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WO2016052386A1 - Procédé de séparation/purification d'acide nucléique, support solide, dispositif, et kit - Google Patents

Procédé de séparation/purification d'acide nucléique, support solide, dispositif, et kit Download PDF

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Publication number
WO2016052386A1
WO2016052386A1 PCT/JP2015/077277 JP2015077277W WO2016052386A1 WO 2016052386 A1 WO2016052386 A1 WO 2016052386A1 JP 2015077277 W JP2015077277 W JP 2015077277W WO 2016052386 A1 WO2016052386 A1 WO 2016052386A1
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Prior art keywords
nucleic acid
solid phase
phase carrier
separating
solution
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English (en)
Japanese (ja)
Inventor
岡本　淳
圭三 米田
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Toyobo Co Ltd
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Toyobo Co Ltd
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Priority to JP2016552006A priority Critical patent/JP6737179B2/ja
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • 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

Definitions

  • the present invention relates to a method for separating and purifying nucleic acid.
  • Genetic testing has attracted a great deal of attention due to advances in genome analysis technology.
  • the market related to genetic testing such as the spread of personalized medicine and POCT (immediate clinical on-site testing) in the field of infectious diseases is expected to grow dramatically, and it is expected that genetic testing will be further spread.
  • a technique called a phenol / chloroform method has been known as a method for obtaining nucleic acid from a sample solution containing nucleic acid.
  • phenol / chloroform is used to insolubilize impurities such as proteins and lipids, and nucleic acids are separated and purified.
  • this conventional technique can separate and purify a large amount of nucleic acid at low cost, there are problems in that the operation is complicated, the recovery rate of nucleic acid is low, and phenol / chloroform is contaminated in the final purified product.
  • phenol / chloroform itself is toxic, so there is a problem that the working environment is limited.
  • This method is a method for separating and purifying nucleic acids by solubilizing proteins, lipids and other contaminants using a chaotropic substance, adsorbing the nucleic acids to a solid support, recovering them, and then dissolving the nucleic acids again.
  • a solid phase carrier used in the boom method various modes such as a silica filter (see, for example, Patent Documents 2 and 3) and silica magnetic beads (for example, see Patent Documents 4 to 6) are known.
  • nucleic acids can be separated and purified quickly and easily.
  • the specimen especially high-viscosity liquid such as blood
  • a high centrifugal force of 10,000 G or more is required for passing the solid phase carrier, there is a problem that automation is difficult and the obtained nucleic acid is easily fragmented by physical external force. It was.
  • silica magnetic beads by using silica magnetic beads, the problem of clogging of the solid phase carrier can be solved and automation is easy, but there is a problem that the recovery rate of nucleic acid is lowered. There is also a problem that silica magnetic particles that cannot be completely removed are contaminated with the final purified product.
  • an object of the present invention is to provide a method for separating and purifying nucleic acid, which is excellent in nucleic acid recovery and liquid permeability.
  • the present inventors use a solid phase carrier (for example, a silica filter) made from a composition containing (2) an organic or inorganic fiber and (3) an organic binder to increase the nucleic acid recovery rate.
  • a solid phase carrier for example, a silica filter
  • the liquid permeability decreases when the basis weight is increased, that is, the nucleic acid recovery rate and the liquid permeability are in a trade-off relationship. Therefore, the present inventors use (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) a solid phase carrier containing a composition comprising an organic binder, so that the nucleic acid recovery rate can be reduced even at low weight. Has been found, that is, it is possible to achieve both recovery of nucleic acid and liquid permeability.
  • the present inventors have found that (3) when a hydrophilic binder generally used for aqueous filtration is used as the organic binder, the nucleic acid recovery rate is lowered.
  • the present inventors have found that the nucleic acid recovery rate is further improved by using a hydrophobic binder such as polyethylene polymer or polyester polymer as the organic binder (3), and completed the present invention.
  • this invention consists of the following structures. 1. Using at least the following steps (A) to (D) in this order, using a solid phase carrier comprising a composition comprising at least (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) an organic binder. A method for separating and purifying nucleic acid characterized by the above.
  • a step of mixing a sample solution containing nucleic acid and a binding solution to obtain a mixed solution (B) A step of bringing the mixed solution into contact with a solid phase carrier and adsorbing the nucleic acid to the solid phase carrier (C) A solid phase 1. Step for washing components other than nucleic acid from carrier (D) Step for desorbing nucleic acid from solid phase carrier 2.
  • nucleic acids in the method for separating and purifying nucleic acid, it is possible to achieve both high nucleic acid recovery rate and high solid phase carrier permeability.
  • nucleic acids can be separated and purified from a highly viscous biological material such as blood with a low centrifugal force.
