JP4825528B2 - Nucleic acid separation and purification method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive method for separating and purifying a nucleic acid which can recover a high yield of RNA from a mixture sample of the RNA with DNA, excellent in separation performance, having high cleaning efficiency, simple, rapid and excellent in suitability for automatization. <P>SOLUTION: The method for separating and purifying the RNA comprises (1) a process for preparing a mixture solution by adding a nucleic acid solubilization reagent to a biomaterial and preparing a sample solution by adding a water-soluble organic solvent or a solution containing the water-soluble organic solvent to the mixture solution, (2) a process for adsorbing the nucleic acid to a nucleic acid-adsorbing porous membrane by passing the sample solution containing the nucleic acid through the membrane, (3) a process for cleaning the nucleic acid-adsorbing membrane in an RNA adsorbed state by passing a cleaning solution through the porous membrane and (4) a process for releasing the RNA from the nucleic acid-adsorbing membrane by passing a recovery solution through the porous membrane. In the method, the nucleic acid-adsorbing porous membrane is a porous membrane which adsorbs the nucleic acid with mutual actions to which ionic bonds are not participating and the sample solution contains the water-soluble organic solvent in the concentration of 10-35 volume%. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a method for separating and purifying nucleic acid. More specifically, the present invention relates to a method for separating and purifying RNA from a nucleic acid mixture containing RNA and DNA. More specifically, the present invention relates to a method for separating and purifying RNA from a nucleic acid mixture containing RNA and DNA using a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane in a container having at least two openings and a pressure generator.

  Nucleic acids are used in various forms in various fields. For example, in the area of recombinant nucleic acid technology, it is required to use nucleic acids in the form of probes, genomic nucleic acids, and plasmid nucleic acids.

  Also in the diagnostic field, nucleic acids are used for various purposes in various forms. For example, nucleic acid probes are routinely used for detection and diagnosis of human pathogens. Similarly, nucleic acids are used to detect genetic disorders. Nucleic acids are also used to detect food contaminants. In addition, nucleic acids are routinely used in the localization, identification and isolation of nucleic acids of interest for a variety of reasons ranging from genetic mapping to cloning and recombinant expression.

In many cases, nucleic acids are available only in very small amounts, and the isolation and purification operations are cumbersome and time consuming. This often time-consuming and cumbersome operation tends to lead to the loss of nucleic acids.
When purifying nucleic acids from samples obtained from serum, urine and bacterial cultures, there is also the risk of contamination and false positive results.

  As one of widely known separation and purification methods, there is a method in which nucleic acid is adsorbed on a solid phase such as silicon dioxide, silica polymer, magnesium silicate, followed by operations such as washing and desorption, followed by separation and purification. (For example, Patent Document 1). Although this method is excellent in separation performance, it cannot be said to be sufficient in terms of simplicity, rapidity, and suitability for automation, and instruments and devices used in this method are not suitable for automation and miniaturization. Particularly, it is difficult to industrially mass-produce the adsorbent with the same performance, and there are problems such as inconvenience in handling and difficulty in processing into various shapes. Furthermore, since the material itself is fragile, a certain thickness or more is required to obtain mechanical strength. Therefore, when RNA is selectively recovered from a mixed sample of DNA and RNA, an expensive reagent such as DNAse is used. There is a problem such as need.

  In addition, as one method for separating and purifying nucleic acid simply and efficiently, it consists of an organic polymer having a hydroxyl group on its surface, using a solution for adsorbing nucleic acid on the solid phase and a solution for desorbing nucleic acid from the solid phase, respectively. A method for separating and purifying nucleic acid by adsorbing and desorbing nucleic acid on a solid phase has been described (Patent Documents 2 and 3).

In addition, conventionally known nucleic acid separation and purification methods include those using a centrifugal method, those using magnetic beads, and those using a filter, and nucleic acid separation and purification apparatuses using these have been proposed. For example, as a nucleic acid separation apparatus using a filter, a large number of filter tubes containing filters are set in a rack, a sample solution containing nucleic acid is dispensed into the rack, and the periphery of the bottom of the rack is placed through a sealing material. The inside is sealed with an air chamber, the inside is depressurized, and all filter tubes are sucked from the discharge side at the same time to allow the sample liquid to pass through to adsorb the nucleic acid to the filter. Thus, a mechanism for cleaning and desorbing has been proposed (see, for example, Patent Document 4).
Japanese Patent Publication No. 7-51065 JP 2003-128691 A JP 2004-49108 A Japanese Patent No. 2832586

  The object of the present invention is a method of separating and purifying nucleic acid by adsorbing nucleic acid in a biomaterial to the surface of the porous membrane and then desorbing it through washing or the like, particularly by reducing clogging in the washing step. The purpose is to increase the amount of biomaterials and increase the amount of RNA recovered as a result.

As a result of intensive studies to solve the above problems, the present inventors have used a porous membrane that adsorbs nucleic acids through an interaction that does not involve ionic bonds and includes nucleic acids in order to reduce clogging in the washing step. It has been found that it is effective to adjust the concentration of the water-soluble organic solvent in the sample solution to 10 to 35% by volume, and the present invention has been completed.
In the present invention, it is preferable to use a nucleic acid separation / purification cartridge in which a porous membrane made of an organic polymer is housed in a container having two openings.
That is, the present invention has the following configuration.
1.
(1) A step of preparing a sample solution by adding a nucleic acid solubilizing reagent to a biomaterial, and further adding a water-soluble organic solvent or a solution containing a water-soluble organic solvent to the mixture,
(2) passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane to adsorb the nucleic acid to the porous membrane;
(3) passing the washing solution through the nucleic acid-adsorbing porous membrane and washing the porous membrane with the RNA adsorbed; and (4) passing the recovered solution through the nucleic acid-adsorbing porous membrane and removing the porous membrane from the porous membrane. In an RNA separation and purification method comprising a step of desorbing RNA,
The nucleic acid-adsorbing porous membrane is a porous membrane obtained by saponifying a porous membrane of triacetyl cellulose and adsorbing nucleic acids by an interaction not involving ionic bonds,
Wherein the water-soluble organic solvent is at least one alcohol methanol, ethanol, is whether we selected propanol及beauty blanking Tano Le,
And the RNA isolation | separation purification method whose water-soluble organic-solvent density | concentration in the said sample solution is 10-25 volume%.
2.
2. The method for separating and purifying RNA according to 1 above, wherein the alcohol is ethanol.
3.
3. The method for separating and purifying RNA according to 1 or 2 above, wherein the biomaterial is a specimen containing cells or viruses.
4).
4. The method for separating and purifying RNA as described in 3 above, wherein the specimen containing cells is animal tissue.
5).
In each of the steps (1), (2), (3) and (4), a container having at least two openings for allowing a sample solution, a washing solution or a recovery solution containing a nucleic acid to pass through the nucleic acid-adsorbing porous membrane. A nucleic acid separation / purification cartridge and a pressure generator containing a nucleic acid-adsorbing porous membrane through which the solution can pass, and the pressure generator can be attached to and detached from one opening of the nucleic acid separation / purification cartridge. 5. The method for separating and purifying RNA according to any one of 1 to 4, which is a pump to be bound.
6).
A nucleic acid separation and purification cartridge and a reagent kit for performing the RNA separation and purification method according to any one of 1 to 5 above .
The present invention has the above-described configurations 1 to 6 , but the following are also described for reference.
[1]
(1) A step of preparing a sample solution by adding a nucleic acid solubilizing reagent to a biomaterial, and further adding a water-soluble organic solvent or a solution containing a water-soluble organic solvent to the mixture,
(2) passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid to the porous membrane;
(3) passing the washing solution through the nucleic acid-adsorbing porous membrane and washing the porous membrane with the RNA adsorbed; and (4) passing the recovered solution through the nucleic acid-adsorbing porous membrane and removing the porous membrane from the porous membrane. In an RNA separation and purification method comprising a step of desorbing RNA,
The method for separating and purifying RNA, wherein the nucleic acid-adsorbing porous membrane is a porous membrane that adsorbs nucleic acids by an interaction that does not involve ionic bonds, and the water-soluble organic solvent concentration in the sample solution is 10 to 35% by volume.
[2]
The method for separating and purifying RNA according to [1], wherein the water-soluble solvent is at least one alcohol selected from methanol, ethanol, propanol and isomers thereof, and butanol and isomers thereof.
[3]
The method for separating and purifying RNA according to [1] or [2] above, wherein the concentration of the water-soluble solvent is 10 to 30% by volume.
[4]
The method for separating and purifying RNA according to any one of [1] to [3], wherein the water-soluble solvent concentration is 10 to 25% by volume.
[5]
The method for separating and purifying RNA according to any one of [1] to [4] above, wherein the biomaterial is a specimen containing cells or viruses.
[6]
The method for separating and purifying RNA according to [5] above, wherein the specimen containing cells is animal tissue.
[7]
In each of the steps (1), (2), (3) and (4), a container having at least two openings for allowing a sample solution, a washing solution or a recovery solution containing a nucleic acid to pass through the nucleic acid-adsorbing porous membrane. A nucleic acid separation / purification cartridge and a pressure generator containing a nucleic acid-adsorbing porous membrane through which the solution can pass, and the pressure generator can be attached to and detached from one opening of the nucleic acid separation / purification cartridge. The method for separating and purifying RNA according to any one of [1] to [6], which is a pump to be bound.
[8]
A nucleic acid separation and purification cartridge and a reagent kit for performing the nucleic acid separation and purification method according to any one of [1] to [7].
[9]
An apparatus for automatically performing the method for separating and purifying nucleic acid according to any one of [1] to [7].

