WO2017159763A1 - 核酸の回収方法 - Google Patents
核酸の回収方法 Download PDFInfo
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- WO2017159763A1 WO2017159763A1 PCT/JP2017/010553 JP2017010553W WO2017159763A1 WO 2017159763 A1 WO2017159763 A1 WO 2017159763A1 JP 2017010553 W JP2017010553 W JP 2017010553W WO 2017159763 A1 WO2017159763 A1 WO 2017159763A1
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- nucleic acid
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- adsorbed
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- water
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
Definitions
- the present invention relates to a method for recovering nucleic acid, a carrier in which a water-soluble neutral polymer is adsorbed on the surface of cerium oxide, and a kit for recovering nucleic acid.
- miRNA is a single-stranded RNA having a length of 18 to 25 bases, and is biosynthesized from a pre-miRNA having a length of 60 to 90 bases. These have a function of regulating protein synthesis and gene expression, and thus are considered to be related to diseases, and are attracting attention as targets for gene analysis.
- the first step required for gene analysis is a step of recovering nucleic acid from a biological sample. If the nucleic acid can be recovered with high purity and high yield, highly sensitive gene detection and gene analysis can be performed in the subsequent detection reaction. Typical methods for recovering nucleic acid include phenol / chloroform extraction, ethanol precipitation, and nucleic acid adsorption onto silica.
- the most versatile method is the boom method described in Patent Document 1 in which nucleic acid is adsorbed and eluted by a metal oxide containing silica.
- This method is characterized in that the nucleic acid can be concentrated simultaneously with the recovery of the nucleic acid from the silica adsorbed with the nucleic acid by centrifugation.
- Patent Document 2 when the boom method is used, the adsorptivity of a nucleic acid having a length of not less than 300 bases and not more than 1000 bases is longer than that of a nucleic acid having a longer length. It is described that it is inferior. From these facts, it is expected that it is difficult to recover pre-miRNA and miRNA having a length of 100 bases or less. Since gene analysis is also used in the medical field, a method capable of recovering nucleic acid without using complicated operations and organic solvents is preferable.
- Patent Document 3 describes a method in which a nucleic acid is adsorbed and recovered using a magnetic inorganic component such as iron oxide as a carrier.
- a magnetic inorganic component such as iron oxide
- this method has a problem in that the inorganic component is eluted in the nucleic acid recovery solution and the purity of the recovered nucleic acid is lowered. Therefore, the method of coating the inorganic component carrier with a polymer or metal oxide is also included.
- Patent Document 4 describes a method for increasing the density of a column by increasing the density of the column by adding particles having a large specific gravity such as titania, ceria, zirconia, hafnia, etc., to a silica gel carrier used for an ion exchange column. ing.
- a small fragment of nucleic acid is not adsorbed to a carrier, and pre-miRNA, which is a very short nucleic acid, It is expected that miRNA will be difficult to recover.
- the present inventors In order to efficiently recover from a very short nucleic acid such as pre-miRNA or miRNA to a long nucleic acid such as a genome, the present inventors have used a carrier that adsorbs nucleic acid at a high adsorption rate, and an adsorbed nucleic acid. We thought that it was necessary to find a method that could be eluted efficiently.
- the present inventors have newly found a cerium oxide carrier as a carrier having a high nucleic acid adsorption rate. Furthermore, it was clarified that by adsorbing a water-soluble neutral polymer on the surface, the elution rate of nucleic acid can be improved without reducing the adsorption rate of nucleic acid, and the present invention has been completed.
- the present invention is as follows. (1) A method for recovering nucleic acid from a biological sample, comprising the following steps: Step a) A step in which a water-soluble neutral polymer adsorbed on the surface of cerium oxide is mixed with a solution containing nucleic acid, and the nucleic acid is adsorbed on the carrier.
- Step b) separating the carrier adsorbed with the nucleic acid from the solution mixed in step a); Step c) a step of adding the eluate to the carrier adsorbed with the nucleic acid separated in step b) and recovering the nucleic acid;
- a method for recovering nucleic acid comprising: (2) The nucleic acid recovery method according to (1), wherein the water-soluble neutral polymer is a polymer having a zeta potential of ⁇ 10 mV to +10 mV in a pH 7 solution.
- the polymer is characterized in that it is polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, poly (2-ethyl-2-oxazoline), polypropylene glycol, polyacrylamide, poly N-isopropylacrylamide or hydroxypropylmethylcellulose (The method for recovering a nucleic acid according to 1) or (2). (4) The method for recovering nucleic acid according to any one of (1) to (3), wherein the eluate is a buffer solution.
- the biological sample is blood, urine, saliva, mucous membrane, sweat, cultured cells, cultured cells, cultured cells, tissue samples, specimens, microorganisms, microbial cultures or viruses ( The method for recovering a nucleic acid according to any one of 1) to (4).
- the carrier according to (6), wherein the water-soluble neutral polymer is a polymer having a zeta potential of ⁇ 10 mV to +10 mV in a pH 7 solution.
- the water-soluble neutral polymer is polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, poly (2-ethyl-2-oxazoline), polypropylene glycol, polyacrylamide, poly N-isopropylacrylamide or hydroxypropylmethylcellulose.
- the carrier according to (6) or (7), wherein (9) A kit for recovering nucleic acid, comprising the carrier according to any one of (6) to (8) and a buffer solution.
- nucleic acids when a carrier having a water-soluble neutral polymer adsorbed on the surface of cerium oxide is used, nucleic acids can be recovered in a high yield by a simple method, and pre- Even very short nucleic acids such as miRNA and miRNA can be recovered in high yield.
- the biological sample used in the present invention can be any sample containing nucleic acid.
- the nucleic acid include RNA, DNA, RNA / DNA (chimera), and artificial nucleic acid.
- DNA include cDNA, micro DNA, cell-free DNA, genomic DNA, and synthetic DNA.
- RNA include total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA or non-coding RNA, their precursors or synthetic RNA.
- Synthetic DNA and synthetic RNA can be artificially produced, for example, using an automatic nucleic acid synthesizer based on a predetermined base sequence (which may be either a natural sequence or a non-natural sequence).
- biological samples include, for example, cultured cells, culture solutions of cultured cells, cell-derived samples such as tissue samples and specimens, microorganism-derived samples such as bacteria, fungi, protists and viruses, blood, urine, saliva, Human-derived animal samples such as mucous membranes, sweat, sputum, semen and other human fluids such as stool, and solutions containing compounds with biological functions such as proteins, sugars and lipids can be used in addition to nucleic acids However, it is not limited to these.
- the biological sample is preferably a cultured cell or body fluid, more preferably blood.
- the blood includes whole blood, plasma, serum, blood cells and the like.
- the present invention may be applied as it is after collection, or may be diluted by adding a solution after collection.
- the biological sample is a solid sample such as a cell pellet or tissue piece, it may be diluted with water or a buffer after collection and used in the present invention.
- the biological sample may be processed as follows, if necessary. This is because nucleic acids are encapsulated in biological samples in compounds such as cell membranes, cell walls, vesicles, liposomes, micelles, ribosomes, histones, nuclear membranes, mitochondria, viral capsids, envelopes, endosomes or exosomes. This is because there are many cases that are interacting with each other. In order to recover the nucleic acid with a higher yield, a treatment intended to be liberated from these may be performed.
- the following treatment can be performed in order to increase the efficiency of recovering nucleic acid from a biological sample containing E. coli.
- a mixture of 0.2 M sodium hydroxide and 1% SDS can be added to a biological sample containing E. coli (alkaline denaturation method), or a 10% sarkosyl solution can be added. Yes (non-denaturing method with sarkosyl).
- lysozyme may be added to these solutions.
- the proteinase K can be treated at 37 ° C. for 1 hour.
- sonication can be performed.
- the following treatment can be performed on the biological sample.
- 10% SDS can be added after treatment with zymolyce commercially available from Seikagaku Corporation or Nacalai Tesque Corporation.
- the following treatment can be performed on the biological sample.
- 1% SDS can be added.
- 4M or more of guanidinium chloride, guanidine thiocyanate, urea and the like can be added. You may add sarcosyl to this solution so that it may become 0.5% or more. Further, mercaptoethanol may be added to a concentration of 50 mM or more.
- an inhibitor of a nucleic acid degrading enzyme may be added in order to suppress degradation of the nucleic acid contained in the biological sample.
- EDTA can be added at a concentration of 1 mM or less.
- commercially available RNasin Plus Ribonuclease Inhibitor Promega Corporation
- Ribonclease Inhibitor Takara Bio Inc.
- RNase Inhibitor Toyobo Co., Ltd.
- DNA and RNA When DNA and RNA are mixed in a biological sample, it can be separated by phenol / chloroform extraction. For example, when phenol / chloroform extraction is performed under acidic conditions, RNA is separated into an aqueous phase and DNA is separated into a chloroform phase, and when neutral conditions are performed, RNA and DNA are distributed into an aqueous phase. Using this property, conditions can be selected according to the type of nucleic acid to be obtained.
- the above chloroform can be substituted with p-bromoanisole.