  • nucleic acid separation and purification method of the present invention form of spin column
  • nucleic acid separation and purification method of the present invention in the form of a syringe
  • solid-phase carrier used for the nucleic acid separation and purification method of the present invention.
  • nucleic acids to be separated and purified The type of nucleic acid to be separated and purified in the present invention is not particularly limited, and examples thereof include DNA or RNA, single-stranded nucleic acid or double-stranded nucleic acid, linear nucleic acid, or circular nucleic acid.
  • the present invention can be used for separation and purification of RNA that is extremely easily degraded by physical external force, and is preferably used for separation and purification of total RNA.
  • the principle of separation and purification of nucleic acid in the present invention is the same principle as the nucleic acid extraction method using silica widely known as the BOOM method (Non-patent Document 1), and specific binding of nucleic acid to a solid phase carrier, The nucleic acid is separated and purified by washing and elution.
  • a solid phase carrier is passed by applying physical external force to various solutions such as a mixed solution, a washing solution and an eluate.
  • solutions such as a mixed solution, a washing solution and an eluate.
  • QIAamp and RNeasy both Qiagen
  • a spin column with a solid support fixed to the bottom add various solutions to the top of the spin column, and centrifuge (for example, 8000-15000 G) is used from the top to the bottom.
  • nucleic acid can be separated and purified only by applying a weak external force.
  • the external force is a centrifugal force
  • the degree is not particularly limited, but the nucleic acid is preferably separated and purified by a centrifugal force of 4000 G or less, more preferably 3000 G or less, and even more preferably 2000 G or less.
  • a centrifugal operation exceeding 4000 G is to be performed, a large centrifugal separator is generally required, and it is difficult to perform quick and simple nucleic acid extraction.
  • the nucleic acid may be decomposed due to physical damage.
  • suction and discharge are performed by using a desktop small centrifuge (for example, Chibitan (registered trademark)) capable of performing centrifugation with a centrifugal force of 4000 G or less, or by changing the pressure of air such as a syringe or pipette.
  • a desktop small centrifuge for example, Chibitan (registered trademark)
  • the use of a device is preferred.
  • the lower limit of the centrifugal force is not particularly limited, but 1000 G or more is preferable.
  • the container for fixing the solid phase carrier is a spin column when using a centrifuge, or a pipette tip or similar when using a device that performs suction and discharge by changing the pressure of air, such as a syringe or pipette.
  • Shaped containers eg, luer fittings
  • the shape of the pipette tip is not particularly limited, but the tip is cut in a generally used conical shape, and a cylindrical tip is preferable. Needless to say, the shape is not limited to the pipette tip, and any shape can be selected as long as it can be in close contact with the mating portion with the suction / discharge mechanism.
  • a composition comprising at least (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) an organic binder as a solid phase carrier.
  • a silica filter generally used as a solid support although a nucleic acid recovery rate is excellent, a centrifugation step with a centrifugal force exceeding 4000 G is indispensable, and a large-sized centrifuge is required.
  • the filter may be clogged. In the present invention, papermaking is preferred.
  • the composition of the porous inorganic particles is not particularly limited as long as it can adsorb nucleic acid, and examples thereof include silica, glass, alumina, zeolite, and clay mineral. Among these, silica is preferable and porous silica particles are more preferable.
  • the lower limit of the amount of the porous inorganic particles contained in the solid phase carrier is preferably 1% by weight, more preferably 2% by weight, and even more preferably 3% by weight.
  • the upper limit is preferably 60% by weight, more preferably 40% by weight, and even more preferably 20% by weight. If the amount contained in the solid support is less than 1% by weight, the nucleic acid recovery rate may decrease. On the other hand, when the amount contained in the solid phase carrier is more than 60% by weight, the strength of the solid phase carrier is lowered, and there is a possibility that tearing may occur during the separation and purification of the nucleic acid. In addition, liquid permeability may be reduced and clogging may occur.
  • the shape of the porous inorganic particles is preferably spherical. If the shape is crushed or sheet-like, the nucleic acid recovery rate may decrease. In addition, liquid permeability may be reduced and clogging may occur.
  • the lower limit of the average particle diameter of the porous inorganic particles is preferably 1 ⁇ m, more preferably 2 ⁇ m, and even more preferably 3 ⁇ m.
  • the upper limit is preferably 50 ⁇ m, more preferably 30 ⁇ m, and even more preferably 20 ⁇ m.
  • the average particle size is smaller than 1 ⁇ m, liquid permeability is lowered and clogging may occur.
  • the average particle diameter is larger than 50 ⁇ m, the strength of the solid phase carrier is remarkably lowered, and there is a possibility that the breakage may occur during the separation and purification of the nucleic acid.
  • the organic or inorganic fibers are not particularly limited, but are preferably made of organic fibers such as cellulose or inorganic fibers such as glass, and glass fibers are more preferable.
