entire surface of the nucleic acid reaction unit may be used. The nucleic acid reaction part preferably has a porous structure, and the nucleic acid probe is preferably immobilized on a porous surface. The “porous surface” includes not only the surface inside the pores but also the surface of the nucleic acid reaction part in the portion where no pores are present. An example of the porous structure is a sheet with pores.
　核酸プローブを核酸反応部に間接的に固定する例として、核酸反応部の表面に保持された担体の表面に固定される場合が挙げられる。本明細書において「担体」は、核酸反応部と核酸プローブを連結する介在物質で、核酸プローブをその表面に固定し、自身も核酸反応部の内部表面に、必要に応じて解離可能な状態で固定されている。担体の素材は、限定はしない。例えば、樹脂材料(ポリスチレンなど)、酸化物(ガラス、シリカなど)、金属(鉄、金、白金、銀など)、高分子多糖支持体(例えば、セファロース若しくはセファデックス)、セラミックス、ラテックス及びこれらの組み合わせで構成される。担体の形状は、特に限定はしないが、ビーズのような球体粒子は、結合表面積が大きく、操作性も高いことから好ましい。それ故、磁性ビーズは、担体として好適である。 An example of indirectly fixing the nucleic acid probe to the nucleic acid reaction part is a case where the nucleic acid probe is fixed to the surface of a carrier held on the surface of the nucleic acid reaction part. In the present specification, the “carrier” is an intervening substance that links the nucleic acid reaction part and the nucleic acid probe, fixes the nucleic acid probe to the surface thereof, and is itself dissociable on the inner surface of the nucleic acid reaction part as necessary. It is fixed. The material of the carrier is not limited. For example, resin materials (polystyrene, etc.), oxides (glass, silica, etc.), metals (iron, gold, platinum, silver, etc.), polymeric polysaccharide supports (e.g., Sepharose or Sephadex), ceramics, latex, and these Composed of a combination. The shape of the carrier is not particularly limited, but spherical particles such as beads are preferable because they have a large binding surface area and high operability. Therefore, magnetic beads are suitable as a carrier.
　核酸プローブは、当技術分野で公知の任意の固定方法により固定部を介して核酸反応部に固定されている。固定方法としては、例えば、核酸反応部の表面や担体の表面への生物学的結合、共有結合、イオン結合、又は物理吸着が挙げられる。また、スペーサー配列を介して両プローブを核酸反応部の表面及び担体に固定することも可能である。 The nucleic acid probe is fixed to the nucleic acid reaction part via the fixing part by any fixing method known in the art. Examples of the immobilization method include biological bonding, covalent bonding, ionic bonding, or physical adsorption to the surface of the nucleic acid reaction part or the surface of the carrier. It is also possible to fix both probes to the surface of the nucleic acid reaction part and the carrier via a spacer sequence.
　生物学的結合の例としては、ビオチンとアビジン、ストレプトアビジン又はニュートラアビジンとの結合、抗原と抗体との結合などのような接合分子を介した結合が挙げられる。例えば、アビジン、ストレプトアビジン又はニュートラアビジンが結合した核酸反応部の表面と、ビオチン修飾した核酸プローブを反応させることにより達成できる。 Examples of biological binding include binding via a junction molecule such as binding between biotin and avidin, streptavidin or neutravidin, binding between an antigen and an antibody. For example, it can be achieved by reacting a biotin-modified nucleic acid probe with the surface of a nucleic acid reaction part to which avidin, streptavidin or neutravidin is bound.
　共有結合の場合、例えば、核酸プローブに官能基を導入し、その官能基に対して反応性の官能基を核酸反応部の表面に導入して、両者を反応させることにより達成できる。具体的には、例えば、核酸プローブにアミノ基を導入し、核酸反応部の表面に活性エステル基、エポキシ基、アルデヒド基、カルボジイミド基、イソチオシアネート基又はイソシアネート基を導入することにより共有結合を形成できる。また、核酸プローブにメルカプト基を導入し、細胞保持領域内部の表面に活性エステル基、マレイミド基又はジスルフィド基を導入してもよい。官能基を細胞保持領域内部の表面又は担体表面に導入する方法として、所望の官能基を有するシランカップリング剤によって核酸反応部の表面を処理する方法が挙げられる。カップリング剤の例としては、γ-アミノプロピルトリエトキシシラン、N-β-(アミノエチル)-γ-アミノプロピルトリメトキシシラン、N-β-(アミノエチル)-β-アミノプロピルメチルジメトキシシランなどを用いることができる。官能基を核酸反応部の表面又は担体表面に導入する他の方法として、プラズマ処理が挙げられる。 In the case of covalent bonding, for example, it can be achieved by introducing a functional group into the nucleic acid probe, introducing a functional group reactive to the functional group to the surface of the nucleic acid reaction part, and reacting both. Specifically, for example, an amino group is introduced into a nucleic acid probe, and a covalent bond is formed by introducing an active ester group, epoxy group, aldehyde group, carbodiimide group, isothiocyanate group or isocyanate group on the surface of the nucleic acid reaction part. it can. Further, a mercapto group may be introduced into the nucleic acid probe, and an active ester group, maleimide group or disulfide group may be introduced into the surface inside the cell holding region. Examples of the method for introducing the functional group into the surface inside the cell holding region or the surface of the carrier include a method of treating the surface of the nucleic acid reaction part with a silane coupling agent having a desired functional group. Examples of coupling agents include γ-aminopropyltriethoxysilane, N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane, N-β- (aminoethyl) -β-aminopropylmethyldimethoxysilane, etc. Can be used. As another method for introducing the functional group onto the surface of the nucleic acid reaction part or the surface of the carrier, plasma treatment may be mentioned.
　物理吸着の例としては、核酸反応部の表面をポリ陽イオン(ポリリシン、ポリアリルアミン、ポリエチレンイミンなど)で表面処理し、核酸プローブの荷電を利用して静電結合させる方法などが挙げられる。なお、核酸反応部や担体は、他の物質(核酸やタンパク質など)が吸着しないように、予め表面コーティングを行うことが好ましい。 Examples of physical adsorption include a method in which the surface of the nucleic acid reaction part is surface-treated with a polycation (polylysine, polyallylamine, polyethyleneimine, etc.) and electrostatically coupled using the charge of the nucleic acid probe. The nucleic acid reaction part and the carrier are preferably pre-coated with a surface so that other substances (such as nucleic acids and proteins) do not adsorb.
１－５．第2のプローブ
　本発明の核酸反応デバイスにおいて、「第2のプローブ」とは、第1のプローブと同様に核酸で構成されたプローブである。第2のプローブも原則としてDNAで構成されるが、それに限定するものではなく、例えば、RNAや人工核酸を含んでいてもよい。 1-5. Second Probe In the nucleic acid reaction device of the present invention, the “second probe” is a probe composed of nucleic acid in the same manner as the first probe. The second probe is also composed of DNA in principle, but is not limited thereto, and may include, for example, RNA or artificial nucleic acid.
　第2のプローブは、核酸反応部ごとに固有の塩基配列からなるタグ配列、及び該タグ配列の3’末端側に、前記第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列を含む。また、第2のプローブは、必要に応じて共通配列並びに／又はステムセンス配列及びステムアンチセンス配列を含む。以下、第2のプローブを構成する各配列について説明する。 The second probe is a tag sequence consisting of a unique base sequence for each nucleic acid reaction part, and a part of the base sequence of the single-stranded nucleic acid captured by the first probe on the 3 ′ end side of the tag sequence Contains a complementary nucleotide sequence. Further, the second probe includes a common sequence and / or a stem sense sequence and a stem antisense sequence as necessary. Hereinafter, each sequence constituting the second probe will be described.
　「第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列」とは、前記第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列、又はランダムな配列を含み、前記第1のプローブで捕捉された一本鎖核酸にハイブリダイズするように構成されている。該配列の塩基配列は、標的である前記第1のプローブで捕捉された一本鎖核酸とハイブリダイズすることができれば特に限定されない。それ故、核酸の種類及び配列を考慮して適宜設計することができる。捕捉配列の長さは、ターゲット(標的)となる一本鎖核酸にハイブリダイズしうる長さであればよい。特に、「第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列」は、第1のプローブで捕捉された核酸の任意の領域にハイブリダイズすることを可能とするために、例えばランダムヘキサマー等のランダム配列であることが好ましい。 “A base sequence complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe” is complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe. It comprises a basic nucleotide sequence or a random sequence and is configured to hybridize to a single-stranded nucleic acid captured by the first probe. The base sequence of the sequence is not particularly limited as long as it can hybridize with the single-stranded nucleic acid captured by the target first probe. Therefore, it can be designed appropriately in consideration of the type and sequence of the nucleic acid. The capture sequence may be any length as long as it can hybridize to a single-stranded nucleic acid serving as a target (target). In particular, “a base sequence complementary to a part of the base sequence of a single-stranded nucleic acid captured by the first probe” can hybridize to any region of the nucleic acid captured by the first probe. Therefore, a random sequence such as a random hexamer is preferable.
　「タグ配列」は、第1のプローブに記載のタグ配列と同一の構成であることから、ここでは具体的な説明を省略する。前述のようにタグ配列は、同一核酸反応部由来であることを示す識別タグであることから、一の核酸反応部内に配置される第1のプローブと第2のプローブのタグ配列は、原則として同一の配列で構成される。 Since the “tag sequence” has the same configuration as the tag sequence described in the first probe, a specific description is omitted here. As described above, since the tag sequence is an identification tag indicating that it is derived from the same nucleic acid reaction part, the tag sequences of the first probe and the second probe arranged in one nucleic acid reaction part are in principle Consists of the same sequence.
　「共通配列」は、選択配列であって、本発明の遺伝子解析システムを用いた遺伝子解析方法の核酸増幅工程において、切断断片を増幅する際のフォワード(Fw)プライマー配列として機能し得る配列であり、第1のプローブに記載の共通配列と基本的には同一の構成である。第2のプローブが共通配列を含む場合、共通配列は5’末端に含まれるのが好ましい。この場合、第2のプローブは、5’末端側から順番に、共通配列、核酸反応部ごとに固有の塩基配列からなるタグ配列、及び前記第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列を含む。 A `` common sequence '' is a selected sequence that can function as a forward (Fw) primer sequence for amplifying a cleaved fragment in the nucleic acid amplification step of the gene analysis method using the gene analysis system of the present invention. The configuration is basically the same as the common sequence described in the first probe. Where the second probe includes a consensus sequence, the consensus sequence is preferably included at the 5 'end. In this case, in order from the 5 ′ end side, the second probe has a common sequence, a tag sequence consisting of a unique base sequence for each nucleic acid reaction part, and a base of the single-stranded nucleic acid captured by the first probe. A base sequence complementary to a part of the sequence is included.
　「ステムセンス配列及びステムアンチセンス配列」は、第2のプローブに特有の選択配列であって、互いに相補的な塩基配列からなる。各配列の塩基長は、原則として同一である。その長さは、両配列が安定したステム構造を形成できれば、特に制限はしない。例えば、塩基長は、3～7塩基の範囲にあればよい。各配列を構成する塩基配列も互いが相補的であれば、特に制限はしない。第2のプローブにおいて、ステムセンス配列とステムアンチセンス配列は、一対がその間に配置された任意の塩基配列によって分離した状態で存在してよい。任意の塩基配列には、タグ配列及び／又は共通配列が挙げられる。ステムセンス配列とステムアンチセンス配列は、第2のプローブ内で互いに塩基対合することによってステム構造を形成し得る。これによって、前記任意配列はループ構造となり、第2のプローブは全体としてループ・ステム構造を形成する。ループ・ステム構造を形成することにより、「第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列」以外の配列が一本鎖核酸にハイブリダイズすることを避け、非特異的な反応を抑制することができる。 The “stem sense sequence and stem antisense sequence” are selection sequences unique to the second probe, and are composed of complementary base sequences. In principle, the base length of each sequence is the same. The length is not particularly limited as long as both sequences can form a stable stem structure. For example, the base length may be in the range of 3 to 7 bases. The base sequences constituting each sequence are not particularly limited as long as they are complementary to each other. In the second probe, the stem sense sequence and the stem antisense sequence may exist in a state where a pair is separated by an arbitrary base sequence arranged therebetween. Arbitrary base sequences include tag sequences and / or common sequences. The stem sense sequence and the stem antisense sequence can form a stem structure by base pairing with each other in the second probe. Accordingly, the arbitrary sequence has a loop structure, and the second probe forms a loop stem structure as a whole. By forming a loop / stem structure, it is possible that sequences other than the “base sequence complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe” hybridize to the single-stranded nucleic acid. It can avoid and suppress non-specific reactions.
　本発明の核酸反応デバイスにおいて、第2のプローブ、又は第2のプローブの固定部は、切断部を含む。切断部を切断することにより、第2のプローブが遊離し、遊離した第2のプローブが、第1のプローブで捕捉された一本鎖核酸にハイブリダイズすることが可能になる。 In the nucleic acid reaction device of the present invention, the second probe or the fixing part of the second probe includes a cutting part. By cleaving the cleaved portion, the second probe is liberated, and the liberated second probe can hybridize to the single-stranded nucleic acid captured by the first probe.