- Phenol / chloroform extraction is performed using commercially available reagents such as ISOGEN (registered trademark: Nippon Gene Co., Ltd.), TRIzol (registered trademark: Life Technologies Japan Co., Ltd.), RNAiso (Takara Bio Inc.), 3D-Gene (registered trademark) RNA extraction reagent. From liquid sample kit (Toray Industries, Inc.) can also be used. The above processing may be performed only in one step, or may be combined with steps in other operations. Further, the concentration of the solution used therefor can be changed as necessary.
- the nucleic acid-containing solution includes a nucleic acid, an artificial nucleic acid, a solution in which a nucleic acid modified with a dye or a phosphate group is dissolved, or a liquid sample such as a body fluid when using a biological sample, Diluents, dilutions of solid samples such as cell pellets and tissue pieces can be used.
- a solution obtained after performing any of the above treatments on a liquid sample or a biological sample including a solid sample may be used as it is, or may be diluted as necessary.
- the solution to dilute is not specifically limited, It is preferable to use the solution generally used for the solution containing nucleic acids, such as water and a Tris-hydrochloric acid buffer.
- the solution containing the nucleic acid is preferably a biological sample to which, for example, 4M or more of guanidinium chloride, guanidine thiocyanate or urea is added.
- the length of the nucleic acid to be recovered is not particularly limited, and it can be recovered in a high yield from a short nucleic acid having a length of several tens of bases to a long nucleic acid such as a genome.
- short-chain nucleic acids having a length of 1000 bases or less which were difficult with conventional techniques, can be recovered in high yield, and pre-miRNA and miRNAs having a length of 100 bases or less can also be recovered in high yield.
- the carrier of the present invention is a carrier in which a water-soluble neutral polymer is adsorbed on the surface of cerium oxide.
- the carrier of the present invention is a carrier in which a water-soluble neutral polymer is adsorbed on the surface of cerium oxide.
- the nucleic acid recovery using the carrier of the present invention is characterized in that the nucleic acid is first adsorbed on the carrier of the present invention, and the carrier of the present invention has excellent nucleic acid adsorption ability.
- the nucleic acid adsorption ability of the carrier of the present invention can be evaluated by the adsorption rate of the nucleic acid adsorbed on the carrier of the present invention, and can be calculated as follows. First, the amount of nucleic acid in a solution containing nucleic acid is calculated.
- the carrier of the present invention and a solution containing nucleic acid are mixed, the amount of nucleic acid in the mixture after the nucleic acid is adsorbed on the carrier of the present invention is calculated, and the difference from the amount of nucleic acid in the solution containing nucleic acid is calculated.
- Ask. The obtained value is used as the amount of nucleic acid adsorbed on the carrier of the present invention, and the nucleic acid adsorption rate can be calculated by dividing the amount of nucleic acid adsorbed on the carrier of the present invention by the amount of nucleic acid in the solution containing the nucleic acid.
- Examples of the method for quantifying the amount of nucleic acid include absorbance measurement, fluorescence measurement, luminescence measurement, electrophoresis, PCR, RT-PCR, analysis using a microarray, analysis using a sequencer, and the like.
- the amount of nucleic acid can be quantified by measuring the absorbance at 260 nm.
- the fluorescent dye is a modified nucleic acid
- the amount of nucleic acid can be quantified by comparing the fluorescence intensity derived from the fluorescent dye with the fluorescence intensity in a solution having a known concentration.
- it can carry out by electrophoresis.
- the method of calculating the collection rate by electrophoresis can be determined by running a sample that has been collected simultaneously with a sample of known concentration, staining the gel, and comparing the band concentration by image analysis.
- the nucleic acid is recovered by eluting the nucleic acid adsorbed on the carrier of the present invention from the carrier with the eluent. Elution ability.
- the nucleic acid elution ability of the carrier of the present invention can be evaluated by the nucleic acid elution rate from the carrier of the present invention to the eluate, and can be determined as follows. The eluate is added to the carrier of the present invention to which the nucleic acid has been adsorbed, the amount of nucleic acid in the solution after elution is calculated, and the amount of nucleic acid eluted is calculated.
- the elution rate can be calculated by dividing the nucleic acid elution amount by the nucleic acid amount adsorbed on the carrier of the present invention calculated above.
- the nucleic acid recovery ability in the nucleic acid recovery method of the present invention can be evaluated by the nucleic acid recovery rate calculated by the product of the nucleic acid adsorption rate and elution rate calculated by the above method.
- nucleic acid recovery method of the present invention can be compared by comparing the yield of nucleic acid recovered by the method of the present invention and the yield of nucleic acid recovered by a method other than the method of the present invention without quantifying the yield of nucleic acid.
- the nucleic acid recovery ability of can be evaluated.
- Examples of a method for recovering nucleic acid other than the method of the present invention include a method for recovering using a commercially available kit.
- a specific method for comparing the recovered amount of nucleic acid first, nucleic acid is adsorbed and eluted from the same amount of biological sample using the carrier of the present invention and a carrier other than the carrier of the present invention. Recover.
- the volumes of the eluates may be different or the same, but are preferably the same. If they are different, the recovered nucleic acid is diluted or concentrated to the same volume. Both samples are electrophoresed, the gel is stained, an image is obtained with a fluorescence scanner, etc., and the concentration of the band corresponding to the target nucleic acid fraction is compared by image analysis. The yield of nucleic acid recovered by a method other than the method of the present invention can be compared.
- a polymer is a general term for a monomer that is a basic unit or a compound in which a large number of repeating units called monomers are connected.
- the polymer used for the carrier of the present invention includes both a homopolymer composed of one kind of monomer and a copolymer composed of two or more kinds of monomers, and also includes a polymer having an arbitrary degree of polymerization.
- either a natural polymer or a synthetic polymer is included in the polymer used in the carrier of the present invention.
- the water-soluble neutral polymer used for the carrier of the present invention has a property of being soluble in water, and the solubility in water is at least 0.0001 wt% or more, preferably 0.001 wt% or more.
- the polymer is preferably 0.01 wt% or more, more preferably 0.1 wt% or more.
- the water-soluble neutral polymer used in the present invention is preferably a polymer having a zeta potential of ⁇ 10 mV to +10 mV in a pH 7 solution.
- the polymer is more preferably ⁇ 8 mV or more and +8 mV or less, further preferably ⁇ 7 mV or more and +7 mV or less, and particularly preferably ⁇ 4.0 mV or more and +6.5 mV or less.
- Zeta potential is one of the values representing the electrical properties of the colloidal interface in the solution.
- an electric double layer is formed on the surface of the colloid by counter ions for the surface charge of the colloid.
- the potential of the colloid surface at this time is called a surface potential. Since the electric double layer is formed by electrostatic interaction of the surface charge of the colloid, ions are more strongly fixed toward the colloid side.
- a layer in which counter ions are strongly fixed to the colloid surface by electrostatic interaction is called a fixed layer, and a potential of the fixed layer is called a fixed potential.
- a slip surface or a slip surface there is a boundary surface that moves together with the colloid due to the viscosity of the solution outside the fixed layer as viewed from the colloid.
- this slip surface potential is defined as the zeta potential.
- the zeta potential changes depending on the surface charge of the colloid, and the surface charge changes due to the attachment / detachment of protons depending on the pH, the value in a solution of pH 7 is used as a reference in the present invention.
- the distance to the slip surface is generally smaller than the size of the colloid, the surface of the colloid can be expressed approximately as a slip surface.
- the surface potential of the colloid dispersed in the solution can be regarded as the zeta potential.
- the value measured by laser Doppler electrophoresis is used as the zeta potential of the present invention.
- the zeta potential can be measured by using a zeta potential measuring device.
- Zeta potential measurement devices are available from Otsuka Electronics Co., Ltd., Malvern Instruments Ltd. , Ranko Brother Ltd. , PenKem Inc. Etc. are commercially available.
- a polymer solution When measuring the zeta potential of a polymer, a polymer solution can be prepared as a colloidal dispersion solution and the zeta potential can be measured.
- a polymer solution is prepared by dissolving a polymer in an electrolyte such as phosphate buffer, sodium chloride solution, or citrate buffer, and measurement is performed by detecting scattered light or reflected light of the polymer dispersed in the solution. Do. As the size of the colloid increases, scattered light and reflected light can be detected at a lower concentration.
- the condition for measuring the zeta potential of the polymer by the laser Doppler method is to dissolve the polymer in a phosphate buffer solution (10 mM, pH 7) so that the concentration of the polymer is 0.1 wt% or more and 10 wt% or less.
- a zeta potential measuring device based on the principle of laser Doppler electrophoresis and measured at room temperature.
- a zeta potential measuring device ELS-Z from Otsuka Electronics Co., Ltd. can be used.
- water-soluble neutral polymer used in the carrier of the present invention include the following.