  According to the present invention, a porous membrane having excellent separation performance, good cleaning efficiency, simple, rapid, excellent in automation and downsizing, and capable of mass production of substantially the same separation performance. The method for separating and purifying nucleic acid used can increase the amount of RNA recovered without clogging in the washing step.

  In the method for separating and purifying nucleic acid of the present invention, (1) a sample solution is prepared by adding a nucleic acid solubilizing reagent to a biological material, and further adding a water-soluble organic solvent or a solution containing a water-soluble organic solvent to the mixture solution. A step of creating a solution, (2) a step of passing a sample solution containing nucleic acid through the nucleic acid-adsorbing porous membrane, and adsorbing the nucleic acid to the porous membrane, (3) passing a washing solution through the nucleic acid-adsorbing porous membrane, It includes at least a step of washing the porous membrane with RNA adsorbed, and (4) a step of passing the recovered liquid through the nucleic acid-adsorbing porous membrane to desorb RNA from the porous membrane.

  Preferably, in each of the above steps (1), (2) and (3), the sample solution, the washing solution or the recovery solution containing the nucleic acid mixture containing DNA and RNA is applied to the nucleic acid-adsorbing porous membrane using a pressure generator. More preferably, in the steps (1), (2) and (3), the nucleic acid separation and purification cartridge in which the nucleic acid-adsorbing porous membrane is housed in a container having at least two openings. A sample solution containing nucleic acid, a washing solution, or a recovery solution is injected into one opening of the cartridge, and the cartridge is pressurized by using a pressure difference generator connected to the one opening of the cartridge. The liquid is allowed to pass through and discharged from other openings. By passing the sample solution, the cleaning liquid, or the recovery liquid through the porous membrane under pressure, the apparatus can be automated in a compact manner, which is preferable. The pressurization of the pump is preferably performed in the range of 10 to 300 kPa, more preferably 40 to 200 kPa.

More preferably, RNA can be separated and purified by the following steps using a nucleic acid separation and purification cartridge containing the nucleic acid-adsorbing porous membrane.
That is, (a) a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane in which a sample solution containing a nucleic acid mixture containing DNA and RNA is allowed to pass through the inside of a container having at least two openings. A step of injecting into one opening; (b) using a pressure difference generator connected to the one opening of the nucleic acid separation and purification cartridge to pressurize the inside of the nucleic acid separation and purification cartridge, and A step of adsorbing nucleic acid in the nucleic acid adsorbing porous membrane by passing through the adsorbing porous membrane and discharging from the other opening of the nucleic acid separating and purifying cartridge; (c) a washing liquid in the one opening of the nucleic acid separating and purifying cartridge; (D) the nucleic acid separation / purification cartridge is pressurized using a pressure difference generator connected to the one opening of the nucleic acid separation / purification cartridge, and the injected washing is performed. (E) the step of washing the nucleic acid-adsorbing porous membrane in a state where the nucleic acid is adsorbed by passing the purified solution through the nucleic acid-adsorbing porous membrane and discharging it from another opening; (F) injecting the recovered solution into the opening of the cartridge, and (f) applying pressure inside the nucleic acid separation / purification cartridge using a pressure difference generator coupled to one opening of the nucleic acid separation / purification cartridge. A step of allowing the nucleic acid to be desorbed from the inside of the nucleic acid-adsorbing porous membrane by passing through the adsorptive porous membrane and discharging from other openings and discharging out of the cartridge for nucleic acid separation and purification can be mentioned.

  In the above-mentioned nucleic acid separation and purification step, the steps from injection of the sample solution containing the first nucleic acid to obtaining the nucleic acid outside the nucleic acid separation and purification cartridge can be completed within substantially 30 minutes, and in a suitable situation within 5 minutes. It is.

  In the nucleic acid fraction purification step, the measurement value 1 (260 nm / 280 nm) with an ultraviolet-visible spectrophotometer is 1.8 to 2.2, and the measurement value 2 (260 nm / 230 nm) is 1.5 or more. RNA having a certain purity can be recovered, and a high-purity nucleic acid with a small amount of impurities can be steadily obtained. Furthermore, it is possible to recover a nucleic acid having a purity with a measurement value 1 (260 nm / 280 nm) of around 2.0 and a measurement value 2 (260 nm / 230 nm) of 1.7 or more with an ultraviolet-visible spectrophotometer.

  In the above process, examples of the pressure difference generating device include a pump capable of pressurization such as a syringe, a pipetter, and a peristaltic pump, and a device capable of depressurization such as an evaporator. Of these, a syringe is suitable for manual operation, and a pump is suitable for automatic operation. Also, the pipetter has the advantage that it can be easily operated by one hand. Preferably, the pressure difference generator is detachably coupled to one opening of the nucleic acid separation / purification cartridge.

  Further, in the above step, the inside of the nucleic acid separation / purification cartridge can be suitably put into a reduced pressure state using a pressure difference generator connected to another opening of the nucleic acid separation / purification cartridge. Moreover, it can implement suitably also by making a centrifugal force act on a nucleic acid isolation | separation purification cartridge.

  The specimen that can be used as the biomaterial in the present invention is not particularly limited as long as it contains a nucleic acid. For example, in the diagnostic field, whole blood, plasma, serum, urine, stool, semen, saliva and the like collected as a specimen are used. Of interest are bodily fluids or solutions prepared from biological materials such as plants (or parts thereof), animals (or parts thereof), bacteria, viruses, cultured cells, etc., or lysates and homogenates thereof. Animal tissue is used as an animal (or part thereof) specimen. All tissues constituting an individual such as liver, kidney, spleen, brain, heart, lung, thymus, large intestine, stomach, esophagus, lymph node, and skin that can be collected when the animal is dissected can be used. Examples of cultured cells include suspension cells and adhesion cells. The floating cell refers to a cell that grows and proliferates while drifting without adhering to the container wall in the culture solution, and examples thereof include HL60, U937, HeLaS3, and the like. Adhesive cells refer to cells that grow on the bottom of the container wall and grow and proliferate in the culture medium. For example, NIH3T3, HEK293, HeLa, COS, and CHO cells are typical cell lines.