　切断部は、第2のプローブを任意の位置で核酸反応部から遊離させることができるものであれば、特に限定せず、当技術分野で公知のものを用いればよい。例えば、切断部は、化学的反応、特定の波長の光、及び振動(超音波)により切断されるものであってよく、特に化学的反応により切断される化学結合であることが好ましい。切断部を切断し得る化学的反応の例として、加水分解反応、酸化反応、還元反応、及び酵素反応などが挙げられる。 The cleavage part is not particularly limited as long as it can release the second probe from the nucleic acid reaction part at an arbitrary position, and a known part in this technical field may be used. For example, the cleaving part may be cleaved by a chemical reaction, light of a specific wavelength, and vibration (ultrasonic waves), and is preferably a chemical bond cleaved by a chemical reaction. Examples of chemical reactions that can cleave the cleavage site include hydrolysis reactions, oxidation reactions, reduction reactions, and enzyme reactions.
　例えば、第2のプローブが切断部を含む例として、第2のプローブの配列内に制限酵素認識部位を含む態様が挙げられる。制限酵素を用いる切断では、二本鎖DNA、一本鎖DNA、DNA/RNA鎖、いずれかのDNA鎖を切断することが可能な酵素を用いる。一本鎖DNAを切断できる制限酵素として、例えば、AccI、AccII、AvaII、BspRI、CfoI、DdeI、EcoRI、HaeIII、HapII、HhaI、HinfI、MspI、MboI、MboII、MspI、Sau3AI、SfaI、TthHB81が挙げられる。捕捉する一本鎖核酸がDNAの場合には、そのDNAとそれを鋳型として得られるDNA相補鎖からなる二本鎖DNAを切断できる制限酵素を用いることができる。より好ましくは二本鎖DNAのうち一方のDNA鎖のみを切断できる制限酵素(ニックを作製する酵素)である。 For example, as an example in which the second probe includes a cleavage site, an embodiment in which a restriction enzyme recognition site is included in the sequence of the second probe can be mentioned. In the cleavage using a restriction enzyme, an enzyme capable of cleaving either a double-stranded DNA, a single-stranded DNA, or a DNA / RNA strand is used. Examples of restriction enzymes capable of cleaving single-stranded DNA include AccI, AccII, AvaII, BspRI, CfoI, DdeI, EcoRI, HaeIII, HapII, HhaI, HinfI, MspI, MboI, MboII, MspI, Sau3AI, SfaI, and TthHB81. It is done. When the single-stranded nucleic acid to be captured is DNA, a restriction enzyme capable of cleaving double-stranded DNA comprising the DNA and a DNA complementary strand obtained using the DNA as a template can be used. More preferably, it is a restriction enzyme (enzyme producing enzyme) capable of cleaving only one DNA strand of double-stranded DNA.
　さらに、第2のプローブの切断部を切断する方法として、紫外線照射を用いてもよい。例えば、切断部の配列をvinylC-Tとすることで、312nmの紫外線を照射することによって配列間の結合を切断することができる(図8)。この反応の反応機構の詳細については、Y. Yoshimura and K. Fujimoto, Organic Letters. , vol. 10, no. 5, pp. 3227-3230, 2008を参照されたい。 Furthermore, ultraviolet irradiation may be used as a method for cutting the cutting portion of the second probe. For example, by using vinylC-T as the cutting part array, the bond between the arrays can be cut by irradiating 312 nm ultraviolet rays (FIG. 8). For details of the reaction mechanism of this reaction, see Y. Yoshimura and K. Fujimoto, Organic Letters., Vol. 10, no. 5, pp. 3227-3230, 2008.
　また、第2のプローブと核酸反応部の固定部を含む例として、例えば固定部がジスルフィド結合である態様が挙げられ、この場合、還元剤による還元反応によってジスルフィド結合を切断し、第2のプローブを遊離させることができる。 In addition, as an example including the fixing part of the second probe and the nucleic acid reaction part, for example, an aspect in which the fixing part is a disulfide bond can be mentioned. In this case, the disulfide bond is cleaved by a reduction reaction with a reducing agent, and the second probe Can be liberated.
１－６．第2の開口部
　本発明の核酸反応デバイスは、任意に、第2の開口部を含む。「第2の開口部」は、第1の開口部とは異なる位置で外界に開いた孔により構成される。「第2の開口部」の大きさ、形状、及び個数などは制限しない。核酸反応デバイスに2つの開口部を設けることで、これらの開口部を介して溶液を通過させながら、連続的にサンプルを処理することが可能となる。 1-6. Second Opening The nucleic acid reaction device of the present invention optionally includes a second opening. The “second opening” is constituted by a hole opened to the outside at a position different from the first opening. The size, shape, and number of “second openings” are not limited. By providing two openings in the nucleic acid reaction device, it is possible to continuously process the sample while passing the solution through these openings.
２．フローセルデバイス
　一態様において、本発明は、フローセルデバイスに関する。本発明のフローセルデバイスは、上記核酸反応デバイス及び流路を含む基板からなる。基板の構成については、上記核酸反応デバイスと同一であるため、ここでは説明を省略する。 2. Flow cell device In one aspect, the invention relates to a flow cell device. The flow cell device of the present invention comprises a substrate including the nucleic acid reaction device and a flow path. Since the configuration of the substrate is the same as that of the nucleic acid reaction device, description thereof is omitted here.
　一実施形態において、本発明のフローセルデバイスは、第1の開口部のみを有する本発明の核酸反応デバイス、及び第1の開口部に接する第1の流路を含む基板からなる。この場合、核酸反応部への溶液の流入及び流出は、いずれも第1の流路に対して、第1の開口部を介して行われる。 In one embodiment, the flow cell device of the present invention comprises a nucleic acid reaction device of the present invention having only a first opening and a substrate including a first flow channel in contact with the first opening. In this case, both inflow and outflow of the solution to the nucleic acid reaction unit are performed via the first opening with respect to the first channel.
　別の実施形態において、本発明のフローセルデバイスは、第1の開口部及び第2の開口部を有する本発明の核酸反応デバイス、第1の開口部に接する第1の流路、及び第2の開口部に接する第2の流路を含む基板からなる。この場合、2つの開口部を介して溶液を通過させながらサンプルを処理することができる。例えば、第1の流路を流入のための流路とし、第2の流路を流出のための流路とすることで、溶液の流れを一方向にして、連続的にサンプルを処理することが可能となる。 In another embodiment, the flow cell device of the present invention comprises a nucleic acid reaction device of the present invention having a first opening and a second opening, a first flow path in contact with the first opening, and a second The substrate includes a second flow path in contact with the opening. In this case, the sample can be processed while passing the solution through the two openings. For example, the first flow path is a flow path for inflow and the second flow path is a flow path for outflow, so that the sample can be processed continuously with the flow of the solution in one direction. Is possible.
　上記核酸反応デバイスを複数含むことによって、本発明のフローセルデバイスは、多数のサンプルを並列処理することができる。また、本発明のフローセルデバイスは、任意に溶液を送液するために、送液ポンプなどの送液手段を含んでよい。 By including a plurality of the nucleic acid reaction devices, the flow cell device of the present invention can process a large number of samples in parallel. In addition, the flow cell device of the present invention may include liquid feeding means such as a liquid feeding pump in order to arbitrarily send a solution.
３．遺伝子解析方法
　本発明の遺伝子解析方法について説明する。 3. Gene Analysis Method The gene analysis method of the present invention will be described.
　一態様において、本発明は、本発明の核酸反応デバイスを用いて、細胞由来の一本鎖核酸から、前記タグ配列が付加された核酸を得る方法であって、任意に、本発明の核酸反応デバイスの基板上に複数の細胞を流し、細胞保持領域に1細胞ずつ保持させる工程(細胞保持工程)、前記細胞保持領域に保持された細胞から一本鎖核酸を抽出し、抽出された一本鎖核酸を、前記捕捉配列とのハイブリダイゼーションにより前記第1のプローブに捕捉させる工程(一本鎖核酸捕捉工程)、前記第2のプローブの切断部を切断し、第2のプローブを遊離させる工程(第2のプローブの遊離工程)、遊離した前記第2のプローブを、前記第1のプローブによって捕捉された一本鎖核Aの一部にハイブリダイズさせる工程(第2のプローブのハイブリダイズ工程)、及びハイブリダイズした前記第2のプローブをプライマーとして、前記第1のプローブによって捕捉された一本鎖核酸の一部に相補的な塩基配列を含む核酸を合成することにより、前記タグ配列が付加された核酸を得る工程(核酸合成工程)、を含む前記方法に関する。 In one aspect, the present invention provides a method for obtaining a nucleic acid to which the tag sequence is added from a single-stranded nucleic acid derived from a cell using the nucleic acid reaction device of the present invention, and optionally, the nucleic acid reaction of the present invention. A step of flowing a plurality of cells on the substrate of the device and holding each cell in the cell holding region (cell holding step), extracting a single-stranded nucleic acid from the cells held in the cell holding region, A step of allowing the first probe to capture a strand nucleic acid by hybridization with the capture sequence (single-stranded nucleic acid capturing step), a step of cleaving the cleavage portion of the second probe, and releasing the second probe (Second probe releasing step), the step of hybridizing the released second probe to a part of the single-stranded nucleus A captured by the first probe (hybridizing step of the second probe) ) And hive A nucleic acid to which the tag sequence is added by synthesizing a nucleic acid containing a base sequence complementary to a part of a single-stranded nucleic acid captured by the first probe, using the soybean second probe as a primer A method (nucleic acid synthesis step).
　以下、各工程について説明をする。
(細胞保持工程)
　「細胞保持工程」は、本発明の方法に任意に含まれ得る工程であって、本発明の核酸反応デバイスの基板上に複数の細胞を流し、細胞保持領域に細胞を保持させる工程である。 Hereinafter, each step will be described.
(Cell retention process)
The “cell holding step” is a step that can optionally be included in the method of the present invention, and is a step of flowing a plurality of cells on the substrate of the nucleic acid reaction device of the present invention and holding the cells in the cell holding region.
　本発明において解析に用いる試料は、遺伝子発現を解析しようとする生体由来試料であれば特に限定されるものではなく、細胞試料、組織試料、液体試料などの任意の試料を用いることができる。具体的には、1細胞からなるサンプル、複数の細胞を含む試料、組織切片試料、複数の個々の細胞を2次元的に保持しアレー状に配列させた試料などが挙げられる。 The sample used for analysis in the present invention is not particularly limited as long as it is a biological sample to be analyzed for gene expression, and any sample such as a cell sample, a tissue sample, a liquid sample, or the like can be used. Specific examples include a sample composed of one cell, a sample containing a plurality of cells, a tissue slice sample, a sample in which a plurality of individual cells are two-dimensionally held and arranged in an array.
　また試料の由来となる生体も特に限定されるものではなく、脊椎動物(例えば、哺乳類、鳥類、爬虫類、魚類、両生類など)、無脊椎動物(例えば、昆虫、線虫、甲殻類など)、原生生物、植物、真菌、細菌、ウイルスなどの任意の生体に由来する試料を用いることができる。 The living organism from which the sample is derived is not particularly limited, and vertebrates (for example, mammals, birds, reptiles, fish, amphibians, etc.), invertebrates (for example, insects, nematodes, crustaceans, etc.), protozoa A sample derived from any living body such as an organism, a plant, a fungus, a bacterium, or a virus can be used.
(一本鎖核酸捕捉工程)
　「一本鎖核酸捕捉工程」は、細胞保持領域に保持した細胞から核酸を抽出し、得られた一本鎖核酸を細胞保持領域内の第1のプローブで捕捉する工程である。 (Single-stranded nucleic acid capture step)
The “single-stranded nucleic acid capturing step” is a step of extracting nucleic acid from the cells held in the cell holding region and capturing the obtained single-stranded nucleic acid with the first probe in the cell holding region.
　この工程において、細胞保持領域に保持した細胞から抽出された一本鎖核酸は、直下の核酸反応部に固定された第1のプローブにハイブリダイズにより捕捉される。 In this step, the single-stranded nucleic acid extracted from the cells held in the cell holding region is captured by hybridization with the first probe fixed to the nucleic acid reaction part immediately below.
　本工程において、捕捉すべきターゲットとなる一本鎖核酸としては、限定されるものではないが、生体組織を構成する細胞内のメッセンジャーRNA(mRNA)、非コードRNA(ncRNA)、microRNA、及び一本鎖DNA、並びにそれらの断片、特にmRNAが挙げられる。細胞からの核酸の抽出は、当技術分野で公知の方法により行えばよい。例えば、Proteinase Kのようなタンパク質分解酵素、チオシアン酸グアニジン・グアニジン塩酸といったカオトロピック塩、Tween及びSDSといった界面活性剤、あるいは市販の細胞溶解用試薬を用いて、細胞を溶解し、それに含まれる核酸、すなわちDNA及びRNAを溶出することができる。 In this step, the target single-stranded nucleic acid to be captured is not limited, but is a messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, and one Examples include double-stranded DNA, and fragments thereof, particularly mRNA. Extraction of nucleic acids from cells may be performed by methods known in the art. For example, using a proteolytic enzyme such as Proteinase K, chaotropic salts such as guanidine thiocyanate and guanidine hydrochloride, surfactants such as Tween and SDS, or commercially available reagents for cell lysis, nucleic acids contained therein, That is, DNA and RNA can be eluted.