- polyvinyl polymer such as polyvinyl alcohol or polyvinyl pyrrolidone
- polyacrylamide polymer such as polyacrylamide, poly (N-isopropylacrylamide) or poly (N- (hydroxymethyl) acrylamide
- polyethylene glycol polypropylene glycol or polytetramethylene ether
- Polyalkylene glycol polymers such as glycol, poly (2-ethyl-2-oxazoline), (hydroxypropyl) methylcellulose, methylcellulose, ethylcellulose, 2-hydroxyethylcellulose, hydroxypropylcellulose, and other celluloses can be used.
- a copolymer containing the above-mentioned polymer can also be used.
- polysaccharides such as ficoll, agarose, chitin and dextran or polysaccharide analogues and proteins and peptides such as albumin are also included in the water-soluble neutral polymer of the present invention.
- a part of the functional group of the water-soluble neutral polymer may be ionized, substituted with a positive or negative functional group, or a functional group that exhibits water solubility such as an acetyl group may be introduced into the side chain.
- a polymer of 0.4 kDa or more and 1000 kDa or less can be preferably used, more preferably 2 kDa or more and 500 kDa or less, more preferably 4 kDa or more and 150 kDa or less, and further preferably 6 kDa or more and 150 kDa or less.
- it is most preferably 6 kDa or more and 10 kDa or less.
- the value measured by gel permeation chromatography is used as the molecular weight.
- the GPC apparatus can be measured, for example, by using an apparatus such as EcoSEC HLC-8320 GPC manufactured by Tosoh Corporation and using a TSK-gel ⁇ column manufactured by Tosoh Corporation.
- the cerium oxide used for the carrier of the present invention is an amphoteric oxide represented by a composition formula of CeO 2 and is also called ceria.
- cerium oxide a naturally occurring product or an industrially produced product may be used.
- the method for producing cerium oxide include a method of thermally decomposing cerium oxide oxalate and carbonate, and a cerium hydroxide oxide precipitate obtained by neutralizing an aqueous solution of cerium oxide nitrate. The method etc. are mentioned.
- Industrially produced cerium oxide can be obtained from a reagent manufacturer, a catalyst chemical manufacturer, a reference catalyst section of the General Catalysis Society of Japan, and the like.
- the cerium oxide used in the carrier of the present invention is preferably granular. Even when the particle diameters are uniform, different particle diameters may be mixed and used.
- the particle size is not limited so long as the mixture of water and cerium oxide particles is centrifuged at 6000 G for 1 minute, so that the particles of cerium oxide precipitate.
- cerium oxide of 100 ⁇ m or less can be preferably used.
- cerium oxide of 50 ⁇ m or less can be used, and more preferably, cerium oxide of 10 ⁇ m or less can be used.
- the particle size is calculated as a value of 50% diameter (D50, median diameter) from the frequency distribution of the equivalent sphere diameter obtained as a result of measurement using a particle size distribution measuring apparatus based on the laser diffraction / scattering method. be able to.
- the eluate used in the present invention is not particularly limited as long as the nucleic acid adsorbed on the carrier of the present invention can be eluted, but a buffer solution is preferable, and the buffer solution may contain a chelating agent.
- EDTA was added to a citrate buffer containing citric acid and sodium citrate, a phosphate buffer containing phosphoric acid and sodium phosphate, or a Tris-hydrochloric acid buffer containing trishydroxyaminomethane and hydrochloric acid. Examples include Tris-EDTA buffer.
- the pH of the buffer solution is preferably pH 4 or more and pH 9 or less, more preferably pH 5 or more and pH 8 or less.
- the chelating agent contained in the buffer solution has a ligand having a plurality of coordination sites, and a substance that binds to a metal ion and forms a complex can be used.
- the chelating agent examples include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), glycol etherdiaminetetraacetic acid (EGTA), polyphosphoric acid, metaphosphoric acid and / or a salt thereof.
- EDTA ethylenediaminetetraacetic acid
- NTA nitrilotriacetic acid
- EGTA glycol etherdiaminetetraacetic acid
- polyphosphoric acid metaphosphoric acid and / or a salt thereof.
- the final concentration of the chelating agent is not particularly limited, it may be 50 mM or more, preferably 100 mM or more, more preferably 500 mM or more.
- examples of the compound that becomes a chelating agent other than the above include anionic polymers. Since a polymer having a carboxylic acid in the side chain coordinates a metal ion, these may be contained in a buffer solution. Examples of the polymer having such a function include polyvinyl sulfonic acid and / or a salt thereof. The final concentration is not particularly limited, but may be 1 wt% or more, preferably 10 wt% or more.
- the present invention is a method for recovering nucleic acid from a biological sample, comprising: a) mixing a carrier having a water-soluble neutral polymer adsorbed on the surface of cerium oxide with a solution containing the nucleic acid, and adsorbing the nucleic acid to the carrier; A step of separating the carrier adsorbed with the nucleic acid from the solution mixed in step a), a step c) a step of collecting the nucleic acid by adding an eluate to the carrier adsorbed with the nucleic acid in step c) including.
- a carrier having a water-soluble neutral polymer adsorbed on the surface of cerium oxide with a solution containing the nucleic acid, and adsorbing the nucleic acid to the carrier
- a step of separating the carrier adsorbed with the nucleic acid from the solution mixed in step a) a step c) a step of collecting the nucleic acid by adding an eluate to the carrier adsorbed with the nucleic acid in step
- the carrier of the present invention is prepared by adsorbing a water-soluble neutral polymer on the surface of cerium oxide.
- the polymer may not be adsorbed on the surface of cerium oxide with a uniform thickness.
- cerium oxide may be washed in advance with a solution such as water or ethanol to remove impurities adsorbed on the surface, or this washing operation may be omitted. Good.
- Examples of the method of adsorbing the water-soluble neutral polymer on the cerium oxide surface include a method of dissolving the water-soluble neutral polymer to prepare a water-soluble neutral polymer solution and bringing it into contact with cerium oxide. Specifically, cerium oxide is immersed in a water-soluble neutral polymer solution, a water-soluble neutral polymer solution is dropped onto cerium oxide, a water-soluble polymer solution is applied to cerium oxide, The polymer solution can be atomized and sprayed onto cerium oxide.
- the method of immersing cerium oxide in a water-soluble neutral polymer solution is not particularly limited.
- the solution may be left standing or stirred.
- a method of stirring with a dispersing machine such as pipetting, inversion mixing, a stirrer, a mixer, a vortex, or a mill, or an ultrasonic treatment apparatus can be used.
- the concentration of the water-soluble neutral polymer is not particularly limited, but is preferably 0.01 wt% or more, and more preferably 0.1 wt% or more.
- the immersion time for standing is not particularly limited, but is preferably 5 minutes or longer.
- the stirring time for stirring is not particularly limited as long as the water-soluble neutral polymer and cerium oxide are uniformly mixed. In the case of vortexing, stirring is preferably performed for 1 minute or more, preferably 5 minutes or more.
- cerium oxide it is also possible to dip coat the surface of cerium oxide with a water-soluble neutral polymer using a sieve or a sieve.
- the mixing time when immersed in the solution is preferably 5 minutes or more, more preferably 30 minutes or more if the polymer concentration is 0.1 wt% or more.
- a dropper When dropping a water-soluble neutral polymer solution, a dropper, a dropping funnel, or the like can be used.
- cerium oxide When the polymer solution is dropped, cerium oxide may be vibrated or rotated, and a spin coater or the like may be used.
- a brush, a roller, or a wire bar When applying a water-soluble neutral polymer solution, a brush, a roller, or a wire bar can be used.
- an air spray or an air brush can be used.
- the centrifugation may be performed to remove the polymer solution as a supernatant, or the centrifugation may not be performed. It may be used for nucleic acid recovery.
- a water-soluble neutral polymer solution is dissolved in a solvent, the water-soluble neutral polymer is adsorbed on cerium oxide, and after removing the solvent, the solution may be dried or without drying. It may be used for recovery.
- the obtained carrier of the present invention may be prepared and stored, or may be prepared and used at the time of use.
- the water-soluble neutral polymer solution can be prepared by dissolving in water or an organic solvent if the obtained water-soluble neutral polymer is solid, and can be prepared by diluting if it is a solution.
- heat treatment or ultrasonic treatment may be performed.
- the organic solvent it is preferable to use an organic solvent that is miscible with water, such as ethanol, acetonitrile, methanol, propanol, tert-butanol, DMF, DMSO, acetone, ethylene glycol, and glycerol.
- an organic solvent that is miscible with water such as ethanol, acetonitrile, methanol, propanol, tert-butanol, DMF, DMSO, acetone, ethylene glycol, and glycerol.
- dissolve in water you may add said organic solvent.
- a carrier prepared by covalently bonding cerium oxide and a water-soluble neutral polymer with a linker molecule or the like does not correspond to the carrier of the present invention.
- Specific examples of the linker molecule include a silane coupling agent.
- Step a) is a step of mixing the carrier of the present invention produced by the above production method and a solution containing the nucleic acid to adsorb the nucleic acid to the carrier of the present invention.
- the method for mixing the solution containing the carrier of the present invention and the nucleic acid is not particularly limited.
- pipetting or inversion mixing may be used, or an apparatus such as a mixer or vortex may be used.
- the mixing time is not particularly limited, but may be about 5 minutes, or may be mixed for a longer time.