  These biomaterials are usually treated with an aqueous solution (nucleic acid solubilizing reagent) containing a reagent that dissolves cell membranes and nuclear membranes to elute nucleic acids. As a result, the cell membrane / nuclear membrane is dissolved and the nucleic acid is dispersed in the aqueous solution to obtain a sample solution containing the nucleic acid.

  Below, the process of dissolving the cell membrane / nuclear membrane and eluting the nucleic acid to obtain a sample solution containing the nucleic acid from the biological material will be described. The step usually includes (I) a step of injecting a biomaterial (a specimen containing cells or viruses) into a container, (II) a nucleic acid solubilizing reagent solution is added to the container, and the specimen and the nucleic acid solubilizing reagent solution are added. And (III) adding a water-soluble organic solvent to the mixed solution obtained above to obtain a sample solution.

A nucleic acid solubilizing reagent is used to dissolve the cell membrane / nuclear membrane to elute the nucleic acid. Examples of the nucleic acid solubilizing reagent include a solution containing at least one of a chaotropic salt, a nucleic acid stabilizer, a surfactant, a buffering agent, and an antifoaming agent. The nucleic acid solubilizing reagent may consist of one solution or may be divided into two or more solutions.

  The concentration of the chaotropic salt in the nucleic acid solubilizing reagent is preferably 0.5 mol / L or more, more preferably 0.5 mol / L to 8 mol / L, still more preferably 1 mol / L to 6 mol / L. is there. The chaotropic salt is not particularly limited, and a known chaotropic salt can be used. As the chaotropic salt, a guanidine salt, sodium isothiocyanate, sodium iodide, potassium iodide or the like can be used. Of these, guanidine salts are preferred. Examples of the guanidine salt include guanidine hydrochloride, guanidine isothiocyanate, and guanidine thiocyanate (guanidine thiocyanate). Among them, guanidine hydrochloride or guanidine thiocyanate is preferable. These salts may be used alone or in combination.

  The surfactant in the nucleic acid solubilizing reagent is, for example, a nonionic surfactant, a cationic surfactant, an anionic surfactant, or an amphoteric surfactant. In the present invention, a nonionic surfactant can be preferably used. As the nonionic surfactant, a polyoxyethylene alkylphenyl ether surfactant, a polyoxyethylene alkyl ether surfactant, or a fatty acid alkanolamide can be used. Preferably, a polyoxyethylene alkyl ether surfactant is used. More preferably, the polyoxyethylene alkyl ether surfactant that can be used is POE decyl ether, POE lauryl ether, POE tridecyl ether, POE alkylene decyl ether, POE sorbitan monolaurate, POE sorbitan monooleate, Polyoxyethylene alkyl ether selected from POE sorbitan monostearate, polyoxyethylene sorbite tetraoleate, POE alkylamine, POE acetylene glycol A surfactant.

  A cationic surfactant can also be preferably used. More preferably, the cationic surfactant is a cationic surfactant selected from cetyltrimethylammonium promide, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, cetylpyridinium chloride. These surfactants may be used alone or in combination. The concentration of these surfactants in the nucleic acid solubilizing reagent solution is preferably 0.1 to 20% by mass.

  The nucleic acid solubilizing reagent is preferably allowed to coexist with a nucleic acid stabilizer. The sample may contain a nuclease or the like that degrades the nucleic acid, and when the sample is homogenized, the nuclease may act on the nucleic acid to reduce the yield. For the purpose of avoiding this, a stabilizer having an action of inactivating nuclease can coexist in the nucleic acid solubilizing solution. As a result, the amount and efficiency of nucleic acid recovery are improved, and the amount of specimen can be reduced and accelerated.

In general, a reducing agent can be preferably used as the nuclease inactivating agent.
Examples of the reducing agent include hydrogen, hydrogen iodide, hydrogen sulfide, lithium aluminum hydride, hydrogenated compounds such as sodium borohydride, alkali metals, metals with high electrical positiveity such as magnesium, calcium, aluminum, and zinc, or Amalgams, aldehydes, saccharides, organic oxides such as formic acid and oxalic acid, and the like, and mercapto compounds are preferred. Examples of the mercapto compound include N-acetylcysteine, mercaptoethanol, and alkyl mercaptan, but are not particularly limited. The mercapto compound can be used as a pretreatment liquid at a concentration of 0.1 to 20% by mass, more preferably 0.5 to 15% by mass.

The nucleic acid solubilizing reagent preferably contains an antifoaming agent. The antifoaming agent preferably includes two components, a silicon-based antifoaming agent and an alcohol-based antifoaming agent, and the alcohol-based antifoaming agent is preferably an acetylene glycol surfactant.

  Specific examples of antifoaming agents include silicon-based antifoaming agents (eg, silicone oil, dimethylpolysiloxane, silicone emulsion, modified polysiloxane, silicone compound, etc.), alcohol-based antifoaming agents (eg, acetylene glycol, heptanol, ethyl). Exanol, higher alcohol, polyoxyalkylene glycol, etc.), ether type antifoaming agents (eg, heptylcellosolve, nonylcellosolv-3-heptylcorbitol, etc.), oils and fats type antifoaming agents (eg, animal and vegetable oils, etc.), fatty acid type Antifoaming agents (eg, stearic acid, oleic acid, palmitic acid, etc.), metal soap type antifoaming agents (eg, aluminum stearate, calcium stearate, etc.), fatty acid ester antifoaming agents (eg, natural wax, tributyl phosphate, etc.) ) Phosphate-based antifoaming agents (for example, sodium octyl phosphate), amine-based antifoaming agents (for example, diamylamine), amide-based antifoaming agents (for example, stearamide), and other antifoaming agents (for example, Ferric sulfate, bauxite, etc.). Particularly preferably, the antifoaming agent can be used in combination of two components, a silicon antifoaming agent and an alcohol antifoaming agent. Moreover, it is also preferable to use an acetylene glycol surfactant as the alcohol-based antifoaming agent.

  The nucleic acid solubilizing reagent solution may contain a water-soluble organic solvent. The water-soluble organic solvent is intended to increase the solubility of various reagents contained in the nucleic acid solubilizing reagent, and includes acetone, chloroform, dimethylformamide and the like, with alcohol being preferred. The alcohol may be any of primary alcohol, secondary alcohol, and tertiary alcohol. As the alcohol, methyl alcohol, ethyl alcohol, propyl alcohol and its isomer, butyl alcohol and its isomer can be used more preferably. These water-soluble organic solvents may be used alone or in combination. The concentration of these water-soluble organic solvents in the nucleic acid solubilizing reagent solution is preferably 1 to 20% by mass.

The nucleic acid solubilizing reagent solution is preferably one having a pH of 3 to 8, more preferably a pH of 4 to 7, and even more preferably a pH of 5 to 7.
Specific examples of the buffer material used in the present invention include a commonly used pH buffer (buffer). Preferably, a biochemical pH buffer is used. Examples of such a buffer include a buffer consisting of citrate, phosphate or acetate, Tris-HCl, TE (Tris-HCl / EDTA), TBE (Tris-Borate / EDTA), TAE (Tris-Acetate). / EDTA), Good buffer. Good buffering agents include MES (2-Morpholinoethanesulfonic acid), Bis-Tris (Bis (2-hydoroxyethyl) iminotris (hydroxymethyl) methane), HEPES (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic asid ), PIPES (Piperaxine-1,4-bis (2-ethanesulfonic acid)), ACES (N- (2-Acetamino) -2-aminoethanesulfonic acid), CAPS (N-Cyclohexyl-3-aminopropanesulfonic acid), TES (N -Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid).
These buffers preferably have a concentration in the nucleic acid solubilizing reagent of 1 to 500 mmol / L.