  • the organic binder is not particularly limited, but is preferably a hydrophobic binder such as a polyethylene polymer or a polyester polymer, and more preferably a binder containing a polyester polymer.
  • the basis weight of the solid phase carrier is not particularly limited, but the lower limit is preferably 25 g / m 2 and more preferably 50 g / m 2 .
  • the upper limit is preferably 200 g / m 2 and more preferably 150 g / m 2 .
  • the basis weight is less than 25 g / m 2 , the strength of the solid phase carrier is lowered, and there is a possibility that tearing may occur during the separation and purification of the nucleic acid. In addition, the nucleic acid recovery rate may be reduced.
  • the basis weight is larger than 200 g / m 2 , the liquid permeability is lowered and clogging may occur.
  • a plurality of solid phase carriers may be used in a stacked manner, and when a plurality of the solid phase carriers are stacked, the overall basis weight is the basis weight of the solid phase carrier.
  • the thickness of the solid phase carrier is not particularly limited, but the lower limit is preferably 100 ⁇ m, more preferably 200 ⁇ m.
  • the upper limit is preferably 500 ⁇ m, more preferably 400 ⁇ m.
  • the thickness is less than 100 ⁇ m, the strength of the solid phase carrier is lowered, and there is a possibility that tearing may occur during the separation and purification of the nucleic acid. In addition, the nucleic acid recovery rate may be reduced.
  • the thickness is greater than 500 ⁇ m, the liquid permeability is lowered and clogging may occur.
  • a plurality of solid phase carriers may be used in a stacked manner, and when a plurality of solid phase carriers are stacked, the total thickness is set to the thickness of the solid phase carrier.
  • FIG. 3 illustrates an example in which “porous inorganic particles” indicated by black circles and “organic or inorganic fibers” indicated by lines are bonded with an organic binder.
  • the density of “porous inorganic particles” and “organic or inorganic fibers” can be adjusted by appropriately setting the respective concentrations at the time of production. Therefore, as a matter of course, the form of the carrier is not limited to that shown in FIG.
  • the method for producing the solid phase carrier is not particularly limited, but for example, it is preferably produced by a wet papermaking method.
  • Step (A) of the present invention is a step of mixing a sample containing nucleic acid and a binding solution.
  • the sample containing the nucleic acid used in the mixing step of the present invention is not particularly limited.
  • cultured tissues, cells, bacteria, viruses and the like can also be mentioned.
  • this also applies to nucleic acids purified by other methods.
  • the present invention is preferably used for separation and purification of nucleic acid from blood that is relatively easily clogged with a column.
  • the binding liquid used in the mixing step of the present invention is preferably an aqueous solution containing a chaotropic substance.
  • the chaotropic substance is a substance having a function (chaotropic effect) of generating chaotropic ions in an aqueous solution and increasing the water solubility of the hydrophobic molecule.
  • the chaotropic substance is not particularly limited as long as it contributes to adsorption of nucleic acid to a solid phase carrier.
  • guanidine thiocyanate, guanidine hydrochloride, guanidine nitrate, guanidine sulfate, sodium iodide, potassium iodide. Sodium perchlorate and urea.
  • guanidine thiocyanate and guanidine hydrochloride are preferable because of the strong chaotropic effect.
  • the chaotropic substances may be used alone or in combination of two or more.
  • the concentration of the chaotropic substance is not particularly limited as long as a sufficient chaotropic effect is obtained, but is 1.0 to 6.0 M in the case of guanidine thiocyanate and 1.0 to 8.0 M in the case of guanidine hydrochloride. Is preferred. If the concentration of guanidine thiocyanate is less than 1.0M, a sufficient chaotropic effect may not be obtained.
  • guanidine thiocyanate if the concentration of guanidine thiocyanate is higher than 6.0M, guanidine thiocyanate may precipitate during storage. Similarly, if the concentration of guanidine hydrochloride is less than 1.0M, there is a possibility that a sufficient chaotropic effect cannot be obtained. On the other hand, if the concentration of guanidine hydrochloride is more than 8.0M, guanidine hydrochloride may be precipitated during storage.
  • the binding solution preferably contains an alcohol to assist the binding between the nucleic acid and the solid phase carrier.
  • an alcohol to assist the binding between the nucleic acid and the solid phase carrier.
  • the alcohols any kind of alcohol may be used as long as the above effects are obtained, but ethanol and isopropanol are preferable, and ethanol is more preferable.
  • the said alcohol may be used independently or may be used in combination of 2 or more type.
  • the alcohol concentration is not particularly limited, but is preferably 10 to 70%, more preferably 10 to 50%. If the alcohol concentration is less than 10%, a sufficient binding assisting effect may not be obtained. On the other hand, when the alcohol concentration is higher than 70%, the binding property between the nucleic acid and the solid phase carrier decreases.