- the carrier of the present invention may be packed in a column and passed through a solution containing nucleic acid.
- Step b) is a step of separating the carrier of the present invention adsorbed with the nucleic acid from the solution mixed in step a).
- the separation method include a method of centrifuging the solution mixed in step a), precipitating the carrier of the present invention adsorbed with nucleic acid, and removing the supernatant. Since the specific gravity of the carrier of the present invention to which nucleic acid is adsorbed is heavier than water, it can be easily precipitated by centrifugation. Centrifugation may be performed at 6000 G for 1 minute, and more preferably at 10000 G for 1 minute. Examples of other separation methods include a method using an ultrafiltration membrane.
- the solution mixed in step a) is passed through an ultrafiltration membrane having a pore size smaller than the particle size of the carrier on which the nucleic acid is adsorbed, and the carrier on which the nucleic acid has been adsorbed is separated.
- an ultrafiltration membrane is made into a kit, and a centrifugal filtration kit represented by Ultra Free (registered trademark) of Merck Co., Ltd. and NanoCep (registered trademark) of Pall Corporation can be obtained and used.
- step b the following processing may be performed as necessary.
- a biological sample-derived material other than the target nucleic acid may be adsorbed on the surface of the carrier of the present invention.
- washing and decomposition treatment can be performed.
- washing with water to remove non-specifically adsorbed compounds washing with surfactants to remove non-specifically adsorbed proteins, to remove ions and low-molecular compounds
- washing with a solution containing a surfactant washing with an organic solvent to remove nonspecifically adsorbed hydrophobic compounds, adding a proteolytic enzyme to degrade nonspecifically adsorbed proteins
- Various treatments can be performed such as adding RNase to isolate only DNA and adding RNase to isolate only RNA.
- Step c) is a step of collecting the nucleic acid by adding an eluate to the carrier of the present invention on which the nucleic acid separated in step b) has been adsorbed.
- the eluate is added to the carrier adsorbed with nucleic acid in step c).
- Examples include a method of centrifuging the obtained mixture, precipitating the carrier of the present invention, and obtaining a supernatant from which nucleic acid is eluted. Since the specific gravity of the carrier of the present invention is heavier than that of water, it can be easily precipitated by centrifugation. The centrifugation may be performed at 6000 G for 1 minute, and preferably at 10000 G for 1 minute.
- Other separation methods include a method using an ultrafiltration membrane.
- the mixture obtained in step c) is passed through an ultrafiltration membrane having a pore size smaller than the particle size of the carrier of the present invention to separate the carrier of the present invention.
- an ultrafiltration membrane is made into a kit, and a centrifugal filtration kit represented by Ultra Free (registered trademark) of Merck Co., Ltd. and NanoCep (registered trademark) of Pall Corporation can be obtained and used.
- the collected nucleic acid can be chemically modified as necessary.
- Chemical modifications include fluorescent dye modification, quencher modification, biotin modification, amination, carboxylation, maleimidation, succinimidation, phosphorylation and dephosphorylation, etc. on the end of nucleic acid, and others by intercalator Dyeing. These modifications may be introduced by chemical reaction or by enzymatic reaction. Rather than introducing these modifying groups before quantification and quantifying the recovered nucleic acid itself, it is possible to indirectly quantitate nucleic acids by quantifying the modified groups introduced through chemical modification. . Nucleic acids are recovered according to the present invention. In particular, even short-chain nucleic acids are recovered with a high yield, so that the above quantification can be performed with high sensitivity.
- the nucleic acid recovery kit of the present invention can be used for efficiently recovering nucleic acids from biological samples.
- the nucleic acid recovery kit of the present invention includes the carrier of the present invention and a buffer as its constituent components. Moreover, the description etc. may be contained in addition to these.
- the carrier of the present invention contained in the kit for nucleic acid recovery of the present invention may be in a dry state or in a state of being immersed in a water-soluble neutral polymer solution.
- a buffer solution that can be used for the eluate of the above step c) can be used.
- Polyethylene glycol was obtained from Merck Co., Ltd., poly (2-ethyl-2-oxazoline) from Alfa Aesar, A Johnson Matthey Company, and cerium oxide (particle size 4.4 ⁇ m) from Daiichi Rare Element Chemical Industries, Ltd.
- the aqueous polymer solution used in the examples was dissolved in water or a mixed solvent of water and ethanol at each concentration.
- cerium oxide was used for experiments without being screened.
- plasmid DNA (2.7 kbp) pUC19 and E. coli E. coli.
- E. coli DH5 ⁇ Competent Cells is from Takara Bio Inc., ethidium bromide, LB medium LB Miller, RPMI medium RPMI 1640 (containing L-glutamine), and zymolyce from Nacalai Tesque Co., Ltd., Buffer RLT, Buffer RLT PlusB, NLT & Tissue Kit from Qiagen, MagMax cell-FreeDNA Isolation kit and TrypLE Express from Thermo Fisher Co., Fetal Bovine Serum Sterile Filtered from Equich Chemical Bio Co., Ltd. Purchased.
- the YPD medium was prepared by mixing 1% Yeast extract (Becton Dickinson Co., Ltd.), 2% Polypetone (Becton Dickinson Co., Ltd.) and 2% D-glucose (Wako Pure Chemical Industries, Ltd.).
- 22-base long nucleic acids known as miRNA let7a sequences were converted to DNA sequences and synthesized as RNA sequences from Eurofin Genomics.
- RNA 22 a synthetic nucleic acid having an RNA sequence
- DNA 22 a synthetic nucleic acid having a DNA sequence
- DNA566 The 566 bp DNA sequence used in the examples was obtained by amplification by a PCR reaction.
- DNA566 An E. coli genomic fragment having a length of 10 kbp or more was obtained from E. coli DH5 ⁇ .
- Other reagents were purchased from Wako Pure Chemicals, Tokyo Kasei Co., Ltd., and Sigma Aldrich Japan GK, and used as they were without purification.
- the mixer is CUTE MIXER CM-1000 from Tokyo Rika Kikai Co., Ltd.
- the fluorometer is Nanodrop 3300 from Thermo Fisher Scientific Co., Ltd. and FLUOROMAX-3 from Horiba Ltd.
- ELS-Z from Otsuka Electronics Co., Ltd. is used to measure zeta potential.
- agarose gel electrophoresis Advanced-eXU of Advance Co., Ltd. was used, and for acrylamide gel electrophoresis, a mini gel slab electrophoresis apparatus of As One Co., Ltd. was used.
- the stained agarose gel was analyzed using Typhoon 9410 from GE Healthcare Japan, which is a fluorescent scanner, and the stained acrylamide gel was analyzed using Typhoon FLA 9500 from GE Healthcare Japan, which is a fluorescent scanner.
- ImageQuant trademark registration
- ImageQuant TL trademark registration
- An iron oxide ⁇ -iron oxide manufactured by Wako Pure Chemical Industries, Ltd. was used.
- a 6M guanidine thiocyanate aqueous solution was used as a nucleic acid solution, and a TE buffer solution or water was used as an eluate.
- the adsorption rate of nucleic acid on the nucleic acid carrier was calculated as follows by fluorescence measurement of Cy3. First, the fluorescence intensity of 100 ⁇ l of 6M guanidine thiocyanate aqueous solution in which 100 pmol of DNA 22 was dissolved before adding cerium oxide was measured, and then the fluorescence intensity after adding and mixing cerium oxide was measured. The fluorescence intensity after adding cerium oxide was divided by the fluorescence intensity before addition, and the product of the amount of nucleic acid before addition (100 pmol) was taken to calculate the amount of nucleic acid in the solution. The difference between these values was calculated from the amount of nucleic acid before addition (100 pmol), and the amount of adsorbed nucleic acid was calculated. The amount of adsorbed nucleic acid was divided by the amount of nucleic acid before adding cerium oxide (100 pmol), and the adsorption rate was calculated.
- the elution rate was calculated as follows by fluorescence measurement of Cy3. 50 ⁇ l of TE buffer was added to cerium oxide adsorbed with nucleic acid, and fluorescence was measured on the eluate after elution. Next, water and TE buffer solution in which 100 pmol of DNA 22 was dissolved were prepared, and fluorescence measurement was performed on each of the solutions. The fluorescence intensity of the eluate was divided by the fluorescence intensity of this solution, and the amount of nucleic acid eluted was calculated. The eluted nucleic acid amount was divided by the adsorbed nucleic acid amount to calculate the elution rate. The recovery rate was calculated by taking the product of the calculated adsorption rate and elution rate. The results are shown in Table 1.
- Patent Document 3 describes a method of coating an inorganic component having magnetism with a metal oxide, but when a metal oxide whose surface is coated with cerium oxide is used as a carrier, Comparative Example 2 is used. Similar to the results, a low recovery rate is expected.