In the process of dissolving the cell membrane / nuclear membrane and solubilizing the nucleic acid to obtain the sample solution containing the nucleic acid from the biological material, the sample can be homogenized before or after the nucleic acid solubilizing reagent is added. improves. The homogenization treatment can be performed by, for example, ultrasonic treatment, using sharp protrusions, using high-speed stirring treatment, treatment extruding from fine voids, treatment using beads such as glass, stainless steel, and zirconia. In order to perform these treatments, for example, a mixer such as a vortex, a homogenizer such as a rotor-stator type, a potter type or a downs type, or a commercially available homogenizer such as a bead mill, a pestle, a French press, a grinder, a blade homogenizer or the like is used. be able to. When the homogenization treatment is performed before the nucleic acid solubilizing reagent is added, the sample can be frozen with liquid nitrogen and then treated with a bead mill, a crusher mill, a mortar, a grinder, or the like.

  The method for mixing the homogenized specimen with a nucleic acid solubilizing reagent reagent containing any one or more of chaotropic salts, nucleic acid stabilizers, surfactants, buffers and antifoaming agents is not particularly limited. When mixing, it is preferable to mix at 30 to 3000 rpm for 1 second to 3 minutes with a stirrer. Thereby, the yield of nucleic acid to be separated and purified can be increased. Or it is also preferable to mix by inversion mixing 5 to 30 times. In addition, mixing can also be performed by performing pipetting operations 10 to 50 times. In this case, the yield of nucleic acid separated and purified by a simple operation can be increased.

When the specimen is a cultured cell, yeast or the like, it is desirable to treat the number of cells of 10 to 1 × 10 ⁸ with 50 to 1000 μl of nucleic acid solubilizing reagent. When the specimen is a tissue such as an animal or a plant, it is desirable to treat the tissue amount of 0.1 mg to 200 mg with 50 to 1000 μl of the nucleic acid solubilizing reagent. When the specimen is bacteria, it is desirable to treat 1 × 10 ⁸ to 1 × 10 ¹⁰ bacteria with 50 to 1000 μl of the nucleic acid solubilizing reagent. The amount of the nucleic acid solubilizing reagent can be changed within a range in which the sample is dissolved and does not exceed the capacity of the cartridge.

  In the step of dissolving the cell membrane / nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the biological material, a water-soluble organic solvent is then added to the mixed solution. As the water-soluble organic solvent added to the mixed solution, an alcohol can be preferably used. The alcohol may be any of primary alcohol, secondary alcohol, and tertiary alcohol, and methanol, ethanol, propanol and its isomer, butanol and its isomer can be preferably used. Two or more kinds of these alcohols may be used. The final concentration of the sample solution containing nucleic acids of these water-soluble organic solvents is preferably 10 to 35% by volume. More preferably, it is 10 to 30% by volume. In order to reduce clogging without reducing the amount of RNA recovered, the content is preferably 10 to 25% by volume. Depending on the type of biomaterial, it is preferably 15 to 20% by volume. When mixing, it is preferable to mix at 30 to 3000 rpm for 1 second to 3 minutes with a stirrer. Thereby, the yield of nucleic acid to be separated and purified can be increased. Or it is also preferable to mix by inversion mixing 5 to 30 times. Mixing can also be performed by performing pipetting operations 10 to 50 times.

Moreover, the sample solution containing the obtained nucleic acid preferably has a surface tension of 0.05 J / m ² or less, a viscosity of preferably 1 to 10000 mPa, and a specific gravity of 0.8 to 1. .2 is preferable. By using a solution having such physical properties, the sample solution residue is easily removed after the sample solution is brought into contact with the nucleic acid-adsorbing porous membrane in the next step.

Hereinafter, the nucleic acid-adsorbing porous membrane used in the present invention and the adsorption step will be described.
The nucleic acid-adsorptive porous membrane of the present invention is a solution through which a solution can pass. Here, “solution can pass through” means that when a pressure difference is generated between the space where one surface of the membrane contacts and the space where the other surface of the membrane contacts, To the side, it means that the solution can pass through the inside of the membrane. Or, when a centrifugal force is applied to the membrane, it means that the solution can pass through the membrane in the direction of the centrifugal force.

  Further, the nucleic acid-adsorbing porous membrane of the present invention is a porous membrane that adsorbs nucleic acids by an interaction that does not substantially involve ionic bonds. This means that it is not “ionized” under the use conditions on the porous membrane side, and it is presumed that the nucleic acid and the porous membrane come to attract each other by changing the polarity of the environment. As a result, the nucleic acid can be isolated and purified with excellent separation performance and good washing efficiency. Preferably, the nucleic acid-adsorptive porous membrane is a porous membrane having a hydrophilic group, and it is presumed that the hydrophilic group of the nucleic acid and the porous membrane are drawn by changing the polarity of the environment.

  Here, the hydrophilic group means a polar group (atomic group) capable of interacting with water, and all groups (atomic groups) involved in nucleic acid adsorption are applicable. As hydrophilic groups, those with moderate interaction with water are good (see Chemical Encyclopedia, Kyoritsu Shuppan Co., Ltd., “Hydrophilic groups”, “Groups that are not very hydrophilic”). Examples thereof include a hydroxyl group, a carboxyl group, a cyano group, and an oxyethylene group. Preferably it is a hydroxyl group.

  In the present invention, the porous film having a hydrophilic group is a porous film having a hydrophilic group introduced by treating or coating a porous film having a hydrophilic group or a material forming the porous film. Means a membrane. The material for forming the porous film may be either organic or inorganic. For example, a porous film in which the material forming the porous film itself is an organic material having a hydrophilic group, a porous film in which a porous film of an organic material having no hydrophilic group is treated to introduce a hydrophilic group, or an organic material having no hydrophilic group A porous film in which a hydrophilic group is introduced by coating with a material having a hydrophilic group on the porous film, a porous film in which the material forming the porous film itself is an inorganic material having a hydrophilic group, a porous film of an inorganic material having no hydrophilic group A porous membrane in which a hydrophilic group is introduced by treating the membrane, a porous membrane in which a hydrophilic group is introduced by coating with a material having a hydrophilic group on a porous membrane of an inorganic material that does not have a hydrophilic group can be used. From the viewpoint of ease of processing, it is preferable to use an organic material such as an organic polymer as the material for forming the porous film.

  Examples of porous membranes having hydrophilic groups include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, acetylcellulose, and acetyl having different acetyl values. The formed porous film can be exemplified by a mixture of cellulose and the like, and in particular, a porous film of an organic material having a hydroxyl group can be preferably used.

  As the porous film of an organic material having a hydroxyl group, a material having a polysaccharide structure is preferable, and an organic polymer porous film made of a mixture of acetylcelluloses having different acetyl values can be more preferably used. As a mixture of acetyl cellulose having different acetyl values, a mixture of triacetyl cellulose and diacetyl cellulose, a mixture of triacetyl cellulose and monoacetyl cellulose, a mixture of triacetyl cellulose, diacetyl cellulose and monoacetyl cellulose, a mixture of diacetyl cellulose and monoacetyl cellulose Can be preferably used. In particular, a mixture of triacetyl cellulose and diacetyl cellulose can be preferably used. The mixing ratio (mass ratio) of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99, and more preferably 90:10 to 50:50.