  • the binding solution contains a buffering agent for adjusting pH and improving nucleic acid adsorption effect.
  • a buffering agent for adjusting pH and improving nucleic acid adsorption effect.
  • any kind of buffer may be used as long as it has a sufficient buffering capacity in a target pH range.
  • tris phosphoric acid, phthalic acid, citric acid, maleic acid, Succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, good buffer (MES, ADA, PIPES, ACES, collamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine
  • MES ADA
  • PIPES pallasine
  • ACES collamine hydrochloride
  • BES TES
  • HEPES acetamidoglycine
  • tricine glycinamide
  • bicine acetamidoglycine
  • Tris, phosphoric acid, MES, PIPES, TES, and HEPES are preferable because they have sufficient buffer capacity at pH 5.0 to 9.0 (preferably pH 6.0 to 8.0).
  • the said buffer may be used independently or may be used in combination of 2 or more type.
  • the concentration of the buffer is not particularly limited, but is preferably about 10 to 100 mM.
  • the binding solution may contain a surfactant for the purpose of breaking the cell membrane or denaturing proteins contained in the cells.
  • a surfactant any type of surfactant may be used as long as the above effects are obtained.
  • polyoxyethylene alkylphenyl ether such as Triton (registered trademark) surfactant
  • polyoxyethylene alkyl Ether such as Brij (registered trademark) surfactant
  • polyoxyethylene sorbitan fatty acid ester such as Tween (registered trademark) surfactant
  • polyoxyethylene fatty acid ester such as Tween (registered trademark) surfactant
  • the said surfactant may be used independently or may be used in combination of 2 or more type.
  • the concentration of the surfactant is not particularly limited, but is preferably 0.1 to 20%. If the surfactant concentration is less than 0.1%, sufficient cell membrane destruction or protein denaturation effects may not be obtained. On the other hand, even if the surfactant concentration is higher than 20%, no improvement in the effect is observed.
  • the binding solution may contain a reducing agent for the purpose of denaturing proteins, particularly nucleases, contained in a sample containing nucleic acids.
  • a reducing agent for the purpose of denaturing proteins, particularly nucleases, contained in a sample containing nucleic acids.
  • the reducing agent any kind of reducing agent may be used as long as the above effects are obtained.
  • 2-mercaptoethanol and dithiothreitol are preferable.
  • the concentration of the reducing agent is not particularly limited, but is preferably 1.0 to 100 mM. If the reducing agent concentration is less than 1.0 mM, a sufficient protein denaturation effect may not be obtained. On the other hand, even if the reducing agent concentration is higher than 100 mM, the effect is not improved.
  • the method of mixing the sample containing the nucleic acid and the binding solution is not particularly limited, and examples thereof include mixing with a vortex mixer, inversion mixing, and pipetting.
  • Step (B) of the present invention is a step of bringing the mixed solution into contact with the solid phase carrier and adsorbing the nucleic acid to the solid phase carrier.
  • the method of bringing the mixed solution into contact with the solid phase carrier and adsorbing the nucleic acid to the solid phase carrier is not particularly limited.
  • a spin column having a solid phase carrier fixed on the bottom surface is used, Method of adsorbing nucleic acid to solid phase carrier by adding mixed solution and letting it flow from top to bottom with a centrifuge (2) Using a syringe with a solid phase carrier fixed to the tip by luer fitting, Method of adsorbing nucleic acid to solid phase carrier by repeatedly sucking and discharging by syringe (3) Using a pipette tip with a solid phase carrier fixed inside, and repeatedly passing the mixed solution by pipette suction and discharging And a method of adsorbing nucleic acid to a solid phase carrier.
  • the liquid passing method is not particularly limited.
  • the liquid can flow from top to bottom using gravity.
  • it can also be performed by applying some external force such as applying centrifugal force with a centrifuge or performing suction and discharge by changing the pressure of air such as a syringe or pipette.
  • Step (C) of the present invention is a step in which a washing solution and a solid phase carrier are brought into contact with each other to wash components other than nucleic acids (for example, proteins, lipids, etc.) from the solid phase carrier.
  • the cleaning step may be performed by one cleaning, or the cleaning may be repeated a plurality of times.
  • the washing solution used in the washing step of the present invention is not particularly limited as long as it does not desorb the nucleic acid adsorbed on the solid phase carrier and desorbs components other than the nucleic acid, but an aqueous solution containing alcohols It is preferable that As the alcohols, any kind of alcohol may be used as long as the above effects are obtained, but ethanol and isopropanol are preferable, and ethanol is more preferable. Moreover, the said alcohol may be used independently or may be used in combination of 2 or more type.
  • the alcohol concentration is not particularly limited, but is preferably 20 to 100%, more preferably 30 to 90%. If the alcohol concentration is less than 20%, the nucleic acid may be detached.
