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WO2016001963A1 - Dispositif en réseau pour l'analyse d'acides nucléiques, dispositif d'analyse d'acides nucléiques, et procédé d'analyse d'acides nucléiques - Google Patents

Dispositif en réseau pour l'analyse d'acides nucléiques, dispositif d'analyse d'acides nucléiques, et procédé d'analyse d'acides nucléiques Download PDF

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Publication number
WO2016001963A1
WO2016001963A1 PCT/JP2014/067344 JP2014067344W WO2016001963A1 WO 2016001963 A1 WO2016001963 A1 WO 2016001963A1 JP 2014067344 W JP2014067344 W JP 2014067344W WO 2016001963 A1 WO2016001963 A1 WO 2016001963A1
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Prior art keywords
nucleic acid
acid analysis
array device
region
analysis
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PCT/JP2014/067344
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English (en)
Japanese (ja)
Inventor
白井　正敬
崇秀 横井
神原　秀記
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株式会社日立製作所
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Priority to PCT/JP2014/067344 priority Critical patent/WO2016001963A1/fr
Publication of WO2016001963A1 publication Critical patent/WO2016001963A1/fr

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to an array device for nucleic acid analysis having a plurality of nucleic acid capture regions for capturing nucleic acid molecules, a nucleic acid analysis apparatus and a nucleic acid analysis method using the array device for nucleic acid analysis.
  • a solution containing a nucleic acid molecule to be analyzed is allowed to act on a probe nucleic acid having a specific base sequence (for example, a poly-T sequence) to capture the nucleic acid molecule contained in the solution with the probe nucleic acid.
  • a probe nucleic acid having a specific base sequence for example, a poly-T sequence
  • Various nucleic acid analysis processes such as sequencing are performed. In such nucleic acid analysis processing, a bulk analysis in which nucleic acids extracted from biological tissue or cultured cells composed of a large number of cells are analyzed, and nucleic acids extracted from individual cells constituting the biological tissue are analyzed. And single cell analysis.
  • Examples of the bulk analysis and single cell analysis include genome analysis such as base sequence analysis, such as Whole Genome Analysis that analyzes all genomes and Whole Exsome Analysis that analyzes only exon regions.
  • genome analysis such as base sequence analysis, such as Whole Genome Analysis that analyzes all genomes and Whole Exsome Analysis that analyzes only exon regions.
  • epigenetics analysis that analyzes transcriptional control of the genome
  • a method for analyzing the methylated position of a nucleic acid molecule is known, and a method for analyzing chromatin modification positions such as modification of histone proteins. Is also known as the Chip-Seq method.
  • a method for analyzing the amount of mRNA transcribed from the genome is known as gene expression analysis.
  • a cDNA library sheet corresponding to each cell or cell site in a two-dimensionally expanded tissue is prepared.
  • Devices have been developed that can be fabricated and used to obtain a two-dimensional distribution of gene expression to achieve gene expression analysis in a large number of cells.
  • gene expression analysis in a single cell is performed using the device disclosed in Patent Document 1, it is not necessary to isolate the cell, and mRNA is directly extracted from cells in a section of living tissue, and gene expression analysis is performed. You can also.
  • an amplification reaction called a template walking reaction described in Non-Patent Document 1 and a DNA quantification method described in Non-Patent Document 2 can be performed.
  • reagents are individually supplied to a plurality of individualized regions, and the analysis process is merely executed individually, and a plurality of samples are not analyzed simultaneously using a single device. .
  • the present invention has a plurality of nucleic acid capture regions for capturing nucleic acid molecules, and an array device for nucleic acid analysis that can simultaneously analyze a plurality of samples using the plurality of nucleic acid capture regions.
  • An object of the present invention is to provide a nucleic acid analyzer and a nucleic acid analysis method.
  • sample contamination can be achieved even when a plurality of nucleic acid capture regions are used by appropriately controlling communication and individualization of the upper space portions of the plurality of nucleic acid capture regions.
  • the inventors have found that a plurality of nucleic acid analysis processes can be carried out by preventing nation, and the present invention has been completed.
  • the present invention includes the following.
  • An array device for nucleic acid analysis comprising:
  • control means sets the nucleic acid capture region to a highly hydrophilic surface when comparing the nucleic acid capture region with a region surrounding the nucleic acid capture region.
  • Array device
  • nucleic acid analysis array device wherein the nucleic acid capturing region has a cell capturing means for capturing one or a plurality of cells.
  • nucleic acid analysis array device (4) The nucleic acid analysis array device according to (3), wherein the nucleic acid capture region is a through-hole having a smaller diameter than the cells formed in the nucleic acid capture region.
  • the nucleic acid capture region is an inner surface of a recess formed on one main surface of the base or a region surrounding the inner surface of the recess and the outer periphery of the recess, and the nucleic acid capture means is on the bottom surface of the recess.
  • the array device for nucleic acid analysis according to (1) which is fixed.
  • the array device for nucleic acid analysis according to (1) further comprising a water supply / drainage mechanism for supplying liquid to the space formed by the base and discharging the liquid in the space.
  • the base is a pair of substrates disposed at a predetermined interval, and the plurality of nucleic acid capture regions are formed in opposing regions on the opposing surfaces of the pair of substrates ( 1) The array device for nucleic acid analysis according to 1).
  • the water supply / drainage control unit supplies a nonpolar solvent to the array device for nucleic acid analysis placed on the device placement unit and then supplies a nonpolar solvent to the array device for nucleic acid analysis.
  • a nucleic acid analysis method comprising: a step of supplying a nucleic acid; and a step of performing a nucleic acid analysis in a state where a plurality of nucleic acid capture regions in the nucleic acid analysis array device are individualized.