A more preferable organic material having a hydroxyl group is a saponified product of acetyl cellulose described in JP-A No. 2003-128691. A saponified acetyl cellulose is a saponified mixture of acetyl cellulose having different acetyl values, a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose, a saponified product of a mixture of triacetyl cellulose and monoacetyl cellulose, and triacetyl cellulose. A saponified product of a mixture of diacetylcellulose and monoacetylcellulose, and a saponified product of a mixture of diacetylcellulose and monoacetylcellulose can also be preferably used. More preferably, a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose is used. The mixing ratio (mass ratio) of the triacetyl cellulose and diacetyl cellulose mixture is preferably 99: 1 to 1:99. More preferably, the mixing ratio of the mixture of triacetyl cellulose and diacetyl cellulose is 90:10 to 50:50. In this case, the amount (density) of hydroxyl groups on the solid surface can be controlled by the degree of saponification treatment (saponification rate). In order to increase the nucleic acid separation efficiency, it is preferable that the amount (density) of hydroxyl groups is large. For example, in the case of acetylcellulose such as triacetylcellulose, the saponification rate (surface saponification rate) is preferably about 5% or more, and more preferably 10% or more. In order to increase the surface area of the organic polymer having a hydroxyl group, it is preferable to saponify the porous membrane of acetylcellulose. In this case, the porous film may be a front and back symmetric porous film, but a back asymmetric porous film can be preferably used.

  The saponification treatment refers to bringing acetyl cellulose into contact with a saponification treatment solution (for example, an aqueous sodium hydroxide solution). As a result, the ester group of the ester derivative of cellulose in contact with the saponification treatment solution is hydrolyzed, and a hydroxyl group is introduced to form regenerated cellulose. The regenerated cellulose thus prepared is different from the original cellulose in terms of crystal state and the like. In order to change the saponification rate, the saponification treatment may be performed by changing the concentration of sodium hydroxide and the treatment time. The saponification rate can be easily measured by NMR, IR, or XPS (for example, it can be determined by the degree of peak reduction of the carbonyl group).

  As a method for introducing a hydrophilic group into a porous film of an organic material having no hydrophilic group, a graft polymer chain having a hydrophilic group in a polymer chain or in a side chain can be bonded to the porous film. As a method of bonding the graft polymer chain to the porous film of the organic material, a method of chemically bonding the porous film and the graft polymer chain, and a graft polymer chain by polymerizing a compound having a polymerizable double bond starting from the porous film. There are two ways.

  First, in the method of attaching the porous membrane and the graft polymer chain by chemical bonding, a polymer having a functional group that reacts with the porous membrane is used at the terminal or side chain of the polymer. It can be grafted by chemically reacting with a group. The functional group that reacts with the porous film is not particularly limited as long as it can react with the functional group of the porous film. For example, a silane coupling group such as alkoxysilane, an isocyanate group, an amino group, a hydroxyl group, and a carboxyl group. Groups, sulfonic acid groups, phosphoric acid groups, epoxy groups, allyl groups, methacryloyl groups, acryloyl groups, and the like. Particularly useful compounds as a polymer having a reactive functional group at the end of the polymer or in the side chain are a polymer having a trialkoxysilyl group at the polymer end, a polymer having an amino group at the polymer end, and a polymer having a carboxyl group at the polymer end. , A polymer having an epoxy group at the polymer end, and a polymer having an isocyanate group at the polymer end. The polymer used at this time is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption. Specifically, polyhydroxyethylacrylic acid, polyhydroxyethylmethacrylic acid and salts thereof , Polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, and polyoxyethylene.

  A method of polymerizing a compound having a polymerizable double bond from a porous film as a base point to form a graft polymer chain is generally called surface graft polymerization. Surface graft polymerization is a method in which active species are given to the substrate surface by methods such as plasma irradiation, light irradiation, and heating, and a compound having a polymerizable double bond disposed so as to be in contact with the porous film is made porous by polymerization. Refers to the method of binding to a membrane A compound useful for forming a graft polymer chain bound to a substrate has a double bond that can be polymerized and has a hydrophilic group that participates in nucleic acid adsorption. It is necessary to be. As these compounds, any polymer, oligomer, or monomer compound having a hydrophilic group can be used as long as it has a double bond in the molecule. Particularly useful compounds are monomers having hydrophilic groups. Specific examples of the particularly useful monomer having a hydrophilic group include the following monomers. For example, a hydroxyl group-containing monomer such as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and glycerol monomethacrylate can be particularly preferably used. Also, carboxyl group-containing monomers such as acrylic acid and methacrylic acid, or alkali metal salts and amine salts thereof can be preferably used.

  As another method for introducing a hydrophilic group into a porous film of an organic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for the coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of easy work. Examples of polymers include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, acetylcellulose, and different acetyl values. Examples thereof include a mixture of acetylcellulose, and a polymer having a polysaccharide structure is preferable.

  In addition, after coating a porous film of an organic material having no hydrophilic group with acetyl cellulose or a mixture of acetyl cellulose having different acetyl values, the coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values is saponified. You can also. In this case, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.

  Examples of the porous film that is an inorganic material having a hydrophilic group include a porous film containing a silica compound. A glass filter can be mentioned as a porous film containing a silica compound. Further, a porous silica thin film as described in Japanese Patent Publication No. 3058342 can be given. This porous silica thin film is an amphiphilic substance by spreading a developing solution of a cationic type amphiphilic substance having a bilayer-forming ability on a substrate and then removing the solvent from the liquid film on the substrate. This multilayer bilayer thin film can be prepared by bringing the multilayer bilayer thin film into contact with a solution containing a silica compound, and then extracting and removing the multilayer bilayer thin film.

  As a method for introducing a hydrophilic group into a porous film of an inorganic material having no hydrophilic group, a method of chemically bonding the porous film and a graft polymer chain, and a monomer having a hydrophilic group having a double bond in the molecule There are two methods for polymerizing the graft polymer chain from the porous membrane as a starting point. When the porous film and the graft polymer chain are attached by chemical bonding, a functional group that reacts with the functional group at the terminal of the graft polymer chain is introduced into the inorganic material, and the graft polymer is chemically bonded thereto. In addition, when polymerizing a graft polymer chain starting from a porous membrane using a monomer having a hydrophilic group having a double bond in the molecule, the starting point for polymerizing a compound having a double bond Is introduced into the inorganic material.

  As a graft polymer having a hydrophilic group and a monomer having a hydrophilic group having a double bond in the molecule, the above-described method of chemically bonding a porous film of an organic material having no hydrophilic group and a graft polymer chain In the above, the described graft polymers having a hydrophilic group and monomers having a hydrophilic group having a double bond in the molecule can be preferably used.

  As another method for introducing a hydrophilic group into a porous film made of an inorganic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for the coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of easy work. Examples of polymers include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, acetylcellulose, and different acetyl values. Examples thereof include a mixture of acetylcellulose.

In addition, after coating a porous film of an inorganic material having no hydrophilic group with acetyl cellulose or a mixture of acetyl celluloses having different acetyl values, the coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values is saponified. You can also. In this case, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.

  Examples of the porous film made of an inorganic material having no hydrophilic group include a porous film produced by processing a metal such as aluminum, ceramics such as glass, cement, and ceramics, or new ceramics, silicon, and activated carbon.

  The nucleic acid-adsorptive porous membrane allows the solution to pass through the inside and has a thickness of 10 μm to 500 μm. More preferably, the thickness is 50 μm to 250 μm. The thinner the thickness, the easier it is to clean.

  The nucleic acid-adsorbing porous membrane through which the above solution can pass has a minimum pore size of 0.22 μm or more. More preferably, the minimum pore diameter is 0.5 μm or more. Further, it is preferable to use a porous membrane having a ratio of the maximum pore diameter to the minimum pore diameter of 2 or more. As a result, a sufficient surface area for adsorbing nucleic acids is obtained and clogging is difficult. More preferably, the ratio of the maximum pore diameter to the minimum pore diameter is 5 or more.