  • the washing solution contains a buffering agent for adjusting pH and improving nucleic acid adsorption effect.
  • a buffering agent for adjusting pH and improving nucleic acid adsorption effect.
  • any kind of buffer may be used as long as it has a sufficient buffering capacity in a target pH range.
  • tris phosphoric acid, phthalic acid, citric acid, maleic acid, Succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, good buffer (MES, ADA, PIPES, ACES, collamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine
  • MES ADA
  • PIPES pallasine
  • TES collamine hydrochloride
  • BES TES
  • HEPES acetamidoglycine
  • tricine glycinamide
  • bicine acetamidoglycine
  • Tris, phosphoric acid, MES, PIPES, TES, and HEPES are preferable because they have sufficient buffer capacity at pH 5.0 to 9.0 (preferably pH 6.0 to 8.0).
  • the said buffer may be used independently or may be used in combination of 2 or more type.
  • the buffer concentration is not particularly limited, but is preferably about 10 to 100 mM.
  • the concentration of the buffer in the washing solution is more preferably lower than the concentration of the buffer in the binding solution.
  • the method for bringing the washing solution into contact with the solid phase carrier and washing components other than the nucleic acid from the solid phase carrier is not particularly limited.
  • (1) using a spin column having a solid phase carrier fixed on the bottom surface A method of washing components other than nucleic acid from the solid phase carrier by adding a washing solution to the top and letting it flow from top to bottom with a centrifuge
  • (2) Using a syringe with a solid phase carrier fixed at the tip by luer fitting A method of washing components other than nucleic acid from the solid phase carrier by repeatedly passing the washing solution by suction and discharge of a syringe
  • (3) Using a pipette tip with a solid phase carrier fixed inside, and using a pipette tip A method of washing components other than the nucleic acid from the solid phase carrier by repeatedly passing the solution is used.
  • the washing liquid remaining on the solid phase carrier can be removed by centrifugation or heating, if necessary.
  • the heating temperature is preferably 50 to 90 ° C, more preferably 60 to 80 ° C. When the heating temperature is lower than 50 ° C., a sufficient cleaning liquid removal effect cannot be obtained. On the other hand, if the heating temperature is higher than 90 ° C., the nucleic acid may be decomposed or denatured.
  • Step (D) of the present invention is a step in which the eluate is brought into contact with a solid phase carrier to desorb nucleic acid from the solid phase carrier.
  • the eluate used in the elution step of the present invention is a solution composition that desorbs nucleic acid adsorbed on a solid phase carrier and does not inhibit reactions after nucleic acid extraction, such as reverse transcription and nucleic acid amplification reactions typified by PCR.
  • a solution composition that desorbs nucleic acid adsorbed on a solid phase carrier and does not inhibit reactions after nucleic acid extraction, such as reverse transcription and nucleic acid amplification reactions typified by PCR.
  • water and Tris-EDTA buffer [10 mM Tris-HCl buffer, 1 mM EDTA, pH 8.0] are preferable.
  • the eluate can be heated as necessary to increase elution efficiency.
  • the heating temperature is preferably 50 to 90 ° C, more preferably 60 to 80 ° C. When the heating temperature is lower than 50 ° C., a sufficient elution rate improvement effect cannot be obtained. On the other hand, if the heating temperature is higher than 90 ° C., the nucleic acid may be decomposed or denatured.
  • the method of bringing the eluate into contact with the solid phase carrier and desorbing the nucleic acid from the solid phase carrier is not particularly limited.
  • a spin column having a solid phase carrier fixed to the bottom surface is used, Method of desorbing nucleic acid from solid phase carrier by adding eluate and letting it flow from top to bottom with a centrifuge (2) Using a syringe with a solid phase carrier fixed to the tip by luer fitting, Method of desorbing nucleic acid from solid phase carrier by repeatedly sucking and discharging by syringe (3) Using a pipette tip with a solid phase carrier fixed inside, and repeatedly passing eluate by sucking and discharging the pipette And a method of desorbing nucleic acids from a solid phase carrier.
  • the present invention also includes a solid phase carrier for use in the above-described method for separating and purifying nucleic acid, the composition comprising (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) an organic binder. It is a solid support.
  • the present invention is also a device for use in the nucleic acid separation and purification method, which holds the solid phase carrier.
  • the form of the device is not particularly limited, but a spin column or a syringe is preferable. The specific form is illustrated by the Example etc. which are mentioned later.
  • the present invention also provides a nucleic acid separation and purification kit for performing the nucleic acid separation and purification method, the solid phase carrier or the device, a binding solution for adsorbing nucleic acid to the solid phase carrier, and a solid phase carrier.
  • a kit for separating and purifying nucleic acid comprising a washing solution for washing components other than nucleic acids from eluate, and an elution solution for eluting nucleic acids from a solid phase carrier.