(第2のプローブの遊離工程)
　「第2のプローブの遊離工程」は、第2のプローブ又は第2のプローブの固定部に含まれる切断部を切断することにより、第2のプローブを遊離させる工程である。切断部の構造は、上記「第2のプローブ」の項目にて記載した通りである。切断部は、切断部の構造に応じて、化学的反応、特定の波長の光、及び振動(超音波)など、当業者に公知の方法を用いて切断することができる。 (Second probe release step)
The “second probe releasing step” is a step of releasing the second probe by cleaving the second probe or the cleavage part included in the fixing part of the second probe. The structure of the cut portion is as described in the item “Second probe” above. The cutting part can be cut using a method known to those skilled in the art, such as chemical reaction, light of a specific wavelength, and vibration (ultrasonic waves), depending on the structure of the cutting part.
(第2のプローブのハイブリダイズ工程)
　「第2のプローブのハイブリダイズ工程」は、切断部の切断によって遊離した第2のプローブが、プローブ内の「第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列」によって、同一の核酸反応部内の第1のプローブによって捕捉された一本鎖核酸の塩基配列の一部とハイブリダイズする工程である。上記の通り、「第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列」が、ランダムヘキサマー等のランダム配列である場合には、第1のプローブで捕捉された核酸の任意の領域の解析を可能とすることが可能となる。上記「第2のプローブの遊離工程」と「第2のプローブのハイブリダイズ工程」は、同時に行われるのが好ましい。 (Second probe hybridization step)
In the “second probe hybridization step”, the second probe released by cleaving the cleavage site is complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe in the probe. Is a step of hybridizing with a part of the base sequence of the single-stranded nucleic acid captured by the first probe in the same nucleic acid reaction part. As described above, when the “base sequence complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe” is a random sequence such as a random hexamer, the first probe It becomes possible to analyze an arbitrary region of the captured nucleic acid. The “second probe releasing step” and the “second probe hybridizing step” are preferably performed simultaneously.
(核酸合成工程)
　「核酸合成工程」とは、ハイブリダイズした前記第2のプローブをプライマーとして、前記第1のプローブによって捕捉された一本鎖核酸を鋳型として、その相補鎖を逆転写酵素やDNAポリメラーゼなどにより相補鎖を合成する工程である。本発明において、相補鎖の合成は、当技術分野で公知の方法により行うことができる。例えば、核酸がmRNAなどのRNAの場合には、例えば、逆転写酵素を用いた逆転写反応によってcDNAを合成することができる。また核酸がDNAの場合には、例えば、DNAポリメラーゼを用いた複製反応によってcDNAを合成することができる。本工程により、タグ配列を含む第2プローブが付加された核酸が合成される。 (Nucleic acid synthesis process)
“Nucleic acid synthesis step” means that the hybridized second probe is used as a primer, the single-stranded nucleic acid captured by the first probe is used as a template, and its complementary strand is complemented by reverse transcriptase or DNA polymerase. It is a step of synthesizing a chain. In the present invention, the complementary strand can be synthesized by a method known in the art. For example, when the nucleic acid is RNA such as mRNA, for example, cDNA can be synthesized by a reverse transcription reaction using reverse transcriptase. When the nucleic acid is DNA, for example, cDNA can be synthesized by a replication reaction using DNA polymerase. By this step, a nucleic acid to which the second probe containing the tag sequence is added is synthesized.
　上記「第2のプローブの遊離工程」、「第2のプローブのハイブリダイズ工程」、及び「核酸合成工程」を同時に行うことによって、第2のプローブが効率的にmRNAにハイブリダイズしてプライマーとして機能するので、これらの工程は同時に行うことが好ましい。 By simultaneously performing the “second probe releasing step”, the “second probe hybridizing step”, and the “nucleic acid synthesis step”, the second probe efficiently hybridizes to the mRNA as a primer. Since these functions, it is preferable to perform these steps simultaneously.
　また、本発明の方法は、任意に前記核酸を増幅する工程を含む。増幅工程は、当業者に公知の方法により行うことができる。例えば、「核酸合成工程」により合成された核酸に対し、ターミナルトランスフェラーゼにより3’末端にポリA配列を付加し、続いてポリA配列に相補的なポリT配列を含むプライマーにより「核酸合成工程」により合成された核酸の相補鎖を合成する。プライマーには、上述した第2のプローブに含まれ得る共通配列に対応する共通のプライマー配列(例えば、第2のプローブに含まれ得る共通のフォワードプライマー配列とは異なる配列を持つ共通のリバースプライマー配列)を5’末端へ付加することによって、共通配列によりPCRを行うことが可能となり、この後の増幅工程を簡便かつ効率的に実施することが可能となる。増幅方法としては当技術分野で公知の任意の方法を用いることができ、例えば、ポリメラーゼ連鎖反応(PCR)、Nucleic Acid Sequence-Based Amplification(NASBA)法、Loop-Mediated Isothermal Amplification(LAMP)法、ローリングサークル増幅(RCA)反応が挙げられる。 In addition, the method of the present invention optionally includes a step of amplifying the nucleic acid. The amplification step can be performed by a method known to those skilled in the art. For example, for a nucleic acid synthesized by the “nucleic acid synthesis step”, a polyA sequence is added to the 3 ′ end by terminal transferase, followed by a “nucleic acid synthesis step” using a primer containing a poly T sequence complementary to the poly A sequence. The complementary strand of the nucleic acid synthesized by is synthesized. The primer includes a common primer sequence corresponding to the common sequence that can be included in the second probe described above (for example, a common reverse primer sequence having a sequence different from the common forward primer sequence that can be included in the second probe). ) To the 5 ′ end, it becomes possible to perform PCR using a common sequence, and the subsequent amplification step can be carried out simply and efficiently. As an amplification method, any method known in the art can be used, for example, polymerase chain reaction (PCR), Nucleic Acid-Sequence-Based Amplification (NASBA) method, Loop-Mediated Isothermal Thermal Amplification (LAMP) method, rolling A circle amplification (RCA) reaction may be mentioned.
　本増幅工程で得られた増幅産物を当技術分野で公知の任意の方法により遺伝子配列を解析することもできる。また、配列解析を行うことにより遺伝子発現解析も可能である。例えば、一実施形態では、増幅産物の配列を決定することにより、解析対象の遺伝子の発現の有無、発現量などを解析することができる。また別の実施形態では、上記遺伝子特異的配列に対して相補的な塩基配列を有する標識したプローブを利用して、cDNA又は得られる増幅産物に該プローブをハイブリダイズさせ、標識に基づいて解析対象の遺伝子発現を検出する(例えば、光学的に検出する)ことができる。そのような検出に使用するプローブは、当業者であれば適宜設計することができる。使用する標識もまた当技術分野で公知の任意の標識を用いることができ、例えば、蛍光標識(Cy3、フルオレセインイソチオシアネート(FITC)、テトラメチルローダミンイソチオシアネート(TRITC)など)、化学発光標識(ルシフェリンなど)、酵素標識(ペルオキシダーゼ、β－ガラクトシダーゼ、アルカリフォスファターゼなど)、放射性標識(トリチウム、ヨウ素125など)が挙げられる。さらに別の実施形態では、上記遺伝子特異的配列に対して相補的な塩基配列を有するプローブを利用して核酸増幅反応を行い、増幅の有無を化学発光又は蛍光に基づいて検出することによって、解析対象の遺伝子の発現を解析することができる。 The gene sequence of the amplification product obtained in this amplification step can also be analyzed by any method known in the art. Gene expression analysis is also possible by performing sequence analysis. For example, in one embodiment, the presence or absence of expression of the gene to be analyzed, the expression level, etc. can be analyzed by determining the sequence of the amplification product. In another embodiment, a labeled probe having a base sequence complementary to the gene-specific sequence is used to hybridize the probe to cDNA or the obtained amplification product, and the analysis target is based on the label. Can be detected (eg, optically detected). Those skilled in the art can appropriately design a probe used for such detection. The label used can also be any label known in the art, such as fluorescent labels (Cy3, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), etc.), chemiluminescent labels (luciferin). Etc.), enzyme labels (peroxidase, β-galactosidase, alkaline phosphatase, etc.), and radioactive labels (tritium, iodine 125, etc.). In yet another embodiment, the nucleic acid amplification reaction is performed using a probe having a base sequence complementary to the gene-specific sequence, and the presence or absence of amplification is detected based on chemiluminescence or fluorescence, thereby analyzing the nucleic acid. The expression of the gene of interest can be analyzed.
　本発明においては、上述のとおり得られた核酸における遺伝子解析結果と、サンプル(細胞、組織など)の二次元位置情報とを対応させることにより、細胞又は組織における特定の位置と遺伝子発現との相関データを得ることも可能である。そのようなサンプルの二次元位置情報は、例えば、細胞サンプル又は組織切片サンプルの顕微鏡像、他の標識法により得られる蛍光像又は化学発光像などである
　本発明の核酸反応デバイス及び本発明の遺伝子解析方法について、図を参照しながら、以下に説明する。 In the present invention, the correlation between a specific position in a cell or tissue and gene expression is obtained by associating the result of gene analysis in the nucleic acid obtained as described above with the two-dimensional position information of the sample (cell, tissue, etc.). It is also possible to obtain data. The two-dimensional position information of such a sample is, for example, a microscopic image of a cell sample or a tissue section sample, a fluorescent image or a chemiluminescent image obtained by other labeling methods, etc. The nucleic acid reaction device of the present invention and the gene of the present invention The analysis method will be described below with reference to the drawings.
　図１に基板上に形成される核酸反応デバイスの一例の模式図を示す。 FIG. 1 shows a schematic diagram of an example of a nucleic acid reaction device formed on a substrate.
　図１(a)に核酸反応デバイスの上面図を示し、A-A’での断面図を図１(b)に示す。基板(101)上に複数の細胞保持領域(103)をアレイ状に形成し、その直下に多孔質材料からなる核酸反応部(104)を細胞捕捉領域に1対1で対応させて形成する。細胞(102)は細胞保持領域に保持され、細胞を保持したまま細胞破砕溶液で破砕することによって細胞中の核酸を個々の細胞ごとに核酸反応部に捕捉する。 Fig. 1 (a) shows a top view of the nucleic acid reaction device, and Fig. 1 (b) shows a cross-sectional view at A-A '. A plurality of cell holding regions (103) are formed in an array on the substrate (101), and a nucleic acid reaction part (104) made of a porous material is formed directly below the cell capturing region in a one-to-one correspondence with the cell capturing region. The cell (102) is held in the cell holding region, and the nucleic acid in the cell is captured in the nucleic acid reaction part for each individual cell by crushing with the cell crushing solution while holding the cell.
　特に、細胞保持領域を直径5μm～10μmとし、1つの細胞の大きさと同程度とし、かつ、核酸反応部からみて細胞領域の反対の側から、細胞を含む懸濁液を吸引することによって、1つの細胞保持領域に1つの細胞を単離して捕捉することが可能である。 In particular, the cell holding region has a diameter of 5 μm to 10 μm, the same size as that of one cell, and the cell-containing suspension is aspirated from the opposite side of the cell region as viewed from the nucleic acid reaction part. One cell can be isolated and captured in one cell retention region.
　もちろん、複数の細胞や組織切片を基板の細胞保持領域に配置してもよい。なお、図１では複数の細胞保持領域と核酸反応部は平面内で分離して配置しているが、複数の細胞保持領域及び核酸反応部を隣接して配置してもよい。このような構成は組織切片の解析に特に有効である。 Of course, a plurality of cells and tissue sections may be arranged in the cell holding region of the substrate. In FIG. 1, the plurality of cell holding regions and the nucleic acid reaction part are arranged separately in a plane, but the plurality of cell holding regions and the nucleic acid reaction part may be arranged adjacent to each other. Such a configuration is particularly effective for analysis of tissue sections.
　核酸反応部の表面の拡大図を図２(a)に示す。核酸反応部には第1のプローブ(201)と第2のプローブ(211)が固定されている。第1のプローブはデバイス中の多孔質材料で構成される核酸反応部の内壁(202)に化学的に強固に固定されている。第1のプローブは配列解析対象となる一本鎖核酸を捕捉するための捕捉配列(203)を含み、必要に応じて核酸反応デバイス中での核酸反応部の位置を特定するためのタグ配列(204)及び、核酸増幅用の共通配列 (205)を含む。 An enlarged view of the surface of the nucleic acid reaction part is shown in FIG. A first probe (201) and a second probe (211) are fixed to the nucleic acid reaction part. The first probe is chemically and firmly fixed to the inner wall (202) of the nucleic acid reaction part composed of a porous material in the device. The first probe includes a capture sequence (203) for capturing a single-stranded nucleic acid to be sequence-analyzed, and a tag sequence (for specifying the position of the nucleic acid reaction part in the nucleic acid reaction device, if necessary) 204) and a common sequence (205) for nucleic acid amplification.