The nucleic acid-adsorbing porous membrane through which the above solution can pass has a porosity of 50 to 95%. More preferably, the porosity is 65 to 80%. Moreover, it is preferable that a bubble point is 0.1-10 kgf / cm < ² >. More preferably, the bubble point is 0.2 to 4 kgf / cm ² .

  The nucleic acid-adsorptive porous membrane that allows the solution to pass through the inside thereof preferably has a pressure loss of 0.1 to 100 kPa. Thereby, a uniform pressure is obtained at the time of overpressure. More preferably, the pressure loss is 0.5 to 50 kPa. Here, the pressure loss is the minimum pressure required to pass water per 100 μm thickness of the membrane.

The nucleic acid-adsorbing porous membrane through which the solution can pass inside has a water permeability of 1 to 5000 mL per minute per cm ² membrane when water is passed at 25 ° C. at a pressure of 1 kg / cm ^2. It is preferable. More preferably, the water permeation amount when water is passed at 25 ° C. and a pressure of 1 kg / cm ² is 5 to 1000 mL per cm ^{2 of} membrane.

  In the nucleic acid-adsorbing porous membrane through which the solution can pass, the nucleic acid adsorption amount per mg of the porous membrane is preferably 0.1 μg or more. More preferably, the amount of nucleic acid adsorbed per 1 mg of the porous membrane is 0.9 μg or more.

  The nucleic acid-adsorbing porous membrane through which the solution can pass is a cellulose that does not dissolve within 1 hour but dissolves within 48 hours when a square porous membrane with a side of 5 mm is immersed in 5 mL of trifluoroacetic acid. Derivatives are preferred. Further, a cellulose derivative that dissolves within 1 hour when immersed in 5 mL of trifluoroacetic acid, but a cellulose derivative that does not dissolve within 24 hours when immersed in 5 mL of dichloromethane is more preferable.

  When passing a sample solution containing nucleic acid through the nucleic acid-adsorbing porous membrane, passing the sample solution from one surface to the other surface allows the liquid to be uniformly contacted with the porous membrane. preferable. When a sample solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane, it is preferable that the sample solution is passed from the side having the larger pore diameter of the nucleic acid-adsorbing porous membrane to the side having a smaller pore size because clogging is difficult.

The flow rate when passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane may be ² to 1500 μL / sec per cm ^{2 of} the membrane in order to obtain an appropriate contact time of the liquid with the porous membrane. preferable.
If the contact time of the liquid with the porous membrane is too short, a sufficient separation and purification effect cannot be obtained. Further, the flow rate is preferably 5 to 700 μL / sec per cm ^{2 of} the membrane area.

  Further, the number of nucleic acid-adsorbing porous membranes through which the solution to be used can pass may be one, or a plurality of nucleic acid-adsorbing porous membranes can be used. The plurality of nucleic acid-adsorptive porous membranes may be the same or different.

  The plurality of nucleic acid adsorbing porous membranes may be a combination of an inorganic material nucleic acid adsorbing porous membrane and an organic material nucleic acid adsorbing porous membrane. For example, a combination of a glass filter and a porous membrane of regenerated cellulose can be mentioned. The plurality of nucleic acid adsorbing porous membranes may be a combination of an inorganic material nucleic acid adsorbing porous membrane and an organic material non-nucleic acid adsorbing porous membrane. For example, a glass filter and nylon or polysulfone porous A combination with a membrane can be mentioned.

  A nucleic acid separation and purification cartridge in which a nucleic acid-adsorbing porous membrane capable of passing the above solution in a container having at least two openings can be preferably used. In addition, a nucleic acid separation and purification cartridge in which a plurality of nucleic acid-adsorbing porous membranes through which the above solution can pass is contained in a container having at least two openings. In this case, the plurality of nucleic acid-adsorbing porous membranes accommodated in a container having at least two openings may be the same or different.

  It is preferable that the nucleic acid separation / purification cartridge does not contain other members in a container having at least two openings other than containing a nucleic acid-adsorbing porous membrane through which the above solution can pass. As a material for the container, plastics such as polypropylene, polystyrene, polycarbonate, and polyvinyl chloride can be used. Biodegradable materials can also be preferably used. In addition, the container may be transparent or colored.

  As the nucleic acid separation / purification cartridge, a nucleic acid separation / purification cartridge provided with means for identifying individual nucleic acid separation / purification cartridges can be used. Examples of means for identifying individual nucleic acid separation and purification cartridges include bar codes and magnetic tapes.

  In addition, a nucleic acid separation and purification cartridge having a structure in which the nucleic acid-adsorbing porous membrane can be easily taken out from a container having at least two openings can be used.

Hereinafter, the cleaning process will be described.
Next, the washing solution is passed through the nucleic acid adsorbing porous membrane.

  In the present invention, the cleaning liquid is preferably an aqueous solution containing a water-soluble organic solvent at a concentration of 55 to 90% by volume, and more preferably contains a water-soluble organic solvent at a concentration of 60% to 90% by volume. It is an aqueous solution. By containing the water-soluble organic solvent in the above range, it is preferable that high-purity RNA can be recovered by washing away impurities such as proteins and low-molecular compounds without clogging in the washing step. The washing liquid needs to have a function of washing out impurities in the sample solution adsorbed together with the nucleic acid on the nucleic acid adsorbing porous membrane. For that purpose, it is necessary to have a composition that does not desorb RNA from the nucleic acid-adsorbing porous membrane but desorbs impurities.

  By setting the ratio of the water-soluble organic solvent contained in the cleaning liquid within the above range, nucleic acids and proteins that cause clogging are contracted, and as a result, clogging in the cleaning process is reduced.

As the water-soluble organic solvent contained in the cleaning liquid, methanol, ethanol, propanol and isomers thereof, butanol and isomers thereof can be used, and ethanol is particularly preferable. A plurality of these alcohols may be used.

The cleaning liquid of the present invention preferably further contains a water-soluble salt. The water-soluble salt is preferably a halide salt, and chloride is particularly preferable. The water-soluble salt is preferably a monovalent or divalent cation, particularly preferably an alkali metal salt or an alkaline earth metal salt. Among them, a sodium salt, a potassium salt and a lithium salt are preferable, and a sodium salt is most preferable.
When a water-soluble salt is contained in the cleaning solution, the concentration in the cleaning solution is preferably 10 mmol / L or more, and the upper limit is not particularly limited as long as it does not impair the solubility of impurities, but it is 1 mol / L or less. It is preferable that it is 0.1 mol / L or less. More preferably, the water-soluble salt is sodium chloride, and further, sodium chloride is contained in an amount of 20 mmol / L to 1 mol / L.
In order to stabilize the pH of the cleaning solution, it is preferable to contain a buffer. Mention may be made of commonly used pH buffers. Preferably, a biochemical pH buffer is used. Examples of such a buffer include a buffer consisting of citrate, phosphate or acetate, Tris-HCl, TE (Tris-HCl / EDTA), TBE (Tris-Borate / EDTA), TAE (Tris-Acetate). / EDTA), Good buffer. Good buffering agents include MES (2-Morpholinoethanesulfonic acid), Bis-Tris (Bis (2-hydoroxyethyl) iminotris (hydroxymethyl) methane), HEPES (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic asid ), PIPES (Piperaxine-1,4-bis (2-ethanesulfonic acid)), ACES (N- (2-Acetamino) -2-aminoethanesulfonic acid), CAPS (N-Cyclohexyl-3-aminopropanesulfonic acid), TES (N -Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid). Of these, Tris-HCl is preferable.
The concentration of these buffering agents in the washing solution is preferably 1 to 500 mmol / L. More preferably, it is 5-100 mmol / L.