  • the configuration other than the solid phase carrier, the binding solution, the washing solution and the eluate is not particularly limited. For example, a known configuration used in the boom method may be included.
  • Shape of porous inorganic particles Each particle was observed with a scanning electron microscope (SEM) and classified into [1] a sphere or an ellipsoidal sphere [2] a cube or a rectangular parallelepiped sheet [3] other random shapes such as a crushed shape.
  • SEM scanning electron microscope
  • Average particle size of porous inorganic particles Each particle was observed with a scanning electron microscope (SEM), the diameter of 100 particles was measured, and the average diameter (average particle diameter) was calculated.
  • Weight of solid support> A 200 mm ⁇ 200 mm solid phase carrier was heated with a dryer at 80 ° C. for 30 minutes, and allowed to stand at room temperature for 30 minutes with a desiccator (desiccant: silica gel). Thereafter, the weight was measured and converted to a weight per 1 m 2 (weight per unit area).
  • Thickness of solid support The thickness when a load of 686 Pa was applied was measured at 10 locations on a 200 mm ⁇ 200 mm solid phase carrier, and the average thickness was calculated.
  • nucleic acid concentration 10 ng / ⁇ L or more>
  • nucleic acid concentration was measured under the following conditions.
  • nano drop (registered trademark) 2000 manufactured by Thermo Scientific Measurement mode Nucleic acid-DNA Sample volume: 2 ⁇ L Blank: Water
  • nucleic acid concentration 10 ng / ⁇ L or less>
  • the nucleic acid concentration was measured under the following conditions.
  • RNA-RNA Sample liquid volume 10 ⁇ L Blank: Water
  • Measurement mode DNA-dsDNA High Sensitivity Sample liquid volume: 10 ⁇ L Blank: Water
  • RNA degradation degree (RIN value) was measured under the following conditions.
  • Device name Agilent 2200 TapeStation manufactured by Agilent Measurement kit: HS RNA Screen Tape manufactured by Agilent, Sample Buffer, Ladder Sample volume: 2 ⁇ L
  • Example 1 A solid support 1 was prepared using a wet papermaking machine (manufactured by Toyobo Engineering Co., Ltd.) at a ratio of 5% by weight of porous silica particles, 66.5% by weight of glass fiber, and 28.5% by weight of polyester binder. . Details of the obtained solid phase carrier 1 are shown in Table 1. Next, the solid phase carrier 1 was punched into a size of 7 mm ⁇ with a belt punch TPO-70 (manufactured by Trusco).
  • the silica membrane and O-ring of Econospin (registered trademark) IIa are removed from the silica membrane filter housing, and two solid phase carriers 1 are placed in the silica membrane filter housing instead of the silica membrane.
  • the spin column 1 (see FIG. 1) was prepared by fixing with an O-ring.
  • the solid phase carrier 1 was punched into a size of 4 mm ⁇ with a belt punch TPO-40 (manufactured by Trusco).
  • a female luer fitting VPRF206 manufactured by Isis was connected to the tip of a Terumo syringe SS-02SZ (manufactured by Terumo) via two solid phase carriers 1 to prepare a syringe 1 (see FIG. 2).
  • Example 2 to 5 Solid phase carriers 2 to 5 were prepared in the same manner as in Example 1 except that the blending ratios of porous silica particles, glass fiber, and polyester binder were different. Details of the obtained solid phase carriers 2 to 5 are shown in Table 1. In the same manner as in Example 1, spin columns 2 to 5 and syringes 2 to 5 were prepared.
  • Example 6 and 7 Solid phase carriers 6 and 7 were prepared in the same manner as in Example 1 except that the average particle size of the porous silica particles was different. The details of the obtained solid phase carriers 6 and 7 are shown in Table 1. In the same manner as in Example 1, spin columns 6 and 7 and syringes 6 and 7 were prepared.
  • Example 8 and 9 Solid phase carriers 8 and 9 were prepared in the same manner as in Example 1 except that the shape of the porous silica particles was different. Details of the obtained solid phase carriers 8 and 9 are shown in Table 2. Also, spin columns 8 and 9 and syringes 8 and 9 were prepared in the same manner as in Example 1.
  • Example 10 A solid support 10 was prepared in the same manner as in Example 1 except that cellulose fibers were used instead of glass fibers. Details of the obtained solid phase carrier 10 are shown in Table 2. Moreover, the spin column 10 and the syringe 10 were created in the same manner as in Example 1.
  • Example 11 and 12 Solid phase carriers 11 and 12 were prepared in the same manner as in Example 1 except that a polyethylene binder and a polypropylene binder were used instead of the polyester binder. Details of the obtained solid phase carriers 11 and 12 are shown in Table 2. Further, spin columns 11 and 12 and syringes 11 and 12 were produced in the same manner as in Example 1.