- ⁇ Comparative Example 4 Nucleic acid recovery using cerium oxide adsorbed with each water-soluble polymer other than water-soluble neutral polymer as a carrier Other than the water-soluble neutral polymer prepared in Comparative Example 3 in a 1.5 ml tube
- water-soluble polymers polyacrylic acid (PAcA, 5.1 kDa, 10 wt%), polystyrene sulfonic acid (PSS, 7.5 kDa, 1 wt%), polyvinyl sulfonic acid (PVSA, 10 wt%), polyallylamine (PAA, 17 kDa) , 10 wt%), 0.5 mg each of cerium oxide adsorbed with poly-L-lysine (PLL, 150 kDa, 1 wt%) was weighed and used as a carrier.
- the eluate used was a phosphate buffer (0.5 M, pH 8). Conditions and operations other than the carrier and the eluate were performed in the same manner as in Comparative Example 1, and the nu
- Example 1 Preparation of carrier having water-soluble neutral polymer adsorbed on the surface of cerium oxide 0.5 mg of cerium oxide was weighed into a 1.5 ml tube. To this, as a polymer aqueous solution, polyvinyl alcohol (11% acetylated, PVA, 18 kDa, 10 wt%), poly (2-ethyl-2-oxazoline) (PEOz, 5 kDa, 10 wt%), which is a water-soluble neutral polymer, 50 ⁇ l each of polyethylene glycol (PEG, 10 kDa, 10 wt%), polyacrylamide (PAAm, 40 kDa, 10 wt%), hydroxypropylmethylcellulose (HPMC, 10 kDa, 10 wt%), polyvinylpyrrolidone (PVP, 10 kDa, 10 wt%) added.
- PVA polyvinyl alcohol
- PEOz poly (2-ethyl-2-oxazoline)
- HPMC
- Example 2 Nucleic acid recovery using a carrier in which a water-soluble neutral polymer is adsorbed on the surface of cerium oxide
- polyvinyl alcohol, poly 0.5 mg of cerium oxide adsorbed (2-ethyl-2-oxazoline), polyethylene glycol, polypropylene glycol, polyacrylamide, poly N-isopropylacrylamide, hydroxypropylmethylcellulose) and polyvinylpyrrolidone was weighed and used as a carrier.
- Other conditions and operations were performed in the same manner as in Comparative Example 4, and the nucleic acid adsorption rate, elution rate, and recovery rate were calculated. The results are shown in Table 3.
- the zeta potential of the water-soluble polymer other than the water-soluble neutral polymer used in Comparative Example 4 was ⁇ 17 mV or less, or +11 mV or more.
- Example 3 Measurement of zeta potential of water-soluble neutral polymer
- -2-oxazoline polyethylene glycol, polypropylene glycol, polyacrylamide, poly N-isopropylacrylamide, hydroxypropylmethylcellulose, and polyvinylpyrrolidone are dissolved in phosphate buffer (10 mM, pH 7), and zeta is prepared in the same manner as in Comparative Example 5.
- the potential was measured.
- Table 4 shows the correlation between the zeta potential obtained by this measurement and the recovery rate of DNA22 using the cerium oxide adsorbed by each polymer as a carrier (result of Example 2). They are arranged.
- the zeta potential of the water-soluble neutral polymer whose nucleic acid recovery rate was improved in Example 2 was ⁇ 4 mV or more and +6.5 mV or less, and ⁇ 17 mV or less and +11 mV or more in the pH 7 solution. It was found that the recovery rate was improved as compared with a water-soluble polymer having a potential.
- Example 4 Relationship between elution of nucleic acid adsorbed on carrier adsorbed with water-soluble neutral polymer on the surface of cerium oxide and eluate
- a carrier having polyethylene glycol adsorbed on the surface of cerium oxide was prepared. 0.5 mg was weighed into a 1.5 ml tube.
- 0.5M citrate buffer (pH 5, 6), 0.5M phosphate buffer (pH 7, 8), 0.5M Tris-EDTA buffer (0.5M Tris-HCl, 0.5M) EDTA, pH 8) was used respectively.
- Other conditions and operations were performed in the same manner as in Comparative Example 1, and the nucleic acid adsorption rate, elution rate, and recovery rate were calculated. The results are shown in Table 5.
- Example 5 Relationship between nucleic acid recovery rate and nucleic acid length using a carrier having a water-soluble neutral polymer adsorbed on the surface of cerium oxide
- a carrier having polyethylene glycol adsorbed on the surface of cerium oxide Prepared and weighed 0.5 mg into a 1.5 ml tube.
- 100 ⁇ l of 6 M guanidine thiocyanate aqueous solution in which 250 ng of DNA 566, 250 ng of pUC19 (2.7 kbp), and 250 ng of E. coli genomic fragment (> 10 kbp) were respectively dissolved was used.
- Other conditions and operations were performed in the same manner as in Comparative Example 3, and the nucleic acid recovery rate was calculated by electrophoresis. The results are shown in Table 6.
- nucleic acids having any length can be efficiently recovered by using a carrier having polyethylene glycol adsorbed on the surface of cerium oxide, which is a water-soluble neutral polymer.
- Example 6 Nucleic acid recovery from fetal bovine serum A carrier having polyethylene glycol adsorbed on the surface of cerium oxide was prepared according to Example 1, and 2.5 mg was weighed into a 1.5 ml tube.
- a solution containing nucleic acid a mixed solution of 100 ⁇ l of 6M guanidine thiocyanate aqueous solution in which 100 pmol of DNA22 was dissolved and 100 ⁇ l of fetal bovine serum having a protein concentration of 30 mg / ml was used.
- Other conditions and operations were performed in the same manner as in Comparative Example 4, and the nucleic acid adsorption rate, elution rate, and recovery rate were calculated.
- a similar experiment was performed on RNA22. The results are shown in Table 7. The protein concentration in the collected liquid was below the detection limit of the Bradford test (0.25 mg / ml or less).
- Example 7 Effect of molecular weight of water-soluble neutral polymer adsorbed on cerium oxide surface
- Polyethylene glycol having molecular weights of 6 kDa, 10 kDa and 500 kDa and molecular weights of 18 kDa, 40 kDa and 150 kDa (all 11% acetylated)
- Polyvinyl alcohol was prepared to 10 wt% each and used as a polymer solution.
- a carrier having polyethylene glycol adsorbed on the surface of each molecular weight of cerium oxide was prepared and used.
- Other conditions and operations were performed in the same manner as in Comparative Example 4, and the nucleic acid adsorption rate, elution rate, and recovery rate were calculated. The results are shown in Table 8.
- nucleic acid can be recovered with any polymer having any molecular weight.
- Example 8 Relationship between the concentration of the water-soluble neutral polymer and the stirring time in the carrier production method of the present invention 0.5 mg of cerium oxide was weighed into a 1.5 ml tube.
- polyethylene glycol (PEG, 10 kDa) which is a water-soluble neutral polymer, was added at a concentration of 0.1 wt%, 1 wt%, and 10 wt%, 50 ⁇ l each. Each concentration was stirred for 1 minute, 5 minutes, and 30 minutes with a mixer. The supernatant was removed by centrifugation (10000 G, 1 min) with a centrifuge to obtain a carrier having polyethylene glycol adsorbed on the surface of cerium oxide. Moreover, it carried out similarly to the comparative example 4, and computed the recovery rate of the nucleic acid. The results are shown in Table 9.
- Example 9 Relationship between concentration of water-soluble neutral polymer and dipping time in the carrier production method of the present invention 0.5 mg of cerium oxide was weighed into a 1.5 ml tube. As a polymer aqueous solution, polyethylene glycol (PEG, 10 kDa), which is a water-soluble neutral polymer, is added at a concentration of 0.1 wt%, 1 wt%, and 10 wt% to each 50 ⁇ l, and stirring operations such as pipetting are performed. It was not carried out at all and left still for 5 minutes and 30 minutes, respectively.
- PEG polyethylene glycol
- the supernatant was removed by centrifugation (10000 G, 1 min) with a centrifuge to obtain a carrier having polyethylene glycol adsorbed on the surface of cerium oxide. Moreover, it carried out similarly to the comparative example 4, and calculated
- Example 10 Relationship between presence / absence of centrifugation operation in production of carrier of the present invention and recovery rate of nucleic acid 0.5 mg of cerium oxide was weighed into a 1.5 ml tube. To this, 50 ⁇ l of polyethylene glycol (PEG, 10 kDa), which is a water-soluble neutral polymer, was added as a polymer aqueous solution at a concentration of 10 wt%, and stirred for 10 minutes with a mixer. As the subsequent operation, in Example 1, the centrifugation operation using a centrifuge and the operation for removing the supernatant were performed, but in Example 10, these operations were not performed. Except for using the carrier thus prepared, the same procedure as in Comparative Example 4 was carried out, and the nucleic acid adsorption rate, elution rate, and recovery rate were calculated. Table 11 shows the results.
- PEG polyethylene glycol
- Example 11 Cell-free DNA recovery from human plasma using the carrier of the present invention 300 ⁇ l of human plasma was weighed into two 1.5 ml tubes, 450 ⁇ l of Buffer RLT was added and mixed by pipetting. Of the obtained two samples, the first sample was collected using the carrier of the present invention, and the second sample was collected using a commercially available kit as a control.