Furthermore, it is preferable that the cleaning liquid does not contain a chaotropic substance. Thereby, the possibility that the chaotropic substance is mixed in the recovery process subsequent to the cleaning process can be reduced. When a chaotropic substance is mixed during the recovery step, an enzyme reaction such as a PCR reaction is often inhibited. Therefore, it is ideal that the washing solution does not contain a chaotropic substance in consideration of the subsequent enzyme reaction or the like. In addition, since chaotropic substances are corrosive and harmful, it is extremely advantageous for the experimenter from the viewpoint of safety of the test operation that the chaotropic substances need not be used. Since a chaotropic substance is not contained, high-purity RNA having a measurement value (260 nm / 230 nm) with an ultraviolet-visible spectrophotometer of 1.5 or more can be recovered. In a suitable situation, high-purity RNA having a measurement value (260 nm / 230 nm) with an ultraviolet-visible spectrophotometer of 1.7 or more, more preferably around 2.0 can be recovered.
Here, the chaotropic substance refers to the above-described chaotropic salts such as guanidine isothiocyanate, guanidine thiocyanate, sodium isothiocyanate, sodium iodide, potassium iodide, and urea.

  Conventionally, during the washing process in the nucleic acid separation and purification process, since the washing liquid has high wettability with respect to a container such as a cartridge, the washing liquid often remains in the container, and the washing liquid is mixed into the recovery process following the washing process. This causes a decrease in nucleic acid purity and a decrease in reactivity in the next step. Therefore, when nucleic acid is adsorbed and desorbed using a container such as a cartridge, the cleaning residual liquid does not remain in the cartridge so that the liquid used for adsorption and cleaning, particularly the cleaning liquid, does not affect the next step. That is important.

Therefore, the surface tension of the cleaning liquid is set to 0.035 J in order to prevent the cleaning liquid in the cleaning process from being mixed into the recovered liquid in the next process and to keep the cleaning liquid remaining in the cartridge to a minimum.
/ M ² is preferable. When the surface tension is low, the wettability between the cleaning liquid and the cartridge is improved, and the remaining liquid volume can be suppressed.

Here, in order to increase the cleaning efficiency, the proportion of water can be increased, but in this case, the surface tension of the cleaning liquid increases and the amount of the remaining liquid increases. When the surface tension of the cleaning liquid is 0.035 J / m ² or more, the remaining liquid amount can be suppressed by increasing the water repellency of the cartridge. By increasing the water repellency of the cartridge, droplets are formed, and the amount of liquid remaining by the droplets flowing down can be suppressed. As a method for increasing the water repellency, there are means such as coating the surface of the cartridge with a water repellent such as silicon, or kneading a water repellent such as silicon when molding the cartridge, but is not limited thereto.

  Further, by automating the cleaning and recovery operation, the operation can be performed easily and quickly. The cleaning step may be completed by one cleaning for speeding up, and when the purity is more important, the cleaning is preferably repeated a plurality of times.

In the washing step, the washing solution is supplied to the nucleic acid separation / purification cartridge containing the nucleic acid-adsorbing porous membrane using a tube, pipette, automatic injection device, or supply means having the same function. The supplied washing solution is supplied from one opening of the cartridge for separating and purifying nucleic acid (opening into which a sample solution containing nucleic acid is injected), and a pressure difference generator (for example, a spoid, a syringe, a pump, a power pipette, etc.) coupled to the opening. ), The inside of the cartridge for separating and purifying nucleic acid is pressurized and passed through the porous nucleic acid-adsorbing porous membrane, and discharged from an opening different from one opening. Also, the cleaning liquid can be supplied from one opening and discharged from the same opening. Furthermore, it is also possible to supply and discharge the cleaning liquid from an opening different from the one opening to which the sample solution containing the nucleic acid in the cartridge for nucleic acid separation and purification is supplied. However, a method of supplying from one opening of the nucleic acid separation and purification cartridge, passing through the nucleic acid-adsorptive porous membrane, and discharging from an opening different from the one opening is more preferable because of excellent cleaning efficiency. The amount of the cleaning solution in the cleaning step is preferably ² μl / mm ² or more. If the amount of the cleaning solution is large, the cleaning effect is improved, but 200 μl / mm ² or less is preferable in order to maintain the operability and suppress the outflow of the sample.

In the washing step, the flow rate when the washing solution is passed through the nucleic acid-adsorbing porous membrane is preferably 2 to 1500 μL / sec, more preferably 5 to 700 μL / sec, per unit area (cm ² ) of the membrane. preferable. If the passage speed is lowered and time is taken, the washing is sufficiently performed. However, since the speed of the separation and purification operation of the nucleic acid is important, the above range is selected.

  In the washing step, the temperature of the washing solution is preferably 4 to 70 ° C. Furthermore, it is more preferable that the temperature of the cleaning liquid is room temperature. In the washing step, the cartridge for separating and purifying nucleic acid can be washed while being subjected to mechanical vibration, stirring by ultrasonic waves, or by centrifugation.

  According to the present invention, the washing process can be simplified by using the nucleic acid-adsorbing porous membrane. That is, (1) the number of times the washing solution passes through the nucleic acid-adsorbing porous membrane may be one, and (2) the washing step can be performed at room temperature. (3) The recovered liquid can be poured into the cartridge immediately after washing. (4) Any one or more of (1), (2), and (3) are also possible. In the conventional method, a drying step is often required in order to quickly remove the organic solvent contained in the cleaning liquid. However, since the nucleic acid-adsorbing porous membrane according to the present invention is a thin film, it can be omitted.

Conventionally, in the nucleic acid separation and purification process, there is a problem that contamination of the sample occurs due to the washing liquid often scattering and adhering to the other during the washing process. This kind of contamination in the washing process can be suppressed by devising the shape of the waste liquid container and the nucleic acid separation and purification cartridge in which the nucleic acid-adsorbing polyporous membrane is housed in a container having two openings.

DNase can be allowed to act during the washing step. In order to selectively separate and purify only RNA from a nucleic acid mixture solution containing DNA and RNA, it was passed through a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane, and nucleic acid was adsorbed to the nucleic acid-adsorbing porous membrane (adsorption After the step), it is preferable to carry out washing (washing step 1) and then undergo a step of allowing DNase to act. Furthermore, in order to remove impurities such as DNase and other proteins / salts, it can be washed after DNase action (washing step 2).
There is no particular limitation on DNase, and any DNase can be used. For example, DNase I derived from animal pancreas such as bovine and recombinant DNase prepared by gene recombination technology can be used.
Divalent metal ions such as magnesium, calcium, and manganese suitable for expression of DNase activity may be added to the DNase solution (hereinafter also referred to as DNase reaction solution) when DNase is allowed to act. Further, sodium salts, lithium salts, potassium salts, and the like can coexist in order to retain nucleic acids in the nucleic acid-adsorptive porous membrane.
In order to adjust to the optimum pH of DNase activity, the DNase reaction solution also contains a buffer. As the buffering agent, commonly used TrisHCl, HEPES, phosphate buffer and the like can be used.
In the method of the present invention, the total amount of the DNase solution in the step of allowing DNase to act on the nucleic acid-adsorbing porous membrane of the nucleic acid separation and purification cartridge is 250 μl or less per 1 cm ² of the nucleic acid-adsorbing porous membrane. In addition, in the step of allowing DNase to act on the nucleic acid-adsorbing porous membrane of the nucleic acid separation and purification cartridge, the DNase concentration in the DNase solution (hereinafter also simply referred to as DNase concentration) is preferably 10 Kunitz U / mL or more and 10,000 Kunitz U / mL or less, and 50 Kunitz U / ML or more and 5000 Kunitz U / mL or less is more preferable. The activity Kunitz U used here is defined as “DNase activity that increases the absorbance of A260 in 1 ml of reaction solution by 0.001 per minute at 25 ° C. and pH 5.0 with 1 Kunitz U using DNA as a substrate”. It is a thing. In addition, the time in the step of allowing DNase to act on the nucleic acid-adsorbing porous membrane of the nucleic acid separation and purification cartridge varies depending on the amount of DNA in the nucleic acid mixture solution containing DNA and RNA and the DNase concentration to be acted, but is preferably 5 seconds to 360 minutes. 30 seconds to 180 minutes is more preferable. In addition, the temperature in the step of allowing DNase to act on the nucleic acid-adsorptive porous membrane of the nucleic acid separation and purification cartridge may be 4 ° C. or higher, preferably 10 to 50 ° C., and is performed at a high temperature, for example, 50 to 70 ° C. to increase the reaction efficiency. You can also. In addition, “to make DNase act on the nucleic acid-adsorbing porous membrane” means to act on the portion of the nucleic acid-adsorbing porous membrane where the nucleic acid is adsorbed and DNase. Includes not only the acid-adsorbing porous membrane but also the pores in the porous membrane and the outlet of the pores on the back side of the membrane.