  • Example 13 and 14 Solid phase carriers 13 and 14 were prepared in the same manner as in Example 1 except that the basis weight and thickness of the solid phase support were different. The details of the obtained solid phase carriers 13 and 14 are shown in Table 2. Also, spin columns 13 and 14 and syringes 13 and 14 were produced in the same manner as in Example 1.
  • Solid phase carriers 15 and 16 were prepared in the same manner as in Example 1 except that the mixing ratios of porous silica particles, glass fibers, and polyester binder were different. The details of the obtained solid phase carriers 15 and 16 are shown in Table 3. Since the solid phase carrier 16 was poor in quality and very brittle, later evaluation could not be performed. Further, a spin column 15 and a syringe 15 were prepared in the same manner as in Example 1.
  • Example 3 A solid support 17 was prepared in the same manner as in Example 1 except that the average particle diameter of the porous silica particles was different. The details of the obtained solid phase carrier 17 are shown in Table 3. Further, in the same manner as in Example 1, a spin column 17 and a syringe 17 were prepared.
  • Example 4 A solid support 18 was prepared in the same manner as in Example 1 except that a PVA binder was used instead of the polyester binder. Details of the obtained solid phase carrier 18 are shown in Table 3. Further, in the same manner as in Example 1, a spin column 18 and a syringe 18 were prepared.
  • ⁇ Liquid permeability of solid support The spin columns 1 to 18 were used to evaluate the liquid permeability of the solid phase carrier.
  • Bovine Blood manufactured by Funakoshi Co., Ltd.
  • MX-307 Tommy Seiko Co., Ltd.
  • Bovine Blood Bovine Blood (Funakoshi Co., Ltd.) evaluated the minimum centrifugal force required for passing the solid phase carrier in the spin column.
  • Table 4 shows the obtained minimum flow centrifugal force.
  • RNA recovery rate of total RNA, total RNA degradation degree spin column method> Using the spin columns 1 to 18, the total RNA recovery rate from the total RNA aqueous solution and the total RNA degradation degree were evaluated.
  • Total RNA recovery rate (%) ⁇ purified total RNA concentration (ng / ⁇ L) ⁇ eluate volume 30 ( ⁇ L) ⁇ ⁇ ⁇ input total RNA concentration 100 (ng / ⁇ L) ⁇ input total RNA solution volume 100 ( ⁇ L) ⁇ ⁇ 100 (2)
  • RNA recovery rate and the total RNA degradation degree from the blood of healthy subjects were evaluated using the spin columns 1-18.
  • 200 ⁇ L of healthy human blood (whole blood) and 200 ⁇ L of PureLink (registered trademark) (manufactured by Life Technologies) as a binding solution were mixed and mixed with a vortex mixer.
  • 200 ⁇ L of ethanol SP manufactured by Nacalai Tesque was mixed, and then applied to spin columns 1 to 18 and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (manufactured by Tommy Seiko). Discarded.
  • Total RNA recovery rate (%) ⁇ purified total RNA concentration (ng / ⁇ L) ⁇ eluate volume 30 ( ⁇ L) ⁇ ⁇ ⁇ total RNA amount per unit blood volume 1.5 (ng / ⁇ L) ⁇ input blood volume 200 ( ⁇ L) ⁇ ⁇ 100 (3)
  • RNA degradation degree syringe method> Using the syringes 1 to 18, the total RNA recovery rate from the total RNA aqueous solution and the total RNA degradation degree were evaluated.
  • 50 ⁇ L of 100 ng / ⁇ L total RNA aqueous solution (RIN value 9.9) separated and purified according to the protocol using Sepasol (registered trademark) RNAI SuperG (manufactured by Nacalai Tesque) from HeLa S3 cells, and binding solution RNeasy (registered trademark) (manufactured by Qiagen) as Buffer RLT 175 ⁇ L was mixed with a vortex mixer.
  • ethanol SP manufactured by Nacalai Tesque
  • suction and discharge were repeated 10 times with syringes 1 to 18, and the filtrate was discarded.
  • 500 ⁇ L of Buffer RPE of RNeasy (registered trademark) (manufactured by Qiagen) as a cleaning solution was repeatedly aspirated and discharged 10 times with syringes 1 to 18, and then the operation of discarding the filtrate was repeated twice.
  • Total RNA recovery rate (%) ⁇ purified total RNA concentration (ng / ⁇ L) ⁇ eluate amount 50 ( ⁇ L) ⁇ ⁇ ⁇ input total RNA concentration 100 (ng / ⁇ L) ⁇ input total RNA solution amount 50 ( ⁇ L) ⁇ ⁇ 100 (4)
  • RNA recovery rate from the blood of healthy subjects and the total RNA degradation degree were evaluated.