  • the array device for nucleic acid analysis the nucleic acid analysis apparatus, and the nucleic acid analysis method according to the present invention, it is possible to prevent sample contamination and simultaneously execute a plurality of nucleic acid analysis processes using a plurality of nucleic acid capture regions. Therefore, by applying the nucleic acid analysis array device, the nucleic acid analysis apparatus, and the nucleic acid analysis method according to the present invention, the efficiency of the analysis processing relating to the nucleic acid molecule to be analyzed can be greatly improved.
  • the array device for nucleic acid analysis has a plurality of nucleic acid capture regions arranged on at least one main surface of the base.
  • the base means a support that forms a plurality of nucleic acid capture regions.
  • a flat plate member can be exemplified, but it is not necessarily a flat plate member, and any shape member such as a sheet, a membrane, a gel thin film, a capillary plate, a bead, or a film is used. be able to.
  • the base material is not particularly limited, and examples thereof include metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium and nickel; alloys such as stainless steel, hastelloy, inconel, monel and duralumin; silicon Glass materials such as glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite and photosensitive glass; polyester resin, polystyrene, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon , Plastics such as acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin and vinyl chloride resin; agarose, dextran, cellulose, polyvinyl Alcohol, nitrocellulose, chitin, chitosan.
  • metals such as gold, silver, copper, aluminum, tungsten, molybdenum,
  • the sheet when a sheet is used as the substrate, the sheet can be produced from alumina, glass, or the like.
  • a gel thin film when a gel thin film is used as the substrate, the gel thin film can be produced using, for example, acrylamide gel, gelatin, modified polyethylene glycol, modified polyvinyl pyrrolidone, hydrogel, or the like.
  • a membrane When a membrane is used as the substrate, for example, cellulose acetate, nitrocellulose or a mixed membrane thereof, nylon membrane or the like can be used.
  • the beads when beads are used as the substrate, the beads can be produced from a resin material (such as polystyrene), an oxide (such as glass), a metal (such as iron), sepharose, and combinations thereof.
  • a flat plate member is used as a base will be described. In this case, the base is referred to as a “substrate”.
  • the nucleic acid capturing region means a region having a nucleic acid capturing means capable of capturing the nucleic acid molecule when a solution containing the nucleic acid molecule to be captured comes into contact.
  • the nucleic acid capture means may be provided over the entire nucleic acid capture region or may be provided in a part of the nucleic acid capture region.
  • a plurality of nucleic acid capture regions means that at least two nucleic acid capture regions are arranged adjacent to each other with a predetermined interval.
  • the nucleic acid capturing means is not particularly limited, and examples thereof include a nucleic acid probe having a predetermined base sequence. More specifically, the nucleic acid probe can be appropriately designed according to the nucleic acid molecule to be captured. Examples of the nucleic acid molecule to be captured include messenger RNA (mRNA), non-coding RNA (ncRNA), DNA (including genomic DNA and mitochondrial DNA), and fragments thereof.
  • the nucleic acid probe is not particularly limited as long as it can hybridize and capture with these nucleic acid molecules, and is known to those skilled in the art. For example, when the nucleic acid molecule to be captured is mRNA, it is preferable to use a DNA probe containing a poly T sequence as the nucleic acid probe.
  • a DNA probe containing a poly-T sequence, that is, oligo (dT) can be synthesized by a conventional method, and the degree of polymerization of oligo (dT) is hybridized with the poly-A sequence of mRNA, and mRNA is oligo- (dT) Any degree of polymerization that can be captured by a fixed substrate.
  • the nucleic acid molecule to be captured can be extracted from the cell by a method 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 array device for nucleic acid analysis has a control means for communicating or individualizing the upper space portions of the plurality of nucleic acid capture regions.
  • the upper space means a space located above the main surface of the substrate on which the nucleic acid capturing region is formed, that is, the main surface on which the nucleic acid capturing means is provided.
  • the communication of the upper space portions of the plurality of nucleic acid capture regions with each other means, for example, that when the liquid is supplied to the main surface of the substrate, the liquid flows between the plurality of upper space portions corresponding to the plurality of nucleic acid capture regions. It means that it is possible.
  • individualizing the upper space portions of the plurality of nucleic acid capture regions means that the liquid cannot flow between the plurality of upper space portions corresponding to the plurality of nucleic acid capture regions, and each upper space portion is independent. It means to put in a state.
  • the nucleic acid capture region has a high hydrophilic surface (hereinafter referred to as the first embodiment). .
  • the nucleic acid capture region having a high hydrophilic surface means that the surface of the nucleic acid capture region is compared with the surface of the region surrounding the nucleic acid capture region. It has a sex.
  • a water droplet contact angle WCA
  • WCA water droplet contact angle
  • the upper spaces of the plurality of nucleic acid capture regions can be communicated with each other by the solution.
  • a nonpolar solvent is supplied to the main surface of the substrate on which a plurality of nucleic acid capture regions are arranged from this state, an aqueous solution remains only in the upper space part of the nucleic acid capture region, and the upper space of the region other than the nucleic acid capture region Part of the aqueous solution replaces the nonpolar solvent.
  • the non-polar solvent after supplying the aqueous solution to the main surface of the substrate on which the plurality of nucleic acid capture regions are arranged, the upper spaces of the plurality of nucleic acid capture regions can be individualized.
  • the array device for nucleic acid analysis includes a pair of transparent substrates 5 and 6, a cell-capturing partition wall 7 disposed between the transparent substrates 5 and 6, and a side wall 10 as shown in FIG.
  • a space 2 is formed in the interior formed by the transparent substrates 5 and 6, the cell capturing partition wall 7 and the side wall 10.
  • This array device for nucleic acid analysis captures floating cells in the cell-capturing partition wall 7, and mRNA liberated from the captured cells in the transparent substrates 5 and 6 and the plurality of nucleic acid-capturing regions 4 formed in the cell-capturing partition wall 7.
  • the captured mRNA is subjected to analysis processing (cDNA synthesis, sequence analysis). That is, gene expression analysis at the single cell level can be performed by using the array device for nucleic acid analysis.