Next, a process of desorbing and recovering RNA from the nucleic acid adsorbing polyporous membrane will be described.
In the recovery step, the recovery liquid is supplied to the nucleic acid separation / purification cartridge equipped with the nucleic acid-adsorbing porous membrane using a tube, pipette, automatic injection device, or supply means having the same function. The recovered liquid is supplied from one opening of the cartridge for separating and purifying nucleic acid (opening into which a sample solution containing nucleic acid is injected), and a pressure difference generator (for example, a spoid, a syringe, a pump, a power pipette, etc.) coupled to the opening is connected to the recovered liquid. By using it, the inside of the cartridge for separating and purifying nucleic acid can be pressurized and passed through the nucleic acid-adsorbing porous membrane, and discharged from an opening different from one opening. Further, the recovery liquid can be supplied from one opening and discharged from the same opening. Furthermore, it is also possible to supply and discharge the recovery liquid from an opening different from the one opening to which the sample solution containing the nucleic acid in the nucleic acid separation and purification cartridge is supplied. However, a method of supplying from one opening of the nucleic acid separation and purification cartridge, passing through the nucleic acid-adsorptive porous membrane, and discharging from an opening different from the one opening is more preferable because of excellent recovery efficiency.

  Nucleic acid can be desorbed by adjusting the volume of the recovered liquid with respect to the volume of the sample solution containing the nucleic acid prepared from the specimen. The amount of the recovered liquid containing the separated and purified nucleic acid depends on the amount of sample used at that time. In general, the volume of recovered liquid used is from several tens to several hundreds of microliters. However, when the amount of specimen is extremely small, or conversely when a large amount of nucleic acid is to be separated and purified, the volume of recovered liquid is from 1 μl to several tens of ml. You can change the range.

  As the recovered solution, preferably, purified distilled water, Tris-HCl buffer, Tris / EDTA buffer, or the like can be used. In addition, when the collected nucleic acid is subjected to PCR (polymerase chain reaction), a buffer solution used in the PCR reaction (for example, an aqueous solution having a final concentration of KCl 50 mmol / L, Tris-HCl 10 mmol / L, MgCl2 1.5 mmol / L) is used. It can also be used.

  The pH of the collected liquid is preferably pH 2-11. Furthermore, it is preferable that it is pH 5-9. In particular, ionic strength and salt concentration have an effect on the elution of adsorbed nucleic acids. The recovered liquid preferably has an ionic strength of 290 mmol / L or less, and more preferably has a salt concentration of 90 mmol / L or less. By doing so, the recovery rate of nucleic acids is improved, and more nucleic acids can be recovered.

  By reducing the volume of the recovery solution compared to the volume of the sample solution containing the original nucleic acid, a recovery solution containing concentrated nucleic acid can be obtained. Preferably, (recovered liquid volume) :( sample solution volume) = 1: 100 to 99: 100, more preferably (recovered liquid volume) :( sample solution volume) = 1: 10 to 9:10 it can. Thus, the nucleic acid can be easily concentrated without performing an operation for concentration in the step after the separation and purification of the nucleic acid. By these methods, it is possible to provide a method for obtaining a nucleic acid solution in which the nucleic acid is more concentrated than the specimen.

  As another method, nucleic acid is desorbed under the condition that the volume of the recovery solution is larger than that of the sample solution containing the initial nucleic acid, whereby a recovery solution containing a desired concentration of nucleic acid can be obtained. A recovery solution containing a nucleic acid at a concentration suitable for PCR (such as PCR) can be obtained. Preferably, (recovered liquid volume) :( sample solution volume) = 1: 1 to 50: 1, more preferably (recovered liquid volume) :( sample solution volume) = 1: 1 to 5: 1. it can. As a result, there is an advantage that there is no need to adjust the concentration after separation and purification of nucleic acid. Furthermore, by using a sufficient amount of the recovery liquid, it is possible to increase the nucleic acid recovery rate from the porous membrane.

  In addition, nucleic acids can be easily recovered by changing the temperature of the recovery solution according to the purpose. For example, the nucleic acid is desorbed from the porous membrane by setting the temperature of the recovered liquid to 0 to 10 ° C., thereby suppressing the function of the nucleolytic enzyme without adding any reagent or special operation for preventing the enzymatic degradation. Thus, nucleic acid solution can be easily and efficiently obtained by preventing nucleic acid degradation.

  In addition, when the temperature of the recovery solution is 10 to 35 ° C., the nucleic acid can be recovered at a general room temperature, and can be separated and purified by removing the nucleic acid without requiring a complicated process. .

  As another method, the temperature of the recovery liquid is set to a high temperature, for example, 35 to 70 ° C., so that the nucleic acid can be easily detached from the porous membrane at a high recovery rate without complicated operations.

  After the recovery liquid is injected, the recovery amount can be increased by immersing the porous membrane in the recovery liquid. The immersion time is preferably 10 seconds or longer, and more preferably 30 seconds or longer.

  The number of injections of the recovery liquid is not limited and may be one time or multiple times. Usually, when the nucleic acid is separated and purified quickly and easily, it is carried out by one collection, but the collection liquid may be injected several times, such as when collecting a large amount of nucleic acid.

  In the recovery step, the nucleic acid recovery solution can be made into a composition that can be used in the subsequent subsequent steps. The separated and purified nucleic acid is often amplified by a PCR (polymerase chain reaction) method. In this case, the separated and purified nucleic acid solution needs to be diluted with a buffer solution suitable for the PCR method. In the recovery step according to this method, a buffer solution suitable for the PCR method is used as the recovery solution, so that the subsequent PCR step can be easily and rapidly transferred.

In the recovery step, a stabilizer for preventing degradation of the recovered nucleic acid can be added to the nucleic acid recovery solution. As a stabilizer, an antibacterial agent, an anti-fouling agent, a nucleic acid degradation inhibitor and the like can be added. EDTA etc. are raised as a nucleolytic enzyme inhibitor.
As another embodiment, a stabilizer may be added to the collection container in advance.

  The collection container used in the collection step is not particularly limited, but a collection container made of a material that does not absorb 260 nm can be used. In this case, the concentration of the recovered nucleic acid solution can be measured without transferring to another container. Examples of materials that do not absorb at 260 nm include, but are not limited to, quartz glass.

  A nucleic acid separation and purification cartridge and a reagent in each step for performing the RNA separation and purification method can be used as a kit.

  The step of separating and purifying nucleic acid from a sample containing nucleic acid using a nucleic acid separation and purification cartridge and a pressure generator, which contain a nucleic acid-adsorbing porous membrane in a container having at least two openings, is performed automatically. It is preferable to carry out using an automatic device. This not only simplifies and speeds up the operation, but also makes it possible