  • 30 ⁇ L of healthy blood (whole blood), 70 ⁇ L of Nuclease-Free Water (Life Technologies), and 100 ⁇ L of PureLink (registered trademark) (Life Technologies) Lysis Buffer as a binding solution were mixed, Mix with a vortex mixer.
  • 100 ⁇ L of ethanol SP manufactured by Nacalai Tesque was mixed, and then suction and discharge were repeated 10 times with syringes 1 to 18, and the filtrate was discarded.
  • Total RNA recovery rate (%) ⁇ purified total RNA concentration (ng / ⁇ L) ⁇ eluate volume 50 ( ⁇ L) ⁇ ⁇ ⁇ total RNA volume per unit blood volume 1.5 (ng / ⁇ L) ⁇ input blood volume 100 ( ⁇ L) ⁇ ⁇ 100 (5)
  • ⁇ Recovery rate of Genomic DNA Spin column method> Using the spin columns 1 to 18, the recovery rate of Genomic DNA from the Genomic DNA aqueous solution was evaluated.
  • 100 ⁇ L of 100 ng / ⁇ L Human Genomic DNA (manufactured by Roche) and 350 ⁇ L of a dissolved adsorption solution of Gene Cube pretreatment set (manufactured by Toyobo Co., Ltd.) as a binding solution were mixed and mixed with a vortex mixer.
  • Genomic DNA recovery (%) ⁇ purified Genomic DNA concentration (ng / ⁇ L) ⁇ eluate volume 30 ( ⁇ L) ⁇ ⁇ ⁇ input Genomic DNA concentration 100 (ng / ⁇ L) ⁇ input Genomic DNA volume 100 ( ⁇ L) ⁇ ⁇ 100 (6)
  • ⁇ Recovery rate of Genomic DNA Using the syringes 1 to 18, the recovery rate of Genomic DNA from the Genomic DNA aqueous solution was evaluated.
  • As a sample solution 50 ⁇ L of 100 ng / ⁇ L Human Genomic DNA (manufactured by Roche) and 175 ⁇ L of a dissolved adsorption solution of Gene Cube pretreatment set (manufactured by Toyobo Co., Ltd.) as a binding solution were mixed and mixed with a vortex mixer.
  • Genomic DNA recovery rates are shown in Tables 5-7.
  • the Genomic DNA recovery rate was calculated from the following formula (7).
  • Genomic DNA recovery rate (%) ⁇ purified Genomic DNA concentration (ng / ⁇ L) ⁇ eluate volume 50 ( ⁇ L) ⁇ ⁇ ⁇ input Genomic DNA concentration 100 (ng / ⁇ L) ⁇ input Genomic DNA volume 50 ( ⁇ L) ⁇ ⁇ 100 (7)
  • the present invention it is possible to achieve both a high recovery rate of nucleic acid and a high liquid permeability of a solid phase carrier, to enable more rapid and simple nucleic acid extraction, and it is very easy to automate the nucleic acid separation and purification process. Therefore, it is expected to greatly contribute to the industry.

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Abstract

Le problème posé concerne un procédé de séparation/purification d'acide nucléique qui permet d'obtenir un excellent taux de récupération d'acide nucléique et une excellente perméabilité aux liquides. La solution selon l'invention concerne un procédé de séparation/purification d'acide nucléique caractérisé par l'utilisation d'un support solide qui comprend une composition constituée au moins (1) de particules inorganiques poreuses, (2) de fibres organiques ou inorganiques et (3) d'un liant organique, et également caractérisé en ce qu'il comprend au moins les étapes mentionnées ci-après de (A) à (D) dans cet ordre : (A) une étape consistant à mélanger une solution échantillon contenant un acide nucléique avec une solution de liaison pour produire une solution mélangée; (B) une étape consistant à mettre la solution mixte en contact avec le support solide afin de provoquer l'adsorption de l'acide nucléique sur le support solide; (C) une étape d'élimination par lavage des composants, autres que l'acide nucléique, du support solide; et (D) une étape de désorption de l'acide nucléique du support solide.
PCT/JP2015/077277 2014-09-30 2015-09-28 Procédé de séparation/purification d'acide nucléique, support solide, dispositif, et kit Ceased WO2016052386A1 (fr)

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WO2020090900A1 (fr) 2018-10-31 2020-05-07 東レ株式会社 Colonne pour une utilisation de collecte d'acide nucléique
JPWO2021100801A1 (fr) * 2019-11-20 2021-05-27
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WO2020090900A1 (fr) 2018-10-31 2020-05-07 東レ株式会社 Colonne pour une utilisation de collecte d'acide nucléique
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JP7627423B2 (ja) 2019-11-20 2025-02-06 東レ株式会社 核酸の分離方法、検出方法、核酸精製カラム及びその製造方法
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