- cell-free DNA was collected from the second sample according to the protocol using a commercially available kit (MagMax cell-FreeDNA Isolation kit, Thermo Fisher Co., Ltd.). At this time, the volume of the eluate was 50 ⁇ l.
- the cell-free DNA eluate collected with the carrier of the present invention and a commercially available kit was electrophoresed (10% acrylamide, 100 V, 35 min) and stained with SYBR Gold.
- the fluorescence image of the gel was measured with a fluorescence scanner, and the band density ratio of the cell-free DNA fraction (160 to 200 bp) was determined by image analysis. Since the absolute amount of cell-free DNA contained in human plasma is unknown, the ratio of the recovery amount between the carrier of the present invention and the commercial kit was calculated instead of the recovery rate. The results are shown in Table 12.
- cell-free DNA can be recovered with a yield of 9.9 times that of a commercially available kit even when human plasma is used as a biological sample.
- E. coli (DH5 ⁇ ) was cultured in a liquid medium of LB.
- Buffer RLT Plus 100 ⁇ l of Buffer RLT Plus was added to the first sample and vortexed. This solution was mixed with 5 mg of cerium oxide adsorbed with polyethylene glycol prepared under the same conditions as in Example 1. The mixture was stirred with a mixer for 15 minutes, centrifuged (10000 G, 1 min), and the supernatant was discarded. 400 ⁇ l of 0.05% Tween water was added to the remaining carrier and vortexed. This operation was performed twice more. Thereafter, 50 ⁇ l of a phosphate buffer (0.5 M, pH 8) was added and stirred with a mixer for 30 minutes. The supernatant was collected as a nucleic acid solution by centrifugation (10000 G, 1 min) with a centrifuge.
- a phosphate buffer 0.5 M, pH 8
- Genome recovery was performed from the second sample according to the protocol using a commercial kit (DNeasy blood & Tissue kit, QIAGEN). At this time, the volume of the eluate was 50 ⁇ l. Genome eluate collected with the carrier of the present invention and a commercially available kit was electrophoresed (1% agarose, 100 V, 60 min) and stained with ethidium bromide. The fluorescence image of the gel was measured with a fluorescence scanner, and image analysis of the band concentration ratio of the genomic fraction (around 25 kbp) was performed. Since the absolute amount of the genome contained in E. coli is unknown, the ratio of the recovered amount between the carrier of the present invention and the commercial kit was calculated instead of the recovery rate. The results are shown in Table 12.
- the genome can be recovered with a yield of 3.8 times that of a commercially available kit even when Escherichia coli (DH5 ⁇ ) in a culture solution is used as a biological sample.
- zymolyce solution (1 M sorbitol, 100 mM EDTA, 14 mM mercaptoethanol, 100 U / ml zymolyce, pH 7.4) was added to the first sample, and the mixture was allowed to stand at 30 ° C. for 30 minutes. Thereafter, the supernatant was removed by centrifugation (5000 G, 10 min) to obtain a pellet. 200 ⁇ l of Buffer RLT Plus was added thereto and vortexed. This solution was mixed with 5 mg of cerium oxide adsorbed with polyethylene glycol prepared under the same conditions as in Example 1. The mixture was stirred with a mixer for 15 minutes, centrifuged (10000 G, 1 min), and the supernatant was discarded.
- Genome eluate collected with the carrier of the present invention and a commercially available kit was electrophoresed (1% agarose, 100 V, 60 min) and stained with ethidium bromide. The fluorescence image of the gel was measured with a fluorescence scanner, and image analysis of the band concentration ratio of the genomic fraction (around 25 kbp) was performed. Since the absolute amount of genome contained in fission yeast is unknown, the ratio of the recovered amount between the carrier of the present invention and the commercial kit was calculated instead of the recovery rate. The results are shown in Table 12.
- the genome can be recovered with a yield of 3.8 times that of a commercially available kit even if fission yeast (NBRC1628) is used as a biological sample.
- Example 14 Genome recovery from budding yeast (NBRC0216) using the carrier of the present invention Other conditions and operations were performed in the same manner as in Example 13 except that fission yeast (NBRC0628) was changed to budding yeast (NBRC0216). . The results are shown in Table 12.
- the detached cells were transferred to a 15 ml tube, centrifuged (1500 rpm, 5 min, room temperature) to give a pellet, and the supernatant was removed by aspiration. Thereafter, 2 ml of medium was added and resuspended. The number of cells was counted, 5 ⁇ 10 4 cells were collected from this cell suspension, transferred to two 1.5 ml tubes, centrifuged (300 G, 5 min), pelleted, and the supernatant was removed. Of the obtained two samples, the first sample was collected using the carrier of the present invention, and the second sample was collected using a commercially available kit as a control.
- Buffer RLT Plus 100 ⁇ l of Buffer RLT Plus was added to the first sample and vortexed. This solution was mixed with 5 mg of cerium oxide adsorbed with polyethylene glycol prepared under the same conditions as in Example 1. The mixture was stirred with a mixer for 15 minutes, centrifuged (10000 G, 1 min), and the supernatant was discarded. 400 ⁇ l of 0.05% Tween water was added to the remaining carrier and vortexed. This operation was performed twice more. Thereafter, 50 ⁇ l of a phosphate buffer (0.5 M, pH 8) was added and stirred with a mixer for 30 minutes. The supernatant was collected as a nucleic acid solution by centrifugation (10000 G, 1 min) with a centrifuge.
- a phosphate buffer 0.5 M, pH 8
- Genome eluate collected with the carrier of the present invention and a commercially available kit was electrophoresed (1% agarose, 100 V, 60 min) and stained with ethidium bromide. The fluorescence image of the gel was measured with a fluorescence scanner, and image analysis of the band concentration ratio of the genomic fraction (around 25 kbp) was performed. Since the absolute amount of the genome contained in the adherent cells is unknown, the ratio of the recovered amount between the carrier of the present invention and the commercial kit was calculated instead of the recovery rate. The results are shown in Table 12.
- Floating cells were prepared by adding fetal bovine serum to RPMI medium to 10% (v / v) and adding 25 cm. They were cultured in a 2 cell culture flasks. For genome collection, the cells were transferred to a 15 ml tube, centrifuged (1500 rpm, 5 min, room temperature) to give a pellet, and the supernatant was removed by aspiration. 2 ml of medium was added and resuspended. Other conditions and operations were the same as in Example 15. The results are shown in Table 12.
- Example 17 Nucleic acid recovery from urine A carrier having polyethylene glycol adsorbed on the surface of cerium oxide was prepared according to Example 1, and 1.5 mg was weighed into a 1.5 ml tube.
- 1 ml of artificial urine (0.2 g / l CaCl 2 , 0.4 g / l MsSO 4 , 8 g / l NaCl, 20 g / l urea, 0.3% ammonia) in which 100 pmol of DNA 22 was dissolved was prepared as a solution containing nucleic acid. .
- 200 ⁇ l of 1M acetate buffer (pH 4) was added to the artificial urine and vortexed, and the mixture was added to the carrier.
- Other conditions and operations were performed in the same manner as in Comparative Example 4, and the nucleic acid adsorption rate, elution rate, and recovery rate were calculated. The results are shown in Table 13.
- DNA 22 can be efficiently recovered even when urine is used as a biological sample.
- the present invention it is possible to efficiently recover from a very short nucleic acid such as pre-miRNA or miRNA to a long nucleic acid such as a genome from a biological sample by a simple method.