  • the pair of transparent substrates 5 and 6 and the cell-capturing partition wall 7 have a plurality of nucleic acid-capturing regions 4 to which DNA probes (poly T probes) are fixed as nucleic acid-capturing means. Each is formed.
  • the plurality of nucleic acid capture regions 4 formed on the transparent substrate 5, the plurality of nucleic acid capture regions 4 formed on the cell capture partition 7, and the plurality of nucleic acid capture regions 4 formed on the transparent substrate 6 are , Facing each other.
  • the size of the nucleic acid capture region 4 can be appropriately set as long as adjacent nucleic acid capture regions 4 do not contact each other.
  • the nucleic acid capturing region 4 has a circular shape with a diameter of 200 ⁇ m, but can have any shape within a range from about 5 ⁇ m to several mm.
  • the plurality of nucleic acid capture regions 4 have a higher hydrophilic surface than the surrounding region.
  • a plurality of nucleic acid trapping regions 4 are formed on one main surface of a quartz substrate having a thickness of 0.2 mm, for example, which is a material for the transparent substrates 5 and 6 and the cell trapping partition 7.
  • a resist film is formed in the region using a general lithography technique. Next, plasma fluorine treatment (plasma ashing (300 W) in a CHF 3 , C 2 F 6 atmosphere) is performed for 5 minutes, and water repellent treatment is performed on the region not covered with the resist film.
  • the water-repellent treatment may be performed by coating a fluororesin (for example, trade name: Cytop (Asahi Glass Co., Ltd.)). Then, by removing the resist film, the region where the resist film is formed is not subjected to water repellent processing, and the region other than the region where the resist film is formed is subjected to water repellent processing. . Next, a resist film is formed in the water-repellent processed region to expose only the regions that become the plurality of nucleic acid capturing regions 4, and in this state, a 172 nm deep ultraviolet treatment is performed for 3 minutes on one main surface of the substrate. Thereby, the process which provides hydrophilicity to the area
  • a fluororesin for example, trade name: Cytop (Asahi Glass Co., Ltd.
  • a hydrophilic surface is applied to the regions corresponding to the plurality of nucleic acid capturing regions 4 on each of the transparent substrates 5 and 6 and the cell capturing partition walls 7 by performing a water repellent process and a treatment for imparting hydrophilicity. Can be formed. Note that either the water repellent treatment or the treatment for imparting hydrophilicity may be performed first.
  • the plurality of nucleic acid capture regions 4 only need to have high hydrophilicity when compared with the region surrounding the plurality of nucleic acid capture regions 4, the above-described water-repellent treatment and the treatment for imparting hydrophilicity Do only one of them.
  • a DNA probe (poly T probe: SEQ ID NO: 1) is immobilized on the hydrophilic surface.
  • a method for immobilizing the DNA probe will be described later.
  • one DNA probe can be fixed at an average ratio of 30 to 100 nm 2 , 1.2 to 1.2 ⁇ 10 7 DNA probes are fixed to one nucleic acid capture region 4. Copy number of the mRNA in a single cell, it is possible to fix a sufficient number of DNA probes to capture all of the mRNA for is 10 6 or less.
  • a through-hole 1 is formed as a cell trapping means in the approximate center of the cell trapping partition 7.
  • the through-hole 1 captures floating cells and is not particularly limited, but has a diameter (for example, 3 to 30 ⁇ m, specifically 8 ⁇ m) according to the size of the cell to be captured. It is preferable to do.
  • the through-hole 1 has a shape having a small diameter in the thickness direction from both main surfaces of the cell-capturing partition wall 7.
  • the number of through-holes 1 is not limited at all, and can be, for example, several tens to several millions on one main surface of the cell-capturing partition wall 7. In the example shown in FIG.
  • the through holes 1 are one-dimensionally arranged on one main surface of the cell trapping partition 7.
  • the interval between the through holes 1 is 300 ⁇ m, but it can be set freely from several tens of ⁇ m to several millimeters.
  • the distance between the transparent substrates 5 and 6 and the cell-capturing partition wall 7 is 200 ⁇ m, but it can be set freely from several tens of ⁇ m to several millimeters.
  • the through hole 1 is not particularly limited, but a laser processing method can be applied. Laser processing can be performed from both surfaces of the cell-capturing partition wall 7 to form a shape (hourglass type) having a smaller diameter in the thickness direction from both main surfaces.
  • the through hole 1 may be formed by applying a dry or wet etching method.
  • an inlet 11 and an outlet 13 are formed on the transparent substrate 5, and an inlet 12 and an outlet 14 are formed on the transparent substrate 6.
  • These inlets 11 and 12 are connected to a liquid supply mechanism (not shown).
  • the outlets 13 and 14 are connected to a drainage mechanism (not shown).
  • a single cell is captured in the through-hole 1.
  • a cell solution suspended in a culture solution, physiological saline or serum is allowed to flow from the inlet 11 toward the outlet 14.
  • the cell solution flows from the inlet 11 to the outlet 14 through the through-hole 1 serving as a cell trapping means. Since the diameter of the through-hole 1 is set to 3 to 30 ⁇ m, the cells 3 larger than the diameter of the cell solution pass through the through-hole 1. Cannot pass through and is captured.
  • the cell solution is filled in the entire space 2 including the upper space portion of the nucleic acid capturing region 4, and the upper space portions of the plurality of nucleic acid capturing regions are communicated with each other by the cell solution.
  • a single cell is captured by each through-hole 1 and no cell exists in other regions.
  • the diameter of the through hole 1 is smaller than the diameter of the cell to be captured, and when one cell is captured, the flow through the cell capturing portion of the solution is inhibited. Therefore, if the flow of the cell solution from the inlet 11 to the outlet 14 is continued, a single cell is trapped in almost all the through holes 1. Excess cells can be removed from the outlet 13.
  • the cell disrupting solution is allowed to flow from the inlet 11 toward the outlet 14 while maintaining a constant pressure in the direction from the inlet 11 to the outlet 14.
  • the solution for cell disruption is filled in the entire space 2 including the upper space part of the nucleic acid capture region 4 in the same manner as when the cell solution is supplied. It will be in the state which mutually connected the upper space part of the area
  • a buffer solution containing a surfactant can be used. More specifically, a Tris-HCl (pH 8.0) solution containing 0.5% Tween 20 and 1M NaCl can be used as the cell disrupting solution. Note that the cell disruption solution having the composition can be used as a solvent for the cell solution used for capturing cells since the surfactant has a weak cell disruption effect at room temperature.
  • FIG. 3 shows the procedure from capturing mRNA in individual cells to a nucleic acid capture region 4 with a DNA probe (poly T probe) 31 immobilized via streptavidin 33 and synthesizing cDNA (ie, until reverse transcription). Show.
  • FIG. 3 shows a partial cross-sectional view of the cell-capturing partition wall 7, and enlarged views of the surface are shown in (a) to (c).
  • a washing buffer (10 mM Tris-HCl containing 0.1% Tween-20, pH 8.0) is introduced from inlets 11 and 12 in an amount 20 times the internal volume of space 2, and discharged from outlets 13 and 14. Thereby, the mineral oil which is a nonpolar solvent in the upper space 9 is discharged.
  • RNaseOUT Invitrogen
  • Superscript III reverse transcriptase, Invitrogen mixed in 58.5: 4: 22.5: 4: 4
  • a cDNA chain 34 is synthesized using the mRNA 32 captured by the DNA probe 31 as a template.
  • the reaction solution is slowly heated to 50 ° C. and a complementary strand synthesis reaction is carried out for about 50 minutes (FIG. 3 (b)).
  • the mixture is kept at 85 ° C. for 1.5 minutes to inactivate the reverse transcriptase and cooled to 4 ° C.
  • excess polyprobe is decomposed by introducing an enzyme solution that selectively decomposes single-stranded DNA in an amount equal to the internal volume of the space 2.
  • a cDNA library sheet can be constructed simultaneously in a plurality of nucleic acid capture regions 4.
  • FIG. 4 shows a sample preparation method for performing single-molecule sequencing that does not require amplification.
  • the example shown here shows the sequencing method of 5 'end of mRNA (3' end of cDNA).
  • a solution containing terminal transferase is uniformly introduced into the space 2
  • the poly A sequence 35 is added to the 3 'end of the cDNA.
  • a solution containing the poly-T probe 36 as a sequencing primer is introduced into the space 2.
  • sequencing becomes possible as shown in FIG. 4 (d-2).
  • dATP dATP
  • dCTP dCTP
  • dGTP dGTP
  • dTTP dTTP
  • the base sequence is determined by measuring each molecule to determine whether or not the As soon as the extension of one kind of base is completed, the phosphor is decomposed and desorbed so that the next base can be introduced. By repeating such a reaction, the base sequence of cDNA 34 can be determined in the direction of the arrow in FIG. 4 (d-3).
  • an enzyme solution necessary for cDNA fragmentation and poly A addition reaction is introduced into the space 2 in a state where the activity of these enzymes is low (eg, 4 ° C.). Then, the fragmented cDNA is captured by the DNA probe 31 in the same manner as the mRNA in FIG. 3 (a), the complementary strand is synthesized in the same manner as in FIG. 3 (b), and then alkali-denatured or heat-denatured. By flowing the washing solution, the state shown in FIG. 3 (c) can be produced for the fragmented cDNA. By making the base length of the fragmented cDNA shorter than the base length that can be sequenced, it becomes possible to analyze the sequence of the entire cDNA region.
  • the upper space 8 of the plurality of nucleic acid capture regions 4 is individualized, Prevents contamination of mRNA derived from cells. Thereafter, in a state where the upper space portions 8 of the plurality of nucleic acid capture regions 4 are in communication with each other, the cDNA library can be adjusted using the captured mRNA and the base sequence of the cDNA can be determined. That is, after the nucleic acid molecules are captured in the plurality of nucleic acid capture regions 4, the nucleic acid analysis process can be simultaneously performed in all the nucleic acid capture regions 4. Therefore, even if a plurality of nucleic acid capture regions 4 are used, the reagents can be used in common and the supply and discharge operations of the reagents can be shared. Can be improved.
  • FIG. 5 shows a sample preparation method for performing gene expression analysis by selectively sequencing only such specific genes.
  • 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27 and OAZ1 20 ⁇ 5 bases upstream of 109 ⁇ 8 bases from the polyA tail of the target gene can be used for the gene-specific sequence of () (SEQ ID NOs: 3 to 22).
  • the nucleic acid molecule to be captured is mRNA, but the capture target may be genomic DNA or microRNA.
  • genomic DNA when genomic DNA is targeted, a restriction enzyme that cleaves DNA is mixed in a solution for cell disruption, and the solution is introduced together with cells at a sufficiently low temperature for restriction enzyme and cell disruption activity, and then raised to a suitable temperature. That's fine.
  • a random 6-base mixed probe may be used in place of the poly-T probe in order to capture the genome sequence, or a mixed probe having a specific sequence of restriction enzyme may be used as the nucleic acid capturing means.
  • the measurement target is microRNA
  • the cell disruption solution is the same as that of mRNA, and a mixed probe having a complementary sequence may be fixed to a part of the microRNA to be measured instead of the poly T probe.
  • the array device for nucleic acid analysis has an excellent effect when immobilizing a DNA probe.
  • a region to be a plurality of nucleic acid capture regions 4 is made hydrophilic as described above, and then 0.3 mg / ml silanized pulling agent GTMSi (GTMSi: 3-Glycidoxypropyltrimethoxysilane)
  • GTMSi silanized pulling agent
  • An aqueous solution containing 0.02% acetic acid (Shin-Etsu Chemical) and acid catalyst is introduced into space 2, and a nonpolar solvent such as mineral oil is immediately introduced.
  • the DNA probe can be fixed to a plurality of regions having a hydrophilic surface, and a plurality of nucleic acid capturing regions 4 can be formed on the transparent substrates 5 and 6 and the cell capturing partition wall 7.
  • a state where the upper spaces of the plurality of hydrophilic surfaces are in communication with each other and an individualized state are created so that the DNA probe is not fixed to a region other than the hydrophilic surface. Can be.
  • FIG. 6 shows the flow of the entire analysis
  • FIGS. 7 and 8 show the configuration of the entire system.
  • a cell suspension solution is introduced into the reaction device, and the cells are captured by the through-hole 1 that is a cell capturing means.
  • a microscopic image of the captured cells is acquired.
  • the microscopic image is preferably an optical image that can be obtained without significantly destroying the cell shape, such as a fluorescent image, a bright field image, or a non-linear microscopic image.
  • reaction solution is separated for each single cell by introducing a nonpolar solvent such as mineral oil into the array device for nucleic acid analysis.
  • a nonpolar solvent such as mineral oil
  • This is synonymous with individualizing the upper spaces 8 of the plurality of nucleic acid capture regions 4 by introducing a nonpolar solvent such as mineral oil into the array device for nucleic acid analysis.
  • a sample that can be sequenced In the case of this example, cDNA is synthesized by reverse transcription, then mRNA is degraded, and sequencing primers are hybridized. Thereafter, sequencing of the sample is simultaneously performed in a plurality of nucleic acid capture regions 4. By associating the sequencing data with the microscopic image, it is possible to take correspondence between imaging and gene analysis.
  • the nucleic acid analysis apparatus includes a device mounting unit 20 on which the above-described nucleic acid analysis array device is mounted, and liquid supply to the nucleic acid analysis array device mounted on the device mounting unit 20. And the result of nucleic acid analysis performed using the array device for nucleic acid analysis placed on the device placement unit 20 and the water supply / drainage control unit 30 for controlling the discharge of the liquid supplied to the array device for nucleic acid analysis.
  • the detection part 40 to be provided.
  • the device mounting unit 20 is not particularly limited as long as the device mounting unit 20 is equipped with an array device for nucleic acid analysis to enable predetermined nucleic acid analysis processing.
  • the device placement unit 20 includes a Peltier element 74 that adjusts the temperature of the array device for nucleic acid analysis placed on the upper surface, and a rotating stage 75 that rotates the array device for nucleic acid analysis in-plane together with the Peltier element 74. And an XYZ stage 76.
  • the rotation stage 75 and the XYZ stage 76 are controlled by a control system 77.
  • the water supply / drainage control unit 30 may supply and discharge liquid to and from the nucleic acid analysis array device mounted on the device mounting unit 20 according to a predetermined nucleic acid analysis process, and the configuration thereof is not particularly limited.
  • the water supply / drainage control unit 30 supplies the reagent reservoir 83 in which a plurality of reagent tubes 81 are arranged, the reagents in the reagent tube 81 into the array device for nucleic acid analysis, and the solution in the array device for nucleic acid analysis.
  • a syringe 85 for discharging and a waste liquid bottle 86 for storing the solution are provided.
  • the reagent reservoir 83, the syringe 85, the nucleic acid analysis array device, and the waste liquid bottle 86 are connected to each other via a pipe, and the pipes are connected by a plurality of pinch valves 87 to 93 arranged on the pipe. Communication and blockage can be controlled.
  • a sipper 82 for aspirating reagents from a plurality of reagent tubes 81 is connected to the syringe 85.
  • the reagent reservoir 83 is attached to the XYZ stage 84 so as to be accurately positioned with respect to the shipper 82.
  • the detection unit 40 is not particularly limited as long as it can detect an analysis result corresponding to a predetermined nucleic acid analysis process.
  • the detection unit 40 includes an excitation light irradiation optical system including a mercury lamp 71, an irradiation lens 72, an excitation filter 73, a dichroic mirror 78, and an objective lens 74, and a fluorescence detection optical system including an imaging lens 79 and an image sensor 80. It consists of.
  • a cell to be measured is excited and irradiated through the lens 74.
  • Annexin V FITC and Hoechst Dye are used as the fluorescent dyes of cells, the former shows 535 nm emission with 488 nm excitation, and the latter shows 465 nm emission with 355 nm excitation.
  • the former fluorescence can stain the cell membrane in the apoptotic process, and the latter can stain the nucleus.
  • the dichroic mirror 78 suitable for such imaging has an edge wavelength of 505 nm when FITC fluorescence is imaged, and has an edge wavelength of 385 nm when Hoechst fluorescence is imaged.
  • An imaging lens 79 arranged so as to form an image at a magnification of 40 on the image sensor 80 was arranged. Since only one cell image fits on the imaging area of the imaging device 80, the cell is handled by moving the array device for nucleic acid analysis using the XYZ stage 76 in order to capture a large number of cells.
  • a reaction system for sample preparation will be described with reference to FIG.
  • a method for introducing a cell solution and various reagents into the two inlets 11 and 12 of the array device for nucleic acid analysis will be described.
  • the cell solution and the various reagents are respectively dispensed into the reagent tube 81, and the temperature is adjusted to be an appropriate temperature by the reagent reservoir 83 (for example, 4 ° C., 37 ° C., and 60 ° C. for each reagent type). Setting).
  • This reagent reservoir 83 is mounted on the XYZ stage 84, and after adjusting the XY position so that a necessary reagent or sample can be selected and sucked from the sipper 82, the reagent reservoir 83 is moved in the Z direction in the figure to Allow the tip to sink into the solution.
  • the pinch valve 87 is opened, the pinch valves 88 and 89 are closed, and the syringe 85 is pulled.
  • pinch valves 89, 90, 91, 92, and 93 are opened, pinch valves 87 and 88 are closed, and syringe 85 is pushed.
  • the solution discharged from the nucleic acid analysis array device is stored in the waste liquid bottle 86. Further, the excessively aspirated reagent is discharged to the waste liquid bottle 86 by opening the pinch valve 88, closing the pinch valves 87 and 89, and pushing the syringe 85. In this way, it is possible to automatically execute the various reactions shown in FIGS. 3, 4, and 5 under the control of the control system 77 for supplying and discharging reagents to and from the array device for nucleic acid analysis.
  • single molecule sequencing refers to fluorescence measurement of the base extension process for each molecule of cDNA immobilized on the nucleic acid capture region 4, as shown in FIGS. 4 (d-3) and 5 (e-2). To determine the sequence of full length or part 34 thereof.
  • a laser 97 is incident from the transparent substrates 5 and 6 and the side surface of the cell-capturing partition wall 7 through the coupling lens 96 to obtain multiple reflected light.
  • the phosphor on the DNA immobilized on the surface is excited in TIR (Total Internal Reflection) mode.
  • An image of a firefly spot for each molecule on the nucleic acid capture region 4 is taken by the image sensor 80, and which of the four types of bases (dATP, dGTP, dTTP, and dCTP) is introduced by the control system 77. Sequencing is performed for each spot in light of the above information.
  • the TIR excitation rotates the rotary stage 75 by 90 degrees, and the fluorescence images at the time of excitation from two directions of 0 degrees and 90 degrees are divided into two images.
  • the reason for doing this is that it is difficult to determine the base sequence that exists on the same straight line as the through-hole 1 because it is the through-hole 1 that is a cell trapping means. Of course, this is not the case when the cell trapping means is not a through-hole 1 but a chemical action by surface treatment as will be shown later.
  • reagents are injected from inlets 11 and 12 and discharged from outlets 13 and 14. Thereby, the reagents supplied to the space 2 can be kept at a uniform concentration.
  • a PCR product 63 having a cell recognition sequence for each cell is synthesized. May be. Specifically, as shown in FIG. 9, first, a cell recognition sequence having a different sequence for each position of the nucleic acid capture region 4 and a PCR amplification common sequence (Forward direction) are sequentially connected to the 5 ′ end of the poly-T sequence. A DNA probe having an oligonucleotide having a base sequence thus prepared is immobilized according to the method described above.
  • a PCR product 63 having a cell recognition sequence for each cell can be obtained as a result of the process shown in FIGS. 9 and 10, and cell identification information can be obtained even when sequencing is performed outside the array device for nucleic acid analysis. You can avoid losing.
  • FIGS. 9 and 10 The reaction of FIGS. 9 and 10 will be described below.
  • the process up to the synthesis of the cDNA of FIG. 9 is the same as that shown in FIG.
  • a solution containing the fragmenting enzyme and the poly-A-added enzyme is introduced into the space 2 from the inlets 11 and 12 at a low temperature (4 ° C.), and mineral oil is immediately introduced in the same manner.
  • region 4 can be individualized similarly to the state shown in FIG.
  • fragmentation of the synthesized cDNA at a predetermined reaction temperature and addition of poly A to the fragment are simultaneously performed, and a fragment obtained by adding poly A51 to the fragmented cDNA 34 as shown in FIG.
  • the PCR solution 63 is obtained by substituting the inner solution and mineral oil and adding 20 thermal cycles of 96 ° C. (30 seconds) and 55 ° C. (1 minute 30 seconds). This is discharged from the outlets 13 and 14 to the outside of the device, collected, and then processed for sequencing. Since the sequence information obtained as a result includes cell recognition sequence information, it is possible to know which nucleic acid capture region 4 of the plurality of nucleic acid capture regions 4 belongs to the cDNA.
  • the above-described array device for nucleic acid analysis includes transparent substrates 5 and 6 and a cell-capturing partition wall 7 as shown in FIG. Although the region 4 is formed, the array device for nucleic acid analysis according to the present invention is not limited to this configuration.
  • the array device for nucleic acid analysis may be one in which a plurality of nucleic acid capture regions 4 are formed only in the cell capture partition wall 7.
  • the transparent substrates 5 and 6 do not need to be subjected to a treatment for imparting hydrophilicity or a water repellent treatment, and can have a simpler configuration.
  • a nonpolar solvent such as mineral oil is introduced so that droplets of the aqueous solution cover a plurality of nucleic acid capture regions 4.
  • the upper space 8 of the plurality of nucleic acid capture regions 4 can be individualized.
  • the cell capturing partition wall 7 sandwiched between the transparent substrates 5 and 6 is flat as shown in FIG.
  • the configuration is not limited.
  • the array device for nucleic acid analysis may include a rib 1801 and a cell capturing partition wall 7 in which a nucleic acid capturing region 4 is formed in a recess surrounded by the rib 1801. . That is, in the array device for nucleic acid analysis shown in FIG. 12, the inner surface (that is, the side surface and the bottom surface) constituting the recess and the outer edge region surrounding the recess are high hydrophilic surfaces, and the DNA probe is immobilized on the bottom surface in the recess. Yes.
  • the nonpolar solvent such as mineral oil
  • the nonpolar solvent is held in the space formed by the upper surface of the rib 1801 and the transparent substrates 5 and 6, and The upper space part 8 of the nucleic acid capture region 4 will be individualized.
  • the thickness of the portion where the nonpolar solvent such as mineral oil is accumulated is reduced, and the individualized state can be formed more stably.
  • the array device for nucleic acid analysis described above has a configuration including a cell-capturing partition wall 7 sandwiched between transparent substrates 5 and 6, as shown in FIG.
  • the configuration is not limited.
  • a space 2 is constituted by a pair of transparent substrates 5 and 6, and a single cell is captured by any one of these transparent substrates 5 and 6.
  • the array device for nucleic acid analysis has a cell capture processing unit 1301 at substantially the center of the plurality of nucleic acid capture regions 4 formed on the transparent substrate 6.
  • the size of the cell capture processing unit 1301 can be appropriately set according to the diameter of the cell to be captured. More specifically, the cell capture processing unit 1301 can be obtained by fixing fibronectin at a high density over a diameter of 1 ⁇ m.
  • the array device for nucleic acid analysis can supply and discharge the liquid to and from the space 2 by the inlet 11 and the outlet 14, and the configuration can be simplified.
  • the reaction procedure using these inlet 11 and outlet 14 is as follows.
  • a cell solution suspended in a culture solution, physiological saline or serum is allowed to flow from the inlet 11 to the outlet 14.
  • the cells come into random contact with the cell capture region by Brownian motion, and the cells are captured by selective binding between the cell membrane and the substance fixed to the cell capture processing unit 1301.
  • the size of the cell is larger than the size of the cell capture processing unit 1301, the possibility that two or more cells are captured by the same cell capture processing unit 1301 is small.
  • a cell disrupting solution is flowed from the inlet 11 toward the outlet 14. It is common to use a buffer solution containing a surfactant.
  • Tris-HCl (pH 8.0) containing 0.5% Tween 20 and 1M NaCl can be used.
  • a nonpolar solvent such as mineral oil is allowed to flow from the inlet 11 to the outlet 14.
  • the solution containing the surfactant is left only in the upper space 8 of the plurality of nucleic acid capture regions 4 subjected to the hydrophilic treatment.
  • the upper space portion 9 in the region is filled with mineral oil.
  • the upper space portions 8 of the plurality of nucleic acid capture regions 4 can be individualized.
  • single cells can be crushed in an individualized state.
  • the aqueous solution is allowed to flow from the inlet 11 toward the outlet 14, whereby mineral oil (nonpolar solvent) is discharged, separation of the solution is eliminated, and the upper space portions 8 of the plurality of nucleic acid capture regions 4. Can be communicated with the aqueous solution.
  • the array device for nucleic acid analysis shown in FIG. 13 has a configuration in which a plurality of nucleic acid capture regions 4 are formed on each of the transparent substrates 5 and 6, but the array device for nucleic acid analysis according to the present invention has this configuration. It is not limited.
  • the array device for nucleic acid analysis may have a configuration in which a plurality of nucleic acid capture regions 4 are formed only on the transparent substrates 5 and 6 that have cell capture means. That is, the array device for nucleic acid analysis includes a transparent substrate 6 having a plurality of nucleic acid capture regions 4 and a cell capture processing unit 1301 formed substantially at the center of the nucleic acid capture region 4 as shown in FIG.
  • the nucleic acid analysis array device configured as described above can reduce the costs associated with the water-repellent process and the process of imparting hydrophilicity.
  • the nucleic acid analysis array device configured in this way makes it easier to introduce and discharge mineral oil without the aqueous solution droplets coming into contact with the transparent substrate 5.
  • the transparent substrate 6 on which the plurality of nucleic acid capture regions 4 having the cell capture processing unit 1201 is formed is flat, but the array for nucleic acid analysis according to the present invention.
  • the device is not limited to this configuration.
  • the array device for nucleic acid analysis may have a plurality of nucleic acid capture regions 4 each having a cell capture processing unit 1201 formed in a recess.
  • the inner surface (that is, the side surface and the bottom surface) constituting the recess is a highly hydrophilic surface, and the DNA probe is immobilized on the bottom surface in the recess.
  • the nucleic acid analysis array device configured in this way makes it easier to introduce and discharge mineral oil without the aqueous solution droplets coming into contact with the transparent substrate 5.
  • the reaction in the array device for nucleic acid analysis is not limited to the single molecule sequence reaction as shown in FIG. 3 or FIGS. That is, the reaction in the array device for nucleic acid analysis, as shown in FIG. 16, performs amplification using one molecule as a seed, and performs gene expression analysis at the single cell level by sequence analysis (sequencing) of this amplification product. It may be a thing. That is, in this example, instead of using a single molecule sequence, after amplification using a single molecule as a seed, sequencing is performed on the cluster of amplified products.
  • the nucleic acid analysis array device used in this example may be the same as that described above.
  • the reaction procedure shown in this example is the same up to (d-1) in FIG. 3 and FIG.
  • the DNA probe has a higher density, for example, a fixed surface density of 1 molecule or more in a 5 nm square.
  • a fixed surface density for example, a fixed surface density of 1 molecule or more in a 5 nm square.
  • immobilization surface density by immobilizing a gel or polymer such as acrylamide on the surface of the plurality of nucleic acid capturing regions 4 and immobilizing a DNA probe on the polymer.
  • one molecule of DNA probe can be fixed to 1 nm square.
  • FIG. 16 (f-1) is the same as FIG. 4 (d-2), and the poly T probe 36 hybridizes to the poly A probe 35 added to the end (the base length of poly T is 30 bases and the 3 ′ end is VN).
  • Bst polymerase with strand displacement activity and substrate (dNTP) are introduced in an amount equal to the internal volume of space 2 of the array device for nucleic acid analysis, and the temperature is set to 42 degrees (temperature near the melting temperature of poly T hybrid). Hold for 2 hours to react.
  • amplification reaction called template wakening reaction (see PNAS, vol. 110, No. 35, (2013) p. 14320-14323), that is, Fig.
  • the materials that can be used as the transparent substrates 5 and 6 and the cell capturing partition 7 are also resin. A variety of materials can be selected. That is. In this example, a transparent cyclic polyolefin used as a resin material with low background fluorescence can be used, but a transparent polycarbonate may be used.
  • the whole cDNA was amplified. As shown in FIGS. 9 and 10, the full-length cDNA was fragmented and then complementary strand synthesis was performed. Needless to say, the amplification step may be performed by synthesizing the sample in the state of f-1).
  • bridge amplification described in Nucleic Acids Res. Vol. 34, 22e (2006) may be used as an amplification method from one molecule.
  • the DNA sequencing method different from the single molecule sequencing reaction as shown in FIG. 3 or FIGS. 9 and 10 has been described.
  • the array device for nucleic acid analysis according to the present invention The nucleic acid analysis apparatus and the nucleic acid analysis method are not limited to the DNA sequencing method.
  • the array device for nucleic acid analysis, the nucleic acid analysis apparatus, and the nucleic acid analysis method according to the present invention include other quantification methods for quantifying DNA sequences, as shown in Nature biotechnology vol. 26, (3), p. 317 (2008), for example. It goes without saying that the method may be used.
  • the control means for communicating or individualizing the upper space portions 8 of the plurality of nucleic acid capture regions 4 compares the nucleic acid capture region with the region surrounding the nucleic acid capture region, the nucleic acid The capture region was to have a high hydrophilic surface.
  • the control means for communicating or individualizing the upper space portions 8 of the plurality of nucleic acid capture regions 4 at a high level is not limited to such a form.
  • the second embodiment has a first substrate on which a plurality of nucleic acid capture regions 4 are formed, and a second substrate disposed at a position facing the first substrate, and the first substrate and the second substrate. Are in contact with or separated from each other, the upper space portions 8 of the plurality of nucleic acid capture regions 4 are communicated with each other or individualized.
  • the array device for nucleic acid analysis includes a first substrate 1701 in which a plurality of nucleic acid capture regions 4 are formed in a recess, and a first substrate 1701 that can be contacted and separated. And a second substrate 1702 provided.
  • a cell capture processing unit 1703 is formed at substantially the center.
  • the cell capture processing unit 1703 is a substantially circular shape having a diameter of 1 ⁇ m, and fibronectin is fixed at a high density.
  • the second substrate 1702 includes an inlet 11 that supplies a liquid to the space 2 and an elastic member 1704.
  • the elastic member 1704 is formed from a rubber material having waterproofness and elasticity. When the movable stage 1705 pushes down the second substrate 1702 inside the elastic member 1704, the second substrate 1702 can come into contact with the first substrate 1701.
  • the upper space of the plurality of nucleic acid capture regions 4 Will be in communication with the solution.
  • the movable stage 1705 pushes down the second substrate 1702 and the second substrate 1702 comes into contact with the first substrate 1701, the upper space portions of the plurality of nucleic acid capture regions 4 are individualized. At this time, excess liquid is discharged from the outlet 14 to the outside.

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Abstract

La présente invention comprend une pluralité de régions de capture d'acides nucléiques qui capturent des molécules d'acides nucléiques et fait appel à la pluralité de régions de capture d'acides nucléiques pour analyser simultanément une pluralité d'échantillons. Un dispositif en réseau pour l'analyse d'acides nucléiques selon la présente invention comprend une base, une pluralité de régions de capture d'acides nucléiques qui sont disposées sur au moins une surface principale de la base et qui comprennent un moyen de capture d'acides nucléiques qui capture des acides nucléiques, et un moyen de commande qui relie mutuellement ou différencie une partie d'espace supérieur de la pluralité de régions de capture d'acides nucléiques.
PCT/JP2014/067344 2014-06-30 2014-06-30 Dispositif en réseau pour l'analyse d'acides nucléiques, dispositif d'analyse d'acides nucléiques, et procédé d'analyse d'acides nucléiques WO2016001963A1 (fr)

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WO2019079593A1 (fr) * 2017-10-19 2019-04-25 Omniome, Inc. Réduction du bruit de fond et stabilisation de complexes simultanées dans des flux de travaux de dosage par liaison
US10975427B2 (en) 2017-01-20 2021-04-13 Omniome, Inc. Process for cognate nucleotide detection in a nucleic acid sequencing workflow

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JP2008539711A (ja) * 2005-05-03 2008-11-20 オックスフォード・ジーン・テクノロジー・アイピー・リミテッド 個々に細胞を解析するための装置及び方法
JP2011516034A (ja) * 2008-02-29 2011-05-26 ノースウエスタン ユニバーシティ 生物反応促進用バリア
WO2011068088A1 (fr) * 2009-12-04 2011-06-09 株式会社日立製作所 MÉTHODE D'ANALYSE DE L'EXPRESSION DE GÈNES UTILISANT UNE BIBLIOTHÈQUE BIDIMENSIONNELLE D'ADNc
WO2014020657A1 (fr) * 2012-07-30 2014-02-06 株式会社日立製作所 Dispositif de banque d'adnc bidimensionnel lié à une séquence étiquette, et procédé d'analyse d'expression génique et appareil d'analyse d'expression génique l'utilisant

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JP2008539711A (ja) * 2005-05-03 2008-11-20 オックスフォード・ジーン・テクノロジー・アイピー・リミテッド 個々に細胞を解析するための装置及び方法
JP2011516034A (ja) * 2008-02-29 2011-05-26 ノースウエスタン ユニバーシティ 生物反応促進用バリア
WO2011068088A1 (fr) * 2009-12-04 2011-06-09 株式会社日立製作所 MÉTHODE D'ANALYSE DE L'EXPRESSION DE GÈNES UTILISANT UNE BIBLIOTHÈQUE BIDIMENSIONNELLE D'ADNc
WO2014020657A1 (fr) * 2012-07-30 2014-02-06 株式会社日立製作所 Dispositif de banque d'adnc bidimensionnel lié à une séquence étiquette, et procédé d'analyse d'expression génique et appareil d'analyse d'expression génique l'utilisant

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10975427B2 (en) 2017-01-20 2021-04-13 Omniome, Inc. Process for cognate nucleotide detection in a nucleic acid sequencing workflow
WO2019079593A1 (fr) * 2017-10-19 2019-04-25 Omniome, Inc. Réduction du bruit de fond et stabilisation de complexes simultanées dans des flux de travaux de dosage par liaison
US11193166B2 (en) 2017-10-19 2021-12-07 Omniome, Inc. Simultaneous background reduction and complex stabilization in binding assay workflows

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