　一方、第2のプローブ(211)も多孔質材料で構成される内壁に強固に固定されているが、タグ配列(204)を含む少なくとも一部の配列が切断されて遊離するように設計されている。この第2のプローブ(211)は、タグ配列(204)のほかに、第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列(208)、切断部(207)、及び必要に応じて核酸増幅用の共通配列 (205)を含む。この第2のプローブ(211)が酵素などの化学処理によって、所定の位置で切断され遊離することによって、第1のプローブで捕捉した核酸に対し、前記相補配列(208)で複数位置にハイブリダイゼーションし、1st cDNA合成のプライマーとして働くことで核酸捕捉部ごとに固有の塩基配列からなるタグ配列を導入した核酸を調製することが可能となる。その結果、捕捉した一本鎖核酸を、位置情報を含む形で解析することが可能となる。この反応ステップは以下に示すとおりである。 On the other hand, the second probe (211) is also firmly fixed to the inner wall made of a porous material, but at least a part of the sequence including the tag sequence (204) is designed to be cut and released. Yes. In addition to the tag sequence (204), the second probe (211) includes a base sequence (208) complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe, 207), and, if necessary, a common sequence for nucleic acid amplification (205). This second probe (211) is cleaved and released at a predetermined position by chemical treatment such as enzyme, so that the nucleic acid captured by the first probe is hybridized at multiple positions with the complementary sequence (208). and, it is possible to prepare a nucleic acid has been introduced a tag sequence consisting of specific nucleotide sequences for each nucleic acid capture portion by acting as a primer for 1 st cDNA synthesis. As a result, the captured single-stranded nucleic acid can be analyzed in a form including position information. This reaction step is as follows.
　まず、核酸抽出を行う。解析対象となる細胞又は組織を核酸反応デバイス上に展開し、核酸反応部からみて細胞保持領域の反対側から溶液を吸引することによって、細胞保持領域に特定の細胞又は特定の組織切片中の領域を吸引し、固定・保持する。 First, nucleic acid extraction is performed. The cell or tissue to be analyzed is spread on the nucleic acid reaction device, and the solution is aspirated from the opposite side of the cell holding region as seen from the nucleic acid reaction part. Aspirate, fix and hold.
　次に、解析対象の核酸の捕捉を行う。細胞保持領域(103)に細胞を捕捉した後、核酸反応部からみて細胞保持領域の反対側から溶液を吸引することによって、細胞吸引時と同様にして細胞を破砕するための溶液を供給する。これによって、細胞中の核酸は核酸反応部(104)を通過する。核酸反応部(104)の多孔質内壁表面に、前記第1のプローブが固定されているので、第1のプローブの中の捕捉配列(203)は、細胞から抽出された核酸をハイブリダイゼーションによって捕捉することができる。細胞破砕溶液とともに、細胞から抽出された核酸が核酸反応部を通過するため、一般的には核酸抽出と核酸捕捉は同時に行うこととなる。 Next, the nucleic acid to be analyzed is captured. After capturing the cells in the cell holding region (103), the solution is sucked from the opposite side of the cell holding region as viewed from the nucleic acid reaction part, thereby supplying a solution for crushing the cells in the same manner as in the cell aspiration. Thereby, the nucleic acid in the cell passes through the nucleic acid reaction part (104). Since the first probe is immobilized on the porous inner wall surface of the nucleic acid reaction part (104), the capture sequence (203) in the first probe captures the nucleic acid extracted from the cell by hybridization. can do. Since the nucleic acid extracted from the cells passes through the nucleic acid reaction part together with the cell disruption solution, generally, nucleic acid extraction and nucleic acid capture are performed simultaneously.
　特に遺伝子発現解析を行うためにmRNA(209)を捕捉するには、捕捉配列はTの連続配列(ポリT配列)とすれば良いことは前述の通りである。以下の反応過程はmRNAを解析する場合を例として記す。図２(ｂ)中では、捕捉されたmRNAは(209)で示されており、(210)は第1のプローブの3’末端の捕捉配列とハイブリダイズするmRNAのAの連続配列(ポリA配列)である。 In particular, in order to capture mRNA (209) for gene expression analysis, the capture sequence may be a continuous sequence of T (poly T sequence) as described above. The following reaction process is described as an example of analyzing mRNA. In FIG. 2 (b), the captured mRNA is indicated by (209), and (210) is a continuous sequence of mRNA A (polyA) that hybridizes with the capture sequence at the 3 ′ end of the first probe. Array).
　次に、捕捉した核酸の相補鎖を合成する。捕捉された核酸がmRNAの場合には1st cDNAの合成に相当する。 Next, a complementary strand of the captured nucleic acid is synthesized. When the captured nucleic acid is mRNA, this corresponds to the synthesis of 1st cDNA.
　まず、図２(c)に示すように、第2のプローブを遊離させ、捕捉したmRNAに第2のプローブをハイブリダイズさせるために、制限酵素を含む溶液を、細胞破砕溶液と同様の方法で核酸反応部を通過させる。この制限酵素の働きによって、制限酵素の認識配列(206)に対応した切断部(207)で核酸の切断が起きる。切断によって第2のプローブが遊離し、遊離した第2のプローブは、近傍の(同一の核酸反応部内の)mRNAにハイブリダイズする。ハイブリダイズするための、第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列(208)としては6mer程度の短いランダム配列を用いることが好ましい。これによって、mRNAの任意の配列領域に第2のプローブがハイブリダイズできる。第2のプローブは、タグ配列(204)以外に、必要に応じて共通配列(205)、制限酵素の認識配列(206)、並びにステムセンス配列及びステムアンチセンス配列を含む。 ステムセンス配列及びステムアンチセンス配列はランダム配列以外の配列が第1のプローブで捕捉された核酸にハイブリダイズすることを避けるために図２(ｃ)に示すように遊離した第2のプローブがステム構造を形成し安定化するための配列である。 First, as shown in FIG. 2 (c), in order to release the second probe and to hybridize the second probe to the captured mRNA, a solution containing a restriction enzyme was prepared in the same manner as the cell disruption solution. Pass through the nucleic acid reaction part. By the action of this restriction enzyme, the nucleic acid is cleaved at the cleavage site (207) corresponding to the restriction enzyme recognition sequence (206). The second probe is released by the cleavage, and the released second probe hybridizes to nearby mRNA (within the same nucleic acid reaction site). It is preferable to use a short random sequence of about 6 mer as the base sequence (208) complementary to a part of the base sequence of the single-stranded nucleic acid captured by the first probe for hybridization. Thereby, the second probe can hybridize to an arbitrary sequence region of mRNA. In addition to the tag sequence (204), the second probe includes a common sequence (205), a restriction enzyme recognition sequence (206), and a stem sense sequence and a stem antisense sequence as necessary. The stem sense sequence and the stem antisense sequence are formed by the second probe released as shown in FIG. 2 (c) in order to avoid hybridization of the sequence other than the random sequence to the nucleic acid captured by the first probe. An arrangement for forming and stabilizing the structure.
　次に、逆転写酵素を前工程と同様の方法によって核酸反応部に導入し、第1のプローブによって捕捉されたmRNAを鋳型として、1st cDNAを合成する。1st strandの合成方向を矢印で示した。この合成反応のプライマーとして機能するのは第1のプローブ(201)及び第2のプローブ(211)である。第2のプローブ(211)はmRNAの複数の位置にハイブリダイズするランダム配列を含み得、この場合、複数の位置から1st cDNAが合成される。このとき、合成されたすべての1st cDNAの5’末端にタグ配列(204)が導入される。これによって、捕捉されたmRNAのすべての領域において、タグ配列付きの1st cDNA(212((第1のプローブがプライマーとなった場合)又は(213)((第2のプローブがプライマーとなった場合))が合成されることになる。また、合成された1st cDNA(212及び213)の平均の長さはハイブリダイズした第2のプローブの数によって決まるため、配列解析を実行するシーケンサーの解析配列長に対応した数量比(配列解析対象分子数に対する第2のプローブ数比)となるように第2のプローブを固定しておくことが好ましい。mRNA分子数に対して50倍程度の数の第2のプローブを固定し、平均200bpの1st cDNAを合成することが好ましいが、数量比は、配列解析のためのシーケンサーの読み取り塩基長に対応して変更することが可能である。 Then introduced into a nucleic acid reaction unit in a similar manner as the previous step reverse transcriptase, an mRNA captured by the first probe as template to synthesize 1 st cDNA. Synthesis direction of 1 st strand shown by the arrows. The first probe (201) and the second probe (211) function as primers for this synthesis reaction. The second probe (211) may include a random sequence that hybridizes to multiple positions of the mRNA, in which case 1st cDNA is synthesized from the multiple positions. At this time, the tag sequence (204) is introduced into the 5 ′ end of all synthesized 1st cDNAs. As a result, in all regions of the captured mRNA, the 1st cDNA with the tag sequence (212 ((when the first probe serves as a primer) or (213) ((when the second probe serves as a primer) In addition, since the average length of the synthesized first cDNAs (212 and 213) is determined by the number of hybridized second probes, the sequence of the sequencer performing the sequence analysis is analyzed. It is preferable to fix the second probe so that the quantity ratio corresponding to the length (the ratio of the second probe number to the number of molecules to be sequenced) is approximately 50 times the number of mRNA molecules. It is preferable to synthesize two probes and synthesize 1st cDNA having an average of 200 bp, but the quantity ratio can be changed according to the read base length of the sequencer for sequence analysis.
　また、第2のプローブの遊離と1st cDNAの合成を同時に行うことによって、第2のプローブが効率的にmRNAにハイブリダイズしてプライマーとして機能するので、これらの工程は同時に行うことが好ましい。 In addition, since the second probe efficiently hybridizes to mRNA and functions as a primer by simultaneously releasing the second probe and synthesizing the 1st cDNA, these steps are preferably performed simultaneously.
　次に、図２(d)に示すようにmRNAを分解処理する。mRNA処理のためのRNase酵素を、前工程と同様の方法によって核酸反応部に導入する。 Next, mRNA is decomposed as shown in FIG. 2 (d). An RNase enzyme for mRNA treatment is introduced into the nucleic acid reaction part by the same method as in the previous step.
　さらに同時に、核酸増幅のためのリバースプライマーをハイブリダイズさせるための配列の付加反応を行う。図２(d)で示すように、ターミナルトランスフェラーゼ酵素を含む溶液を前工程と同様の方法によって核酸反応部に導入し、図２(c)で合成された1st cDNAの3’末端に連続A配列(214)を付加する。この連続配列はAではなく他の塩基でもよい。 At the same time, a sequence addition reaction for hybridizing a reverse primer for nucleic acid amplification is performed. As shown in Fig. 2 (d), a solution containing a terminal transferase enzyme was introduced into the nucleic acid reaction part by the same method as in the previous step, and a continuous A sequence was placed at the 3 'end of the 1st cDNA synthesized in Fig. 2 (c). (214) is added. This continuous sequence may be other bases instead of A.
　次に、図２(e)に示すように2ndstrand DNAの合成のために、DNAポリメラーゼ、並びに連続A配列に相補的な塩基配列である連続T配列(215)及び共通配列(216)からなるフォワードプライマーを核酸反応部に導入する。2ndstrand 合成の方向を矢印で示した。 Then, for the synthesis of 2 nd strand DNA as shown in FIG. 2 (e), the DNA polymerase, as well as continuous T sequence is a nucleotide sequence complementary to the contiguous A sequence (215) and consensus sequence (216) Into the nucleic acid reaction part. The direction of the 2 nd strand synthesis indicated by arrows.
　最後に図２(f)のリバース方向の共通プライマー(217(205の相補鎖))と図２(ｇ)のフォワード方向の共通プライマー(216)を用いたPCR増幅を行う。PCR増幅に必要な共通(Forward)プライマー(216)、リバース方向の共通プライマー(217)、PCR酵素、及び基質を前工程と同様に核酸反応部に導入することによって反応を完了させる。図２(ｇ)に示す得られた2本鎖のPCR産物(218及び219)は配列解析が可能な配列であり(正確には配列解析の前処理(エマルジョンPCRなど)が可能な配列であり)シーケンシングライブラリーと呼ばれる。この得られたほぼすべてのPCR産物には配列解析したいmRNA由来の配列(212)及び(213)にタグ配列(204)が含まれている。この2つの組み合わせによって、解析した配列がどの核酸反応部、及び細胞に由来するかを識別することが可能となる。 Finally, PCR amplification is performed using the reverse-direction common primer (217 (complementary strand of 205)) in FIG. 2 (f) and the forward-direction common primer (216) in FIG. 2 (g). The reaction is completed by introducing the common primer (216) necessary for PCR amplification, the common primer (217) in the reverse direction, the PCR enzyme, and the substrate into the nucleic acid reaction part as in the previous step. The obtained double-stranded PCR products (218 and 219) shown in FIG. 2 (g) are sequences that can be sequence-analyzed (more precisely, sequences that can be pre-processed for sequence analysis (such as emulsion PCR)). ) Called sequencing library. Almost all of the obtained PCR products contain the tag sequence (204) in the sequences (212) and (213) derived from the mRNA to be sequenced. The combination of the two makes it possible to identify which nucleic acid reaction part and cell originate from the analyzed sequence.
　以下、本発明の実施形態の具体例について説明する。ただし、これらの実施例は本発明を実現するための一例に過ぎず、本発明を限定するものではない。 Hereinafter, specific examples of the embodiment of the present invention will be described. However, these examples are merely examples for realizing the present invention, and do not limit the present invention.
［実施例１］
　本実施例は、磁性ビーズを用いた基板の構造とその作製方法を示す。本基板の模式図を図３に示す。核酸反応部(104)、及び細胞保持領域(103)を形成するPDMS(ポリジメチルシロキサン)製のチップ(303)は、射出成型を用いて作製した。ここで、PDMSチップの代わりにナノインプリント技術や射出成型によって作製した樹脂(ポリカービネート、サイクリックポリオレフィン、及びポリプロピレン)製の基板や、市販のナイロンメッシュやトラックエッチメンブレンを用いてもよい。細孔アレイシートの接着は熱接着を用いることもできる。 [Example 1]
This example shows a structure of a substrate using magnetic beads and a manufacturing method thereof. A schematic diagram of the substrate is shown in FIG. The chip (303) made of PDMS (polydimethylsiloxane) that forms the nucleic acid reaction part (104) and the cell holding region (103) was produced by injection molding. Here, instead of the PDMS chip, a substrate made of a resin (polycarbonate, cyclic polyolefin, and polypropylene) manufactured by nanoimprint technology or injection molding, a commercially available nylon mesh, or a track etch membrane may be used. Thermal adhesion can also be used for adhesion of the pore array sheet.
　細胞保持領域(103)は直径10μmの貫通孔であり、125μm間隔でアレイ状に配置した。チップ(303)の大きさは一辺が1.125mmの正方形であり、その中に細胞保持領域(103)を102個配置した。細胞保持領域(103)の下部では貫通孔の直径が75μmに広くなっており、この部分に磁性ビーズをパッキングし、核酸反応部(104)とした。この貫通孔がアレイ状に配置されたチップ(303)の下に細孔アレイシート(301)を配置した。この細孔アレイシート(301)の細孔の直径は、200nmとした。磁性ビーズの直径は1μmであり、細孔の直径は磁性ビーズの直径よりも小さいため、磁性ビーズが細孔から流出することはない。 The cell holding region (103) is a through-hole having a diameter of 10 μm and arranged in an array at intervals of 125 μm. The size of the chip (303) side is square 1.125 mm, a cell holding area therein the (103) arranged two 10. In the lower part of the cell holding region (103), the diameter of the through hole is as wide as 75 μm, and magnetic beads are packed in this part to form the nucleic acid reaction part (104). A pore array sheet (301) was placed under the chip (303) in which the through holes were arranged in an array. The pore diameter of this pore array sheet (301) was 200 nm. Since the diameter of the magnetic beads is 1 μm and the diameter of the pores is smaller than the diameter of the magnetic beads, the magnetic beads do not flow out of the pores.
　上記磁性ビーズのパッキングはインクジェットプリンタヘッドを用いて核酸反応部ごとに個別に実行する。チップ(303)を上下逆転させた状態で、核酸反応部ごとに異なるタグ配列を含む核酸が固定された磁性ビーズを、5×109個/mLの数密度で含む懸濁溶液を各核酸反応部(104)に2nLずつ充填した。インクジェットプリンタヘッドを用いてビーズ溶液を核酸反応部(104)に加えると、細孔アレイシート(301)中の細孔の内壁が親水表面であるため、毛細管現象によってビーズ溶液中の水分のみが吸収され、ビーズのみが核酸反応部(104)に残る。 The packing of the magnetic beads is performed individually for each nucleic acid reaction unit using an ink jet printer head. In a state of being vertically reversed chip (303), the magnetic beads nucleic acid is fixed comprising a different tag sequence for each nucleic acid reaction unit, 5 × 10 9 cells / mL each nucleic acid reaction suspension containing the number density of 2 nL of each portion (104) was filled. When the bead solution is added to the nucleic acid reaction part (104) using an inkjet printer head, the inner wall of the pores in the pore array sheet (301) is a hydrophilic surface, so that only moisture in the bead solution is absorbed by capillary action. Only the beads remain in the nucleic acid reaction part (104).
　細孔アレイシート(301)としては多孔質のガラスからなるモノリスシート、毛細管を束ねてスライスしたキャピラリープレート、ナイロンメンブレンあるいはゲル薄膜など種々のものを用いることができるが、本実施例ではアルミナを陽極酸化して得た細孔アレイシートを用いた。該細孔シートは、当業者であれば陽極酸化により容易に作製することができるが、孔径20nm～200nm、直径25mmのものが市販品として入手可能である。本実施例では孔径200nmのシート(アノディスク、GEヘルスケア)を使用し、該シート中の細孔が核酸反応部と下部領域をつなぐ流路(302)となる。 As the pore array sheet (301), various materials such as a monolith sheet made of porous glass, a capillary plate obtained by bundling capillaries and sliced, a nylon membrane or a gel thin film can be used. In this embodiment, alumina is used as an anode. A pore array sheet obtained by oxidation was used. Those skilled in the art can easily produce the pore sheet by anodic oxidation, but those having a pore diameter of 20 nm to 200 nm and a diameter of 25 mm are commercially available. In this example, a sheet (Anodisc, GE Healthcare) having a pore diameter of 200 nm is used, and the pores in the sheet serve as a flow path (302) that connects the nucleic acid reaction part and the lower region.
　ここで、磁性ビーズ上にDNAプローブを固定する方法は以下のとおりである。すなわち、タグ配列ごとに別々の反応チューブ中で、ストレプトアビジンを固定した磁性ビーズと5’末端をビオチン基で修飾したDNAプローブ溶液を1.5M NaClを含むTris バッファ(pH 7.4)中で、10分回転させながら混和することによって、ビオチン－ストレプトアビジン反応を介してDNAプローブを磁性ビーズに固定した。 Here, the method for immobilizing the DNA probe on the magnetic beads is as follows. That is, in a separate reaction tube for each tag sequence, a magnetic bead immobilized with streptavidin and a DNA probe solution modified with a biotin group at the 5 'end in Tris buffer (pH 7.4) containing 1.5M NaCl for 10 minutes. By mixing while rotating, the DNA probe was immobilized on the magnetic beads via the biotin-streptavidin reaction.
　ここで、作製したシートは繰り返し利用可能であり、シートを繰り返し利用することによって、高精度な発現分布測定を必要な種類の遺伝子について行うことが可能である。 Here, the produced sheet can be used repeatedly, and by using the sheet repeatedly, it is possible to perform highly accurate expression distribution measurement for a necessary type of gene.
［実施例２］
　本実施例は、核酸反応部に磁性ビーズを含む核酸反応デバイスを用いて、mRNAの任意領域の解析を行った例を示す。 [Example 2]
This example shows an example of analyzing an arbitrary region of mRNA using a nucleic acid reaction device including magnetic beads in the nucleic acid reaction part.
　図３に本発明の核酸反応デバイスの一例の断面図を示す。このデバイスは半導体プロセスを用いて作製した。図３(a)は本発明の核酸デバイスの一例の垂直断面図である。溶液は図中の上側から下に向かって流れる。(103)が細胞保持領域であり、(104)が核酸反応部である。細胞保持領域(103)及び核酸反応部(104)は樹脂製のチップ(303)上に貫通孔を形成することによって形成している。この貫通孔は細胞保持領域(103)で直径が小さく、核酸反応部(104)で直径が大きくなっている。これにより、核酸反応部の領域を大きくして、核酸反応部に含まれ得るビーズの量を増加させ、ビーズに固定された2種類のプローブ(第1のプローブ及び第2のプローブ)の、核酸反応部内の分子数を増加させ、抽出された核酸の捕捉効率を向上させることができる。核酸反応部(104)に含まれる磁性ビーズが流出しないようにするため、上記の通り、細孔アレイシート(301)において流路(302)を構成する孔の直径は、磁性ビーズの直径より小さい。これによって、核酸反応部中のビーズと細胞保持領域の対応関係を維持したまま、様々な酵素を含む溶液を核酸反応部に導入することができる。 FIG. 3 shows a cross-sectional view of an example of the nucleic acid reaction device of the present invention. This device was fabricated using a semiconductor process. FIG. 3 (a) is a vertical sectional view of an example of the nucleic acid device of the present invention. The solution flows from the upper side to the lower side in the figure. (103) is a cell holding region, and (104) is a nucleic acid reaction part. The cell holding region (103) and the nucleic acid reaction part (104) are formed by forming a through hole on a resin chip (303). The through hole has a small diameter in the cell holding region (103) and a large diameter in the nucleic acid reaction part (104). This increases the region of the nucleic acid reaction part, increases the amount of beads that can be contained in the nucleic acid reaction part, and the nucleic acid of two types of probes (first probe and second probe) fixed to the beads. The number of molecules in the reaction part can be increased, and the capture efficiency of the extracted nucleic acid can be improved. In order to prevent the magnetic beads contained in the nucleic acid reaction part (104) from flowing out, the diameter of the holes constituting the flow path (302) in the pore array sheet (301) is smaller than the diameter of the magnetic beads as described above. . As a result, it is possible to introduce solutions containing various enzymes into the nucleic acid reaction part while maintaining the correspondence between the beads in the nucleic acid reaction part and the cell holding region.
　図３(b)は図３(a)のA-A’断面に対応する断面図であり、図３(c)は図３(a)のB-B’断面に対応する断面図である。図３(b)に示すように溶液中に含まれる細胞102を核酸反応デバイス内の細胞保持領域(103)で捕捉することで単離しかつ保持する。細胞保持領域の直下、すなわちB-B’断面では核酸反応部ごとに異なるタグ配列(204)が核酸反応部に含まれるビーズの表面に固定されている。これによって、下記の反応過程によって、一細胞ごとに異なるタグ配列を導入することが可能となる。 3 (b) is a cross-sectional view corresponding to the A-A 'cross section of FIG. 3 (a), and FIG. 3 (c) is a cross-sectional view corresponding to the B-B' cross section of FIG. 3 (a). As shown in FIG. 3 (b), the cells 102 contained in the solution are isolated and held by capturing them in the cell holding region (103) in the nucleic acid reaction device. A tag sequence (204) that is different for each nucleic acid reaction part is fixed to the surface of the bead included in the nucleic acid reaction part immediately below the cell holding region, that is, in the B-B 'cross section. This makes it possible to introduce a different tag sequence for each cell by the following reaction process.
　本実施例では、複数の核酸反応デバイスを含み、細胞溶液や試薬の供給が可能なフローセルデバイスを用いた。このフローセルデバイスの模式図を図４に示す。 In this example, a flow cell device including a plurality of nucleic acid reaction devices and capable of supplying cell solutions and reagents was used. A schematic diagram of this flow cell device is shown in FIG.
　フローセルデバイス(401)は複数の反応室(402)を備えており、各反応室(402)は１つの核酸反応デバイス(105)を含む。この核酸反応デバイスは図３に示したものと同じであり、一つの細胞保持領域(103)を含む核酸反応部(104)を複数含む。フローセルデバイス上の共通流路(403)中に、細胞(102)を上部インレット(404)から上部アウトレット(405)に向かって流す。このとき同時に下部アウトレット(407)に負圧をかけて、吸引流路(406)を介して細胞を細胞保持領域に引き寄せ、細胞の直径よりも小さい開口部で細胞を捕捉する。また、細胞保持領域(103)の直下にビーズを含む核酸反応部(104)が配置されているため、吸引流路(406)を介してかけられる負圧によって、流路(403)中の溶液は、吸引流路(406)に流れる。ビーズの直径を1μmにすることによって、空隙はサブμmオーダーとなり、酵素などのマクロな分子も通過することが可能となる。 The flow cell device (401) includes a plurality of reaction chambers (402), and each reaction chamber (402) includes one nucleic acid reaction device (105). This nucleic acid reaction device is the same as that shown in FIG. 3, and includes a plurality of nucleic acid reaction units (104) including one cell holding region (103). Cells (102) flow from the upper inlet (404) toward the upper outlet (405) in a common channel (403) on the flow cell device. At the same time, a negative pressure is applied to the lower outlet (407), the cells are drawn to the cell holding region via the suction channel (406), and the cells are captured through an opening smaller than the cell diameter. In addition, since the nucleic acid reaction part (104) containing beads is arranged immediately below the cell holding region (103), the solution in the channel (403) is caused by the negative pressure applied through the suction channel (406). Flows into the suction channel (406). By setting the diameter of the beads to 1 μm, the voids are on the order of sub μm, and macromolecules such as enzymes can also pass through.
　次に、核酸反応デバイスの(105)の動作について、反応処理順に説明する。 Next, the operation (105) of the nucleic acid reaction device will be described in the order of the reaction processing.
　1000個の細胞を、傷つけないように500μLの1×PBSで洗浄した後、4℃に冷却した1×PBSバッファ10μLに懸濁し、図４の上部インレット(404)から導入し、反応室(402)をこの溶液で満たすように、上部アウトレット(405)から吸引する。これにより、核酸反応デバイス(105)上は細胞を含むPBSバッファで満たされることとなる。次に、細胞保持領域(103)を通って吸引流路(406)に向かって溶液が流れるように下部アウトレット(407)に負圧1.0気圧を印加して溶液を吸引する。細胞102が溶液の流れに乗って移動し、細胞保持領域(103)まで到達すると、細胞捕保持領域(103)の開口直径が細胞102の直径よりも小さいため、細胞は細胞保持領域に捕捉される。捕捉された細胞は溶液流に対して栓の役割を果たすので、溶液の流れはまだ細胞を捕捉していない細胞保持領域(103)に向かう。そのため、残りの細胞は、まだ細胞が捕捉されていない細胞保持領域(103)へと移動し捕捉される。 1000 cells were washed with 500 μL of 1 × PBS so as not to be damaged, suspended in 10 μL of 1 × PBS buffer cooled to 4 ° C., introduced from the upper inlet (404) of FIG. ) From the top outlet (405) to fill with this solution. As a result, the nucleic acid reaction device (105) is filled with the PBS buffer containing the cells. Next, a negative pressure of 1.0 atm is applied to the lower outlet (407) so that the solution flows through the cell holding region (103) toward the suction channel (406), and the solution is sucked. When the cell 102 moves along the flow of the solution and reaches the cell holding region (103), the cell trapping region (103) has a smaller opening diameter than the cell 102, so the cell is trapped in the cell holding region. The Since the trapped cells act as plugs for the solution flow, the solution flow goes to the cell holding region (103) where the cells have not yet been trapped. Therefore, the remaining cells move to the cell holding region (103) where the cells are not yet captured and are captured.
　細胞が十分な数だけが捕捉されたら、反応室(402)中の捕捉されなかった過剰な細胞とPBSバッファを上部アウトレット(405)から排出する。次に、上部インレット(404)から上部アウトレット(405)に向かってLysis buffer(例えばTween 20などの表面活性剤)を流し、反応室(402)をバッファで満たしたあと、直ちに下部アウトレット(407)に負圧を印加して吸引する。以下すべての溶液は、同様の方法によって核酸反応デバイス中の核酸反応部(104)を通過させる。このとき、核酸反応部の下部の流路(302)は直径0.2μmの多孔質材料から構成された流路であり、圧力損失を抑えるために、Lysis bufferは反応室(402)から細胞保持領域(103)を通って吸引流路(406)に向かって溶液がゆっくりと5分程度流れ続けるようにしてもよい。 When only a sufficient number of cells have been captured, excess cells and PBS buffer in the reaction chamber (402) and PBS buffer are drained from the upper outlet (405). Next, a Lysis buffer (e.g., a surfactant such as Tween 20) is flowed from the upper inlet (404) to the upper outlet (405), and after filling the reaction chamber (402) with the buffer, immediately the lower outlet (407) Apply negative pressure to and suck. In the following, all the solutions are passed through the nucleic acid reaction part (104) in the nucleic acid reaction device by the same method. At this time, the channel (302) below the nucleic acid reaction part is a channel composed of a porous material having a diameter of 0.2 μm, and in order to suppress pressure loss, the Lysis buffer is transferred from the reaction chamber (402) to the cell holding region. The solution may continue to flow slowly through the (103) toward the suction channel (406) for about 5 minutes.
　また、細胞溶解液(Lysis buffer)によって細胞102は破砕され、ｍRNA(209)は細胞外に放出されるが、ｍRNA(209)は周辺に拡散することなく、溶液の流れに従って細胞保持領域(103)を通って核酸反応部(104)まで到達する。このとき、溶液の流れに加えて、この流れの方向に電界を印加し、細胞中の核酸(mRNA(209))を核酸反応部(104)まで電気泳動によって移動させてもよい。mRNA(209)は、核酸反応部(104)に到達すると、捕捉配列とのハイブリダイゼーションによって、核酸反応部(104)に含まれるビーズ上に固定された第1のプローブに捕捉される。 Further, the cell 102 is crushed by the cell lysate (Lysis buffer), and the mRNA (209) is released to the outside of the cell, but the mRNA (209) does not diffuse to the periphery, and the cell holding region (103 ) To reach the nucleic acid reaction part (104). At this time, in addition to the flow of the solution, an electric field may be applied in the direction of the flow, and the nucleic acid (mRNA (209)) in the cell may be moved to the nucleic acid reaction part (104) by electrophoresis. When the mRNA (209) reaches the nucleic acid reaction part (104), it is captured by the first probe immobilized on the beads contained in the nucleic acid reaction part (104) by hybridization with the capture sequence.
　このようにLysis bufferの導入によって、細胞の破砕とmRNA(209)の捕捉が同時に実行される。このときのビーズ表面上の状況を図２(b)に示す。 Thus, by introducing Lysis buffer, cell disruption and mRNA (209) capture are performed simultaneously. The situation on the bead surface at this time is shown in FIG.
　ここで、細胞捕捉された核酸反応デバイス(105)上の位置の情報、すなわちmRNAが核酸反応デバイス(105)のどの細胞保持領域に由来するかの情報を配列情報として保存するために、核酸反応部の位置ごとに異なる配列からなるタグ配列(204)が、第1のプローブ(201)と第2のプローブ(211)に導入されている。 Here, in order to store the position information on the nucleic acid reaction device (105) trapped by the cell, that is, the cell holding region of the nucleic acid reaction device (105) as the sequence information, the nucleic acid reaction is performed. A tag sequence (204) having a different sequence for each position is introduced into the first probe (201) and the second probe (211).
　さらに、第1のプローブ及び第2のプローブは、5’末端側にPCR増幅のための共通配列(205)を含む。(201)の第1のプローブは、具体的には、例えば、5’末端から、30塩基のPCR増幅用共通プライマー(205)、7塩基のタグ配列(204)、18塩基のポリT(dT)配列＋2塩基のVN配列(203)の順に配列している。ここでVはA又はC又はGの塩基をさす。 Furthermore, the first probe and the second probe contain a common sequence (205) for PCR amplification on the 5 ′ end side. Specifically, the first probe of (201) is, for example, from the 5 ′ end, a 30 base PCR amplification common primer (205), a 7 base tag sequence (204), an 18 base poly T (dT ) Sequence + 2 base VN sequence (203). Here, V represents the base of A, C, or G.
　次に、図２(ｃ)に示すように、切断による第2のプローブの遊離と1st cDNA合成を同時に行う。図中の矢印は1st cDNA合成方向を示し、(213)は合成された塩基配列を示す。図５に本実施例で使用した制限酵素の切断部(207)の詳細と制限酵素の認識配列(206)を示す。ここで制限酵素の認識配列(206)中のYはT又はCであり、RはA又はGを示し、NはA、C、G、又はTのいずれかの塩基を示す。また上に線を付したNは、Nと相補的な塩基を示す。ここで(501)はニックであり、この位置にニックを配置することによって6ベースのランダム配列の一本鎖部分(502)を露出することができる(なお、このニックの位置を変更することによって、mRNAにハイブリダイズするランダム配列の長さを5塩基から12塩基までの間で自由に設計することが可能である)。制限酵素の作用を受ける前の第2のプローブはビーズ(503)表面に固定されている。本実施例ではビーズ表面にストレプトアビジンを固定し、ＤＮＡの5’末端及び3’末端をビオチンを修飾して、ビオチン－ストレプトアビジン反応によりプローブの固定を行った。固定化の方法は当該技術者が利用できる他の方法を用いても良い。 Next, as shown in FIG. 2 (c), release of the second probe by cleavage and 1st cDNA synthesis are performed simultaneously. The arrow in the figure indicates the 1st1 cDNA synthesis direction, and (213) indicates the synthesized nucleotide sequence. FIG. 5 shows details of the restriction enzyme cleavage site (207) and restriction enzyme recognition sequence (206) used in this example. Here, Y in the recognition sequence (206) of the restriction enzyme is T or C, R represents A or G, and N represents any base of A, C, G, or T. N with a line on the top represents a base complementary to N. Here, (501) is a nick, and by placing a nick at this position, a single-stranded portion (502) of a 6-base random sequence can be exposed (by changing the position of this nick The length of the random sequence that hybridizes to mRNA can be freely designed between 5 and 12 bases). The second probe before being subjected to the action of the restriction enzyme is immobilized on the surface of the bead (503). In this example, streptavidin was immobilized on the bead surface, biotin was modified at the 5 'end and 3' end of the DNA, and the probe was immobilized by biotin-streptavidin reaction. As the immobilization method, other methods available to the technician may be used.
　第2のプローブの切断と1st cDNA合成を同時に行うために、0.1%Tween20を含む10mM Tris Buffer (pH=8.0) 53.5μL、10mM dNTP 4μL、5xRT Buffer (SuperScript III, Invitrogen社) 22.5μL、0.1M DTT 4μL、RNaseOUT(Invitrogen社) 4μL、BaeI(制限酵素, New England BioLabs社, 5U/uL) 5uL、及びSuperscript III(逆転写酵素, Invitrogen社, 200U/uL) 4μLを混和し、上部インレット(404)から前工程と同様に導入した。上記溶液で核酸反応部のビーズの空隙部分を満たした状態で、上記溶液を上部反応室(402)から吸引流路(406)にゆっくり(1uL/min)流しながら、25℃で3分反応させた後、15分かけて50℃に昇温して50分50℃に保ち、1st cDNA合成反応を行った。 To simultaneously cleave the second probe and 1st cDNA synthesis, 10 mM Tris buffer (pH = 8.0) containing 0.1% Tween20 (pH = 8.0), 53.5 μL, 10 mM dNTP, 4 μL, 5xRT buffer (SuperScript III, Invitrogen), 22.5 μL, 0.1 M Mix DTT 4μL, RNaseOUT (Invitrogen) 4μL, BaeI (restriction enzyme, New England BioLabs, 5U / uL) 5uL, and Superscript III (reverse transcriptase, Invitrogen, 200U / uL) 4μL. ) From the previous step. The above solution is allowed to react at 25 ° C. for 3 minutes while slowly filling (1 uL / min) the above solution from the upper reaction chamber (402) to the suction channel (406) while filling the void portion of the beads in the nucleic acid reaction section. After that, the temperature was raised to 50 ° C. over 15 minutes and kept at 50 ° C. for 50 minutes, and the 1st cDNA synthesis reaction was performed.
　反応終了後、フローセルデバイス(401)全体を85℃にて1.5分保ち、逆転写酵素を失活させた。次に、4℃に冷却後、RNase及び0.1%Tween20を含む10mM Tris Buffer (pH = 8.0) 0.2mLを上部インレット(404)から注入し上部アウトレット(405)から吸引して反応室(402)を溶液で満たした後、下部アウトレット(407)から排出し、反応室中のバッファーを上部アウトレットから取り除くという工程を5回繰り返すことによって、RNAを分解し、核酸反応部中の残存物及び分解物を除去・洗浄した。さらに、アルカリ変性剤を含む液及び洗浄液で同様に５回洗浄した。ここまでのプロセスで図２(d)に示すように細胞保持領域ごとに固有の塩基配列からなるタグ配列が挿入されたcDNAライブラリアレイが構築された。 After completion of the reaction, the entire flow cell device (401) was kept at 85 ° C. for 1.5 minutes to inactivate the reverse transcriptase. Next, after cooling to 4 ° C, 0.2 mL of 10 mM Tris Buffer (pH = 8.0) 含 む containing RNase and 0.1% Tween 20 is injected from the upper inlet (404) and sucked from the upper outlet (405) to evacuate the reaction chamber (402). After filling with the solution, the RNA is decomposed by repeating the process of discharging from the lower outlet (407) and removing the buffer in the reaction chamber from the upper outlet five times, and the remaining and decomposed products in the nucleic acid reaction part are removed. Removed and washed. Further, it was washed five times in the same manner with a solution containing an alkali modifier and a washing solution. In the process so far, as shown in FIG. 2 (d), a cDNA library array was constructed in which a tag sequence consisting of a unique base sequence was inserted for each cell holding region.
　次に、ポリＡ付加反応を行った。図２(ｄ)に付加されたポリＡ配列(214)を示す。RNase free滅菌水22.8uL、10×PCR BufferII 3uL、25mMMgCl2 1.8uL、100mMdATP 0.9uL、及びターミナルトランスフェラーゼ(2U/μL)1.5uLを混和し、前記と同様に上部インレット(404)から核酸反応部(104)に導入する。37℃に昇温し、15分保持することで反応を進行させた。 Next, poly A addition reaction was performed. FIG. 2 (d) shows the added poly A sequence (214). RNase-free sterilized water 22.8 uL, 10 x PCR Buffer II 3 uL, 25 mM MgCl 2 1.8 uL, 100 mM ATP 0.9 uL, and terminal transferase (2 U / μL) 1.5 uL were mixed, and the nucleic acid reaction part (404) from the upper inlet (404) was mixed as described above. 104). The reaction was allowed to proceed by raising the temperature to 37 ° C and holding for 15 minutes.
　次に、図２(e)に示すように、PCR増幅用共通Forward配列(216)とポリT配列(dT30+VN)(215)を含むプライマーを用いて、1st cDNAの相補鎖である2nd DNA鎖を合成する。このために、RNase freeの滅菌水69μL、10 x Ex Taq Buffer (TaKaRa Bio社) 10μL、2.5mM dNTP Mix 100μL、10μMの前記プライマー10μL、及び10μMのEx Taq Hot start version (TaKaRa Bio社)1μLを混和し、この混合溶液を前工程と同様に上部インレット(404)から、核酸反応部(104)に導入した。その後、95℃3分間で保持して核酸の2次構造を解いたあと、44℃2分間で1st cDNA鎖を鋳型としてプライマーのアニーリング部をハイブリダイズさせた。さらに72℃に6分間温度を上げることによって相補鎖伸長反応を行い2nd DNA鎖を合成させた。図２(e)中の矢印は2nd DNA鎖の合成方向を示す。続いて、図２(f)と図２(g)に示すように共通プライマーによるPCR増幅を行った。滅菌水49.μL、10 x High Fidelity PCR Buffer (Invitrogen) 10μL、2.5 mM dNTP mix 10μL、50mM MgSO4 4μL、10μMのPCR増幅用共通配列Fowardプライマー (216) 10μL、10μMのPCR増幅用共通配列Reverseプライマー(217) 10μL、及びPlatinum Taq Polymerase High Fidelity (Invitrogen社) 1.5μLを混和し試薬を調製した後、フローデバイス中の吸引流路(406)を満たしている溶液をアウトレット306及び307から排出する操作を行った後、直ちに上記の調製した試薬を前工程と同様に上部インレット(404)から導入し、(407)から吸引することで核酸反応部に上記試薬を導入した。続いて、フローセルデバイス全体を94℃で30秒間保ち、94℃30秒間→55℃30秒間→68℃30秒間の3段階工程を40サイクル繰り返し、最後に68℃3分間保った後、4℃に冷却してPCR増幅工程を行った。この反応は共通反応であり、すべての核酸反応デバイスについて共通の試薬条件によってPCR増幅が行われ、核酸反応デバイス間での増幅効率の均一化を図った。続いて、溶液中に蓄積されたPCR増幅産物溶液を回収した。この溶液中に含まれるフリーのPCR増幅用共通配列プライマー(Forward/Reverse)や酵素などの残留試薬を除去する目的で、PCR Purification Kit(QIAGEN社)を用いて産物を精製した。 Next, as shown in FIG. 2 (e), using a primer containing a common forward sequence for PCR amplification (216) and a poly-T sequence (dT30 + VN) (215), 2nd DNA which is the complementary strand of 1st cDNA Synthesize a chain. For this purpose, 69 μL of RNase-free sterile water, 10 μL of 10 × Ex Taq Buffer (TaKaRa Bio), 2.5 μm dNTP Mix 100 μL, 10 μM of the primer 10 μL, and 10 μM Ex Taq Hot start version (TaKaRa Bio) 1 μL The mixed solution was introduced into the nucleic acid reaction part (104) from the upper inlet (404) as in the previous step. Thereafter, the nucleic acid secondary structure was solved by holding at 95 ° C. for 3 minutes, and then the primer annealing portion was hybridized at 44 ° C. for 2 minutes using the 1st cDNA strand as a template. Further, by raising the temperature to 72 ° C. for 6 minutes, a complementary strand extension reaction was performed to synthesize a 2nd DNA strand. The arrow in FIG. 2 (e) indicates the direction of synthesis of the 2nd DNA strand. Subsequently, PCR amplification was performed using common primers as shown in FIG. 2 (f) and FIG. 2 (g). Sterile water 49 μL, 10 x High Fidelity PCR Buffer (Invitrogen) 10 μL, 2.5 mM dNTP mix 10 μL, 50 mM MgSO 4 4 μL, 10 μM common primer for PCR amplification Forward primer (216) 10 μL, 10 μM common sequence for PCR amplification Reverse Mix the primer (217) 10 μL and Platinum Taq Polymerase High Fidelity (Invitrogen) 1.5 μL to prepare the reagent, and then discharge the solution filling the suction channel (406) in the flow device from the outlets 306 and 307. Immediately after the operation, the reagent prepared above was introduced from the upper inlet (404) as in the previous step, and the reagent was introduced into the nucleic acid reaction part by aspiration from (407). Subsequently, the entire flow cell device is kept at 94 ° C. for 30 seconds, and the three-stage process of 94 ° C. for 30 seconds → 55 ° C. for 30 seconds → 68 ° C. for 30 seconds is repeated for 40 cycles. The PCR amplification process was performed after cooling. This reaction is a common reaction, and PCR amplification was performed under the same reagent conditions for all nucleic acid reaction devices, so that the amplification efficiency was uniform among the nucleic acid reaction devices. Subsequently, the PCR amplification product solution accumulated in the solution was recovered. For the purpose of removing residual reagents such as free PCR amplification common sequence primers (Forward / Reverse) and enzymes contained in this solution, the product was purified using PCR Purification Kit (QIAGEN).
　得られたPCR産物(218)及び(219)は配列解析が可能な配列であり(正確には配列解析の前処理(エマルジョンPCRなど)が可能な配列であり)シーケンシングライブラリーと呼ばれる。 The obtained PCR products (218) and (219) are sequences that can be sequence-analyzed (more precisely, sequences that can be pre-processed for sequence analysis (such as emulsion PCR)) and are called sequencing libraries.
　このシーケンシングライブラリーを配列解析することによって、タグ配列ごとに遺伝子発現量が得られる。すなわち、フローセルデバイス中に同時に導入されたタグ配列の種類数以下の細胞数についての同時解析が可能となる。 The gene expression level can be obtained for each tag sequence by sequence analysis of this sequencing library. That is, simultaneous analysis can be performed on the number of cells equal to or less than the number of types of tag sequences introduced simultaneously into the flow cell device.
　ここまで、ビーズ表面で合成したcDNA(1st cDNA)をPCRで増幅する例を開示した。もちろんローリングサークル増幅(RCA)やNASBAやLAMP法など他の核酸増幅法を用いても良い。 So far, an example of amplifying cDNA synthesized on the bead surface (1st cDNA) by PCR has been disclosed. Of course, other nucleic acid amplification methods such as rolling circle amplification (RCA), NASBA, and LAMP may be used.
　ここまで本発明のデバイスを用いるmRNAの解析を行うための方法を示してきたが、DNAの配列解析を行うためには、蛋白質分解酵素、適切な長さに断片化するための制限酵素、及びポリAを付加するターミナルトランスフェラーゼを混合した溶液を細胞破砕溶液に加えることによって、mRNAの解析の場合と同様の方法が適用できる。なお、適切な長さの断片が得られる制限酵素の混合溶液を作製する方法は、当業者に一般的に知られている方法を適用すればよい。 So far, a method for analyzing mRNA using the device of the present invention has been shown. In order to perform DNA sequence analysis, a proteolytic enzyme, a restriction enzyme for fragmenting to an appropriate length, and By adding a mixed solution of terminal transferase for adding poly A to the cell disruption solution, the same method as in the analysis of mRNA can be applied. A method generally known to those skilled in the art may be applied as a method for preparing a restriction enzyme mixed solution from which a fragment of an appropriate length can be obtained.
［実施例３］
　本実施例は、ビーズを用いず、DNAプローブを固定した細孔アレイシートを用いた核酸反応デバイスを示す例である。また、本実施例では、第2のプローブの遊離のために光反応を用いた。 [Example 3]
This example is an example showing a nucleic acid reaction device using a pore array sheet to which a DNA probe is fixed without using beads. In this example, a photoreaction was used for releasing the second probe.
　核酸反応デバイスの構造を図６に示した。このデバイスは半導体プロセスを用いて作製した。1つの細胞保持領域(103)を含む核酸反応部(104)を複数含むことは実施例２のデバイスと同じである。ここで図６(ｃ)中の核酸反応部(104)の異なる模様は、核酸反応部ごとに異なるタグ配列が固定されていることを示すものである。図６(a)に基板に垂直な断面図を示し、図６(b)に図６(a)のAA’断面図を図６(c)に図６(a)のBB’断面図を示した。核酸反応部(104)を構成する貫通孔は厚さ5μmのSiO2膜中に直径0.3μｍの貫通孔を0.5μｍ間隔で形成した。その後、シリコン基板をウェットエッチングによって除去した。第1のDNAプローブは、シランカップリング剤を用いて内壁に固定した。細胞保持領域(103)の直径は10μmであり、この捕捉部は、厚さ10μmのポリイミド樹脂膜(601)にリソグラフィとプラズマエッチングによってポリイミドを周期的に取り除いて開口部を形成することによって作製した。 The structure of the nucleic acid reaction device is shown in FIG. This device was fabricated using a semiconductor process. It is the same as the device of Example 2 that a plurality of nucleic acid reaction parts (104) including one cell holding region (103) are included. Here, the different patterns of the nucleic acid reaction part (104) in FIG. 6 (c) indicate that different tag sequences are fixed in each nucleic acid reaction part. 6 (a) shows a cross-sectional view perpendicular to the substrate, FIG. 6 (b) shows a cross-sectional view along AA ′ in FIG. 6 (a), and FIG. 6 (c) shows a cross-sectional view along BB ′ in FIG. 6 (a). It was. The through holes constituting the nucleic acid reaction part (104) were formed at intervals of 0.5 μm in a diameter of 0.3 μm in a SiO 2 film having a thickness of 5 μm. Thereafter, the silicon substrate was removed by wet etching. The first DNA probe was immobilized on the inner wall using a silane coupling agent. The diameter of the cell holding region (103) is 10 μm, and this trapping part was prepared by periodically removing polyimide from the polyimide resin film (601) having a thickness of 10 μm by lithography and plasma etching to form an opening. .
　このとき(601)の凹凸の構造をなくして、核酸反応部の上面を細胞保持領域とすることができる。 At this time, the upper surface of the nucleic acid reaction part can be used as a cell holding region without the (601) uneven structure.
　この場合図１０に示すように一つの細胞保持領域に一つの細胞を単離する機能は失われるが、例えば組織切片のように細胞が互いに接着しており、おおよそのどの位置の細胞から抽出された核酸がどのような配列となっているかを知りたい場合には有効なデバイス構成である。 In this case, as shown in FIG. 10, the function of isolating one cell in one cell holding region is lost, but the cells are adhered to each other as in a tissue section, for example, and are extracted from cells at any approximate position. It is an effective device configuration when it is desired to know the sequence of the nucleic acid.
　次に、本実施例における第1のDNAプローブの核酸反応部への固定方法について説明する。核酸反応部(104)をシラン処理するために、0.3mg/mlのシランカップリング剤GTMSi(GTMSi：3-Glycidoxypropyltrimethoxysilane信越化学)及び、酸触媒である0.02%酢酸を含む水溶液を反応デバイス中に導入し、２時間反応させた。反応デバイス内容積の100倍量のエタノールを反応デバイス内に導入することによってデバイス内部を洗浄後、溶液をすべて排出し、110℃で2時間保持した。次に1μMストレプトアビジン溶液を反応デバイスに導入し、6時間室温にて反応させて、ストレプトアビジンを核酸反応領域(104)に固定する。次に未反応グリシド基をブロックし、過剰なDNAプローブを除去するために、デバイス内容積の10倍量の10ｍMのグリシン、0.01％SDS、及び0.15MのNaClを含むホウ酸バッファ(pH 8.5)を5分間かけて導入、排出し、内容積の10倍量の60℃に加熱された0.01%のSDS及び0.3M NaClを含む30mMクエン酸ナトリウムバッファ(2xSSC, pH 7.0)をデバイス内を通過させる。最後に内容積の100倍量の0.1%Tween 20を含む10mTrisを導入、排出し洗浄を完了する。さらに、内容積と等量の10μMの5’末端をビオチン修飾した２種類のDNAプローブ(第1のプローブ及び第2のプローブ)、１MのNaCl、及び0.1%のTween20を含む10mMのTrisHCl溶液をインクジェット装置を用いて個別の核酸反応部ごとに注入する。１箇所の核酸反応部に注入するプローブ溶液は40ｐLずつ250回に分けて領域を塗りつぶすように注入する。溶液の注入後、30分反応させた後、再度前記10倍量の60℃に加熱された2×SSCバッファを導入、排出後、100倍量の0.1%Tween 20を含むTris HClバッファを核酸反応部中に通過させることによって反応部を洗浄し、固定化反応を完了した。 Next, a method for immobilizing the first DNA probe in the nucleic acid reaction part in this example will be described. In order to silane-treat the nucleic acid reaction part (104), 0.3 mg / ml silane coupling agent GTMSi (GTMSi: 3-Glycidoxypropyltrimethoxysilane Shin-Etsu Chemical) and an aqueous solution containing 0.02% acetic acid as an acid catalyst were introduced into the reaction device. And allowed to react for 2 hours. After washing the inside of the device by introducing 100 times the volume of the reaction device into the reaction device, all the solution was discharged and kept at 110 ° C. for 2 hours. Next, 1 μM streptavidin solution is introduced into the reaction device and reacted at room temperature for 6 hours to immobilize streptavidin in the nucleic acid reaction region (104). Next, in order to block unreacted glycidic groups and remove excess DNA probe, borate buffer (pH 8.5) containing 10 mM glycine, 0.01% SDS, and 0.15 M NaCl in 10 times the internal volume of the device Is introduced and discharged over 5 minutes, and 30 mM sodium citrate buffer (2 x SSC, pH 7.0) containing 0.01% SDS and 0.3 M NaCl heated to 60 ° C, 10 times the internal volume, is passed through the device. . Finally, 10mTris containing 0.1% Tween 20 which is 100 times the internal volume is introduced and discharged to complete the washing. In addition, 10 μM 5 'end biotin-modified DNA probe (first probe and second probe) in the same volume as the internal volume, 1 mM NaCl, and 10 mM TrisHCl solution containing 0.1% Tween20 Injection is performed for each individual nucleic acid reaction unit using an inkjet device. The probe solution to be injected into one nucleic acid reaction part is injected so as to fill the region by dividing 40 pL into 250 times. After injecting the solution, react for 30 minutes, and then introduce and drain the 10 times volume of 2X SSC buffer heated to 60 ° C again. The reaction part was washed by passing through the part to complete the immobilization reaction.
　フローセルデバイスの構造を図７に示した。図６に示した核酸反応デバイス(105)を複数個含み、細胞や反応溶液が導入可能なデバイスである。このような機能及び基本構成は実施例２の場合と同様であるため、省略する。第2のプローブの構造と遊離の方法が異なるのでこの点を以下で説明する。 The structure of the flow cell device is shown in FIG. The device includes a plurality of nucleic acid reaction devices (105) shown in FIG. 6 and is capable of introducing cells and reaction solutions. Since such functions and basic configurations are the same as those in the second embodiment, a description thereof will be omitted. Since the structure of the second probe and the release method are different, this point will be described below.
　核酸反応部の細孔アレーシートの内壁に固定した第2のプローブの模式図を図８に示した。312nmの紫外線を照射することによって切断部(207)でDNAが切断される。この光反応では、ビニル基が付加されたCとTの間の結合が切断される。切断部分の配列はvinylC-Tとして図８に示されている。この部分の光反応時の光反応の詳細を図９に示した。図９(a)の右側が切断前のDNAの構造で図９(a)の左側が切断後の構造である。この変化を化学式で示したものが図９(b)である。(901)はビニル基であり、(902)はチミン(T)のピリミジン部分であり、(903)はシトシン(C)のピリミジン部分である。また、(904)は切断前の(901)-(903)に該当する部分の化学式を示す。 A schematic diagram of the second probe fixed to the inner wall of the pore array sheet of the nucleic acid reaction part is shown in FIG. By irradiating 312 nm ultraviolet light, the DNA is cleaved at the cleaving part (207). In this photoreaction, the bond between C and T to which a vinyl group is added is broken. The arrangement of the cut portions is shown in FIG. 8 as vinylC-T. Details of the photoreaction at the time of the photoreaction of this portion are shown in FIG. The right side of FIG. 9 (a) is the DNA structure before cleavage, and the left side of FIG. 9 (a) is the structure after cleavage. FIG. 9 (b) shows this change in chemical formula. (901) is a vinyl group, (902) is a pyrimidine moiety of thymine (T), and (903) is a pyrimidine moiety of cytosine (C). (904) represents the chemical formula of the portion corresponding to (901)-(903) before cutting.
　第2のプローブのその他の領域は実施例2の場合とほぼ同じである。ただし、図８中、XXXXXXXXXX=TCGCGTATATとし、上に線を付したXは、Xと相補的な塩基を示す。 The other areas of the second probe are almost the same as in the second embodiment. However, in FIG. 8, XXXXXXXXXX = TCGCGTATAT, and X with a line on it represents a base complementary to X.
　本発明によって、生体分子の配列決定や定量、同定が、多数の培養細胞や多数の免疫細胞や（血中）がん細胞などに対して実行でき、どのような状態にある細胞群がどの程度の数だけ生体に存在するかを計測することが可能となる。これにより、がんなどの早期の診断やiPS細胞のヘテロジェナイエティを計測することも可能となる。 According to the present invention, sequencing, quantification, and identification of biomolecules can be performed on a large number of cultured cells, a large number of immune cells, (in blood) cancer cells, and the like, and to what extent the cell group is in what state. It is possible to measure whether there are as many of them in the living body. This makes it possible to measure early diagnosis of cancer and the like and heterogeneity of iPS cells.
101：基板
102：細胞
103：細胞保持領域
104：核酸反応部
105：核酸反応デバイス
201：第1のプローブ
202：核酸反応部の内壁
203：捕捉配列
204：タグ配列
205：共通配列
206：制限酵素の認識配列
207：切断部
208：第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列
209：mRNA
210：ポリA配列
211：第2のプローブ
212：第1のプローブをプライマーとして合成された1st cDNA
213：第2のプローブをプライマーとして合成された1st cDNA
214：ポリA配列
215：ポリT配列
216：共通配列
217：リバース方向の共通プライマー
218：第1のプローブをプライマーとして合成された1st cDNAに由来する二本鎖DNA
219：第2のプローブをプライマーとして合成された1st cDNAに由来する二本鎖DNA
301：細孔アレイシート
302：流路
303：チップ
401：フローセルデバイス
402：反応室
403：共通流路
404：上部インレット
405：上部アウトレット
406：吸入流路
407：下部アウトレット
501：ニック
502：一本鎖部分
503：ビーズ
601：ポリイミド樹脂
901：ビニル基
902：チミンのピリミジン部分
903：シトシンのピリミジン部分
904：切断前の901-903に相当する部分 101: Board
102: Cell
103: Cell retention region
104: Nucleic acid reaction part
105: Nucleic acid reaction device
201: First probe
202: Inner wall of nucleic acid reaction part
203: Capture sequence
204: Tag sequence
205: Common array
206: Restriction enzyme recognition sequence
207: Cutting part
208: Base sequence complementary to part of the base sequence of the single-stranded nucleic acid captured by the first probe
209: mRNA
210: Poly A sequence
211: Second probe
212: 1st cDNA synthesized using the first probe as a primer
213: 1st cDNA synthesized using the second probe as a primer
214: Poly A sequence
215: Poly T array
216: Common sequence
217: Common primer in reverse direction
218: Double-stranded DNA derived from the 1st cDNA synthesized using the first probe as a primer
219: Double-stranded DNA derived from 1st cDNA synthesized using second probe as primer
301: Pore array sheet
302: Flow path
303: Chip
401: Flow cell device
402: Reaction chamber
403: Common flow path
404: Upper inlet
405: Upper outlet
406: Suction channel
407: Lower outlet
501: Nick
502: Single-stranded part
503: Beads
601: Polyimide resin
901: Vinyl group
902: Pyrimidine part of thymine
903: Pyrimidine part of cytosine
904: The part corresponding to 901-903 before cutting
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims (14)

  1. 　核酸反応部(104)を少なくとも1つ含む基板(101)からなる核酸反応デバイスであって、
    　前記核酸反応部(104)は、少なくとも1つの細胞を保持する、第1の開口部からなる1つの細胞保持領域(103)を含み、かつ
    　(1)細胞から抽出される一本鎖核酸の塩基配列の一部に相補的な塩基配列からなる捕捉配列(203)を含む第1のプローブ(201)、並びに、
    　(2)核酸反応部ごとに固有の塩基配列からなるタグ配列(204)、及び該タグ配列の3'末端側に、前記第1のプローブで捕捉された一本鎖核酸の塩基配列の一部に相補的な塩基配列(208)を含む第2のプローブ(211)を含み、
    　前記第1のプローブ及び第2のプローブは固定部を介して前記核酸反応部(104)に固定されており、
    　前記第2のプローブ、又は第2のプローブの前記固定部が切断部(207)を含む、前記核酸反応デバイス。     A nucleic acid reaction device comprising a substrate (101) comprising at least one nucleic acid reaction part (104),
    The nucleic acid reaction part (104) contains one cell holding region (103) consisting of a first opening, holding at least one cell, and (1) a base of a single-stranded nucleic acid extracted from the cell A first probe (201) comprising a capture sequence (203) consisting of a base sequence complementary to a part of the sequence, and
    (2) Tag sequence (204) consisting of a unique base sequence for each nucleic acid reaction part, and a part of the base sequence of the single-stranded nucleic acid captured by the first probe on the 3 ′ end side of the tag sequence A second probe (211) comprising a complementary base sequence (208) to
    The first probe and the second probe are fixed to the nucleic acid reaction part (104) through a fixing part,
    The nucleic acid reaction device, wherein the second probe or the fixing part of the second probe includes a cutting part (207).
  2. 　前記細胞保持領域(104)が、1つの細胞のみを保持する、請求項１に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 1, wherein the cell holding region (104) holds only one cell.
  3. 　前記第2のプローブ(211)が、前記タグ配列の5’末端側に共通配列を含む、請求項１に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 1, wherein the second probe (211) includes a common sequence on the 5 'end side of the tag sequence.
  4. 　前記第1のプローブ(201)の5'末端が核酸反応部に固定されている、請求項１に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 1, wherein the 5 'end of the first probe (201) is fixed to the nucleic acid reaction part.
  5. 　前記第1のプローブ(201)が、前記捕捉配列(203)の5'末端側に、5'末端側から順に、前記共通配列(205)、及び前記タグ配列(204)を含む、請求項４に記載の核酸反応デバイス。 The first probe (201) comprises the consensus sequence (205) and the tag sequence (204) in this order from the 5 'end to the 5' end of the capture sequence (203). The nucleic acid reaction device described in 1.
  6. 　前記核酸反応部が、多孔質構造である、請求項１に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 1, wherein the nucleic acid reaction part has a porous structure.
  7. 　前記第1のプローブ(201)及び／又は第2のプローブ(211)が、前記多孔質の表面に固定されている、請求項６に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 6, wherein the first probe (201) and / or the second probe (211) is fixed to the porous surface.
  8. 　前記核酸反応部が、1以上の担体を有する、請求項１、６、又は７に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 1, 6 or 7, wherein the nucleic acid reaction part has one or more carriers.
  9. 　前記第1のプローブ(201)及び／又は第2のプローブ(211)が、担体の表面に固定されている、請求項８に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 8, wherein the first probe (201) and / or the second probe (211) is fixed on a surface of a carrier.
  10. 　前記核酸反応部(104)が基板上に複数配置されている、請求項１に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 1, wherein a plurality of the nucleic acid reaction units (104) are arranged on a substrate.
  11. 　前記第1の開口部とは異なる第2の開口部を有する請求項１に記載の核酸反応デバイス。 The nucleic acid reaction device according to claim 1, wherein the nucleic acid reaction device has a second opening different from the first opening.
  12. 　請求項１１に記載の核酸反応デバイス、
    　前記第1の開口部に接する第1の流路、及び
    　前記第2の開口部に接する第2の流路を含む、フローセルデバイス。     A nucleic acid reaction device according to claim 11,
    A flow cell device, comprising: a first channel in contact with the first opening; and a second channel in contact with the second opening.
  13. 　請求項１に記載の核酸反応デバイスを用いて、細胞由来の一本鎖RNAから、前記タグ配列(204)が付加されたcDNAを得る方法であって、
    　前記細胞保持領域に保持された細胞から一本鎖RNAを抽出し、抽出された一本鎖RNAを、前記捕捉配列とのハイブリダイゼーションにより前記第1のプローブ(201)に捕捉させる工程、
    　前記第2のプローブ(211)の切断部(207)を切断し、第2のプローブ(211)を遊離させる工程、
    　遊離した前記第2のプローブ(211)を、前記第1のプローブによって捕捉された一本鎖RNAの一部にハイブリダイズさせる工程、及び
    　ハイブリダイズした前記第2のプローブ(201)をプライマーとして、前記第1のプローブ(201)によって捕捉された一本鎖RNAの一部に相補的な塩基配列を含む核酸を合成することにより、前記タグ配列が付加されたcDNAを得る工程、
    を含む前記方法。     A method for obtaining a cDNA to which the tag sequence (204) is added from a single-stranded RNA derived from a cell, using the nucleic acid reaction device according to claim 1,
    Extracting the single-stranded RNA from the cells retained in the cell-retaining region, and allowing the first probe (201) to capture the extracted single-stranded RNA by hybridization with the capture sequence;
    Cutting the cutting portion (207) of the second probe (211) to release the second probe (211);
    Hybridizing the released second probe (211) with a part of the single-stranded RNA captured by the first probe, and using the hybridized second probe (201) as a primer, Synthesizing a nucleic acid containing a base sequence complementary to a part of the single-stranded RNA captured by the first probe (201) to obtain a cDNA to which the tag sequence is added,
    Including said method.
  14. 　請求項１３に記載の方法によりcDNAを得る工程、前記cDNAを増幅する工程、及び増幅された核酸の配列決定を行う工程を含む、遺伝子解析方法。 A gene analysis method comprising a step of obtaining cDNA by the method according to claim 13, a step of amplifying the cDNA, and a step of sequencing the amplified nucleic acid.
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