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Abstract
Description
(1)生物学的試料から核酸を回収する方法であって、以下の工程:
工程a)水溶性の中性ポリマーが酸化セリウムの表面に吸着した担体と核酸を含む溶液を混合し、担体に核酸を吸着させる工程、
工程b)工程a)において混合した溶液から、前記核酸が吸着した担体を分離する工程、
工程c)工程b)において分離した前記核酸が吸着した担体に溶出液を加えて核酸を回収する工程、
を含むことを特徴とする核酸の回収方法。
(2)前記水溶性の中性ポリマーが、pH7の溶液中で-10mV以上+10mV以下のゼータ電位を有するポリマーであることを特徴とする(1)に記載の核酸の回収方法。
(3)前記ポリマーが、ポリビニルアルコール、ポリエチレングリコール、ポリビニルピロリドン、ポリ(2-エチル-2-オキサゾリン)、ポリプロピレングリコール、ポリアクリルアミド、ポリN-イソプロピルアクリルアミド又はヒドロキシプロピルメチルセルロースであることを特徴とする(1)又は(2)に記載の核酸の回収方法。
(4)前記溶出液が緩衝液であることを特徴とする(1)から(3)のいずれかに記載の核酸の回収方法。
(5)前記生物学的試料が、血液、尿、唾液、粘膜、汗、培養細胞、培養細胞の培養液、組織試料、標本、微生物、微生物の培養液またはウイルスであることを特徴とする(1)から(4)のいずれかに記載の核酸の回収方法。
(6)水溶性の中性ポリマーが酸化セリウムの表面に吸着した担体。
(7)前記水溶性の中性ポリマーが、pH7の溶液中で-10mV以上+10mV以下のゼータ電位を有するポリマーであることを特徴とする(6)に記載の担体。
(8)前記水溶性の中性ポリマーが、ポリビニルアルコール、ポリエチレングリコール、ポリビニルピロリドン、ポリ(2-エチル-2-オキサゾリン)、ポリプロピレングリコール、ポリアクリルアミド、ポリN-イソプロピルアクリルアミド又はヒドロキシプロピルメチルセルロースであることを特徴とする(6)または(7)に記載の担体。
(9)(6)から(8)のいずれかに記載の担体と緩衝液を備えることを特徴とする核酸回収用のキット。
ポリエチレングリコールはメルク株式会社より、ポリ(2-エチル-2-オキサゾリン)はAlfa Aesar, A Johnson Matthey Companyより、酸化セリウム(粒径4.4μm)は第一稀元素化学工業株式会社より入手した。実施例中で用いたポリマー水溶液は、それぞれの濃度で水または水とエタノールの混合溶媒に溶解した。また、特に断らない限り、酸化セリウムは、ふるい分けなどせずに購入したまま実験に用いた。
特許文献3の実施例に記載の酸化鉄を担体として用いた核酸の回収方法を検討した。酸化鉄として和光純薬社製のα-酸化鉄を用いた。その他、特許文献3の実施例と同様の条件にするため、核酸溶解液として6Mグアニジンチオシアン水溶液、溶出液としてTE緩衝液または水を使用した。
1.5mlチューブに、0.5mgずつ酸化セリウムを量り取った。これにポリマー溶液として、ポリアクリル酸(PAcA, 5.1kDa, 10wt%)、ポリスチレンスルホン酸(PSS, 7.5kDa, 10wt%)、ポリビニルスルホン酸(PVSA, 10wt%)、ポリアリルアミン(PAA, 17kDa, 10wt%)、ポリ-L-リシン(PLL, 150kDa, 1wt%)をそれぞれ50μlずつ加えて10分間ミキサーで攪拌した。遠心機で遠心(10000G, 1min)して上清を除き、それぞれのポリマーが吸着した酸化セリウム得た。
1.5mlのチューブに比較例3で作製した水溶性の中性ポリマー以外の水溶性のポリマーとして、ポリアクリル酸(PAcA, 5.1kDa, 10wt%)、ポリスチレンスルホン酸(PSS, 7.5kDa, 1wt%)、ポリビニルスルホン酸(PVSA, 10wt%)、ポリアリルアミン(PAA, 17kDa, 10wt%)、ポリ-L-リシン(PLL, 150kDa, 1wt%)が吸着した酸化セリウムを0.5mgずつ量り取り担体として用いた。溶出液はリン酸緩衝液(0.5M, pH8)を用いた。担体と溶出液以外の条件、操作は比較例1と同様に行い核酸の回収率を算出した。結果を表3に示した。
1.5mlチューブに、0.5mgずつ酸化セリウムを量り取った。これに、ポリマー水溶液として、水溶性の中性ポリマーであるポリビニルアルコール(11%アセチル化, PVA, 18kDa, 10wt%)、ポリ(2-エチル-2-オキサゾリン)(PEOz, 5kDa, 10wt%)、ポリエチレングリコール(PEG, 10kDa, 10wt%)、ポリアクリルアミド(PAAm, 40kDa, 10wt%)、ヒドロキシプロピルメチルセルロース)(HPMC, 10kDa, 10wt%)、ポリビニルピロリドン(PVP, 10kDa, 10wt%)をそれぞれに50μlずつ加えた。また、ポリプロピレングリコール(PPG, 4kDa, 10wt%)とポリN-イソプロピルアクリルアミド(pNIPAAm, 30kDa, 10wt%)の水溶液はポリマーが溶解するまでエタノールを加えたのち、0.5mgの酸化セリウムへ50μlずつ加えた。その他の条件、操作は比較例3と同様に行い、それぞれのポリマーが吸着した酸化セリウムの担体を得た。
1.5mlのチューブに実施例1で作製した各水溶性の中性ポリマーとして、ポリビニルアルコール、ポリ(2-エチル-2-オキサゾリン)、ポリエチレングリコール、ポリプロピレングリコール、ポリアクリルアミド、ポリN-イソプロピルアクリルアミド、ヒドロキシプロピルメチルセルロース)、ポリビニルピロリドンが吸着した酸化セリウムを0.5mgずつ量り取り担体として用いた。その他の条件、操作は比較例4と同様に行い、核酸の吸着率、溶出率、回収率を算出した。結果を表3に示した。
比較例4で用いた水溶性の中性ポリマー以外の水溶性のポリマーであるポリアクリル酸、ポリスチレンスルホン酸、ポリビニルスルホン酸、ポリアリルアミン、ポリーL-リシンを終濃度が0.1wt%以上10wt%以下となるようにリン酸緩衝液(10mM, pH7)に溶解し、大塚電子株式会社のELS-Zを用いてゼータ電位を測定した。結果を表4に示す。表4は、本測定によって得られたゼータ電位と、それぞれのポリマーが吸着した酸化セリウムを担体として使ったDNA22の回収率(比較例4の結果)の相関を取り、ゼータ電位の値の低い順に並べたものである。
終濃度が1wt%以上10wt%以下となるよう、実施例2で用いた水溶性の中性ポリマーであるポリビニルアルコール、ポリ(2-エチル-2-オキサゾリン)、ポリエチレングリコール、ポリプロピレングリコール、ポリアクリルアミド、ポリN-イソプロピルアクリルアミド、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドンをリン酸緩衝液(10mM, pH7)に溶解し、比較例5と同様の方法でゼータ電位を測定した。表4は、本測定によって得られたゼータ電位と、それぞれのポリマーが吸着した酸化セリウムを担体として使ったDNA22の回収率(実施例2の結果)の相関を取り、ゼータ電位の値の低い順に並べたものである。
実施例1に従って酸化セリウムの表面にポリエチレングリコールが吸着した担体を作製し、1.5mlチューブに0.5mgずつ量り取った。溶出液として0.5Mクエン酸緩衝液(pH 5, 6)、0.5M リン酸緩衝液(pH 7, 8)、0.5M Tris-EDTA緩衝液(0.5M Tris-HCl, 0.5M EDTA, pH8)をそれぞれ用いた。その他の条件、操作は比較例1と同様に行い、核酸の吸着率、溶出率、回収率を算出した。結果を表5に示した。
実施例1に従って酸化セリウムの表面にポリエチレングリコールが吸着した担体を作製し、1.5mlチューブに0.5mgずつ量り取った。核酸を含む溶液として、250ngのDNA566、250ngのpUC19(2.7kbp)、250ngの大腸菌ゲノム断片(>10kbp)がそれぞれ溶解した6Mグアニジンチオシアン酸塩水溶液を100μl用いた。その他の条件、操作は比較例3と同様に行い、電気泳動により核酸の回収率を算出した。結果を表6に示した。
実施例1に従って酸化セリウムの表面にポリエチレングリコールが吸着した担体を作製し、1.5mlチューブに2.5mgずつ量り取った。核酸を含む溶液として100pmolのDNA22が溶解した6Mグアニジンチオシアン酸塩水溶液100μlと30mg/mlのタンパク質濃度を有するウシ胎児血清100μlの混合溶液を用いた。その他の条件、操作は比較例4と同様に行い核酸の吸着率、溶出率、回収率を算出した。同様の実験をRNA22に対しても行った。結果を表7に示した。なお、回収液中のタンパク質濃度は、Bradford試験の検出限界以下(0.25mg/ml以下)であった。
分子量が 6kDa、 10kDa、500kDaのポリエチレングリコールと、分子量が18kDa、40kDa、 150kDa(いずれも11%アセチル化)のポリビニルアルコールをそれぞれ10wt%になるよう調製しポリマー溶液として用いた。担体は、実施例1と同様にして、各分子量の酸化セリウムの表面にポリエチレングリコールが吸着した担体を調製してこれを用いた。その他の条件、操作は比較例4と同様に行い、核酸の吸着率、溶出率、回収率を算出した。結果を表8に示した。
1.5mlチューブに、0.5mgずつ酸化セリウムを量り取った。これに、ポリマー水溶液として、水溶性の中性ポリマーであるポリエチレングリコール(PEG, 10kDa)を0.1wt%、1wt%、10wt%の濃度でそれぞれに50μlずつ加えた。各濃度に対してミキサーでそれぞれ1分間、5分間、30分間攪拌した。遠心機で遠心(10000G, 1min)して上清を除き、酸化セリウムの表面にポリエチレングリコールが吸着した担体得た。また、比較例4と同様に行い、核酸の回収率を算出した。結果を表9に示した。
1.5mlチューブに、0.5mgずつ酸化セリウムを量り取った。これに、ポリマー水溶液として、水溶性の中性ポリマーであるポリエチレングリコール(PEG, 10kDa)を0.1wt%、1wt%、10wt%の濃度でそれぞれに50μlずつ加えて,ピペッティング等の撹拌操作は一切行わず、それぞれ5分間、30分間静置した。遠心機で遠心(10000G, 1min)して上清を除き、酸化セリウムの表面にポリエチレングリコールが吸着した担体を得た。また、比較例4と同様に行い、核酸の回収率を算出した。結果を表10に示した。
1.5mlチューブに、0.5mgずつ酸化セリウムを量り取った。これに、ポリマー水溶液として、水溶性の中性ポリマーであるポリエチレングリコール(PEG, 10kDa)を10wt%の濃度で50μl加えてミキサーで10分間攪拌した。この後の操作として、実施例1では、遠心機による遠心分離操作及び上清を除く操作を行ったが、実施例10ではこれらの操作を行わなかった。このようにして作製した担体用いた以外は、比較例4と同様に行い、核酸の吸着率、溶出率、回収率を算出し、表11に結果を示した。
2本の1.5mlチューブに、ヒト血漿を300μlずつ量り取り、Buffer RLTを450μlずつ加え、ピペッティングにより混合した。得られた2本の試料のうち、1本目は本発明の担体を用いてゲノムを回収し、2本目はコントロールとして市販キットでゲノムを回収した。
大腸菌(DH5α)は、LBの液体培地で培養した。ゲノム回収にあたり、培養液中の大腸菌濃度をOD600における測定値により算出した。培養液から5×107 cellsずつ分取して1.5mlチューブ2本に移した。得られた2本の試料のうち、1本目は本発明の担体を用いてゲノムを回収し、2本目はコントロールとして市販キットでゲノムを回収した。大腸菌の細胞数は、OD600=1のとき109 cells/mlとして計算した。
本発明の担体と市販キットで回収したゲノム溶出液を電気泳動(1%アガロース、100V、60min)し、臭化エチジウムで染色した。蛍光スキャナーでゲルの蛍光画像を測定し、ゲノム分画(25kbp付近)のバンド濃度比の画像解析を行った。大腸菌に含まれているゲノムの絶対量が不明であるため、回収率の代わりに、本発明の担体と市販キットとの回収量の比を算出した。結果を表12に示した。
分裂酵母(NBRC1628)は、YPB培地に2%アガロースを添加したプレート上で培養し、その後、YPDの液体培地に移して培養した。ゲノム回収にあたり、培養液中の分裂酵母濃度をOD600における測定値により測定することで算出した。培養液から5×105 cellsずつ分取して1.5mlチューブ2本に移した。得られた2本の試料のうち、1本目は本発明の担体を用いてゲノムを回収し、2本目はコントロールとして市販キットでゲノムを回収した。このとき細胞数は、OD600=1のとき107 cells/mlとして計算した。
分裂酵母(NBRC1628)を出芽酵母(NBRC0216)にした以外は、その他条件、操作は実施例13と同様に行った。結果を表12に示した。
<実施例15>本発明の担体を使った接着細胞(Panc10.05)からのゲノム回収
接着細胞(Panc10.05)は、RPMI培地に10%(v/v)となるようウシ胎児血清を添加して、25cm2細胞培養用フラスコで培養した。ゲノム回収にあたり、TrypLE Expressを用いて細胞を培養フラスコから剥離した。剥離した細胞は15mlチューブに移し、遠心(1500rpm, 5min, 室温)してペレットにし、上清を吸引除去した。その後、2mlの培地を入れて再懸濁した。細胞数をカウントし、この細胞懸濁液から5×104 cellsずつ分取して1.5mlチューブ2本に移し、遠心(300G, 5min)してペレットにし、上清を除いた。得られた2本の試料のうち、1本目は本発明の担体を用いてゲノムを回収し、2本目はコントロールとして市販キットでゲノムを回収した。
浮遊細胞(Jurkat)は、RPMI培地に10%(v/v)となるようウシ胎児血清を添加して、25cm2細胞培養用フラスコで培養した。ゲノム回収にあたり、細胞は15mlチューブに移し、遠心(1500rpm, 5min, 室温)してペレットにし、上清を吸引除去した。2mlの培地を入れて再懸濁した。その他の条件、操作は実施例15と同様に行った。結果を表12に示した。
実施例1に従って酸化セリウムの表面にポリエチレングリコールが吸着した担体を作製し、1.5mlチューブに1.5mg量り取った。核酸を含む溶液として100pmolのDNA22が溶解した人工尿(0.2g/l CaCl2、0.4g/l MsSO4, 8g/l NaCl、20g/l 尿素、0.3%アンモニア)1mlを調製した。人工尿に1Mの酢酸緩衝液(pH4)を200μl加えてボルテックスし、混合液を担体に加えた。その他の条件、操作は比較例4と同様に行い核酸の吸着率、溶出率、回収率を算出した。結果を表13に示した。
比較例1におけるα-酸化鉄を四酸化三鉄に変更した以外は、比較例1と同様の条件、操作で核酸を回収した。結果を表13に示した。
Claims (9)
- 生物学的試料から核酸を回収する方法であって、以下の工程:
工程a)水溶性の中性ポリマーが酸化セリウムの表面に吸着した担体と核酸を含む溶液を混合し、担体に核酸を吸着させる工程、
工程b)工程a)において混合した溶液から、前記核酸が吸着した担体を分離する工程、
工程c)工程b)において分離した前記核酸が吸着した担体に溶出液を加えて核酸を回収する工程、
を含むことを特徴とする核酸の回収方法。 - 前記水溶性の中性ポリマーが、pH7の溶液中で-10mV以上+10mV以下のゼータ電位を有するポリマーであることを特徴とする請求項1に記載の核酸の回収方法。
- 前記ポリマーが、ポリビニルアルコール、ポリエチレングリコール、ポリビニルピロリドン、ポリ(2-エチル-2-オキサゾリン)、ポリプロピレングリコール、ポリアクリルアミド、ポリN-イソプロピルアクリルアミド又はヒドロキシプロピルメチルセルロースであることを特徴とする請求項1又は2に記載の核酸の回収方法。
- 前記溶出液が緩衝液であることを特徴とする請求項1から3のいずれかに記載の核酸の回収方法。
- 前記生物学的試料が、血液、尿、唾液、粘膜、汗、培養細胞、培養細胞の培養液、組織試料、標本、微生物、微生物の培養液またはウイルスであることを特徴とする請求項1から4のいずれかに記載の核酸の回収方法。
- 水溶性の中性ポリマーが酸化セリウムの表面に吸着した担体。
- 前記水溶性の中性ポリマーが、pH7の溶液中で-10mV以上+10mV以下のゼータ電位を有するポリマーであることを特徴とする請求項6に記載の担体。
- 前記水溶性の中性ポリマーが、ポリビニルアルコール、ポリエチレングリコール、ポリビニルピロリドン、ポリ(2-エチル-2-オキサゾリン)、ポリプロピレングリコール、ポリアクリルアミド、ポリN-イソプロピルアクリルアミド又はヒドロキシプロピルメチルセルロースであることを特徴とする請求項6または7に記載の担体。
- 請求項6から8のいずれかに記載の担体と緩衝液を備えることを特徴とする核酸回収用のキット。
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WO2020129963A1 (ja) * | 2018-12-18 | 2020-06-25 | 東レ株式会社 | 酸化セリウムのナノ粒子、核酸の分解方法、ポリペプチドの分解方法、酸化セリウムのナノ粒子の製造方法、酸化剤、抗酸化剤、抗カビ剤および抗ウイルス剤 |
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WO2018088502A1 (ja) * | 2016-11-10 | 2018-05-17 | 東レ株式会社 | 核酸の検出方法 |
JPWO2018088502A1 (ja) * | 2016-11-10 | 2019-10-03 | 東レ株式会社 | 核酸の検出方法 |
US11492655B2 (en) | 2016-11-10 | 2022-11-08 | Toray Industries, Inc. | Method of detecting a nucleic acid |
CN111542605A (zh) * | 2017-12-27 | 2020-08-14 | 东丽株式会社 | 核酸的回收方法 |
US20200332277A1 (en) * | 2017-12-27 | 2020-10-22 | Toray Industries, Inc. | Method of collecting nucleic acid |
JPWO2019131760A1 (ja) * | 2017-12-27 | 2020-11-19 | 東レ株式会社 | 核酸の回収方法 |
JP7238766B2 (ja) | 2017-12-27 | 2023-03-14 | 東レ株式会社 | 核酸の回収方法 |
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WO2020129963A1 (ja) * | 2018-12-18 | 2020-06-25 | 東レ株式会社 | 酸化セリウムのナノ粒子、核酸の分解方法、ポリペプチドの分解方法、酸化セリウムのナノ粒子の製造方法、酸化剤、抗酸化剤、抗カビ剤および抗ウイルス剤 |
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Also Published As
Publication number | Publication date |
---|---|
EP3431598A1 (en) | 2019-01-23 |
CN108779455B (zh) | 2022-09-27 |
JPWO2017159763A1 (ja) | 2019-01-17 |
JP6958353B2 (ja) | 2021-11-02 |
US20190085317A1 (en) | 2019-03-21 |
CN108779455A (zh) | 2018-11-09 |
BR112018068429A2 (pt) | 2019-01-29 |
CA3015610A1 (en) | 2017-09-21 |
EP3431598A4 (en) | 2019-11-13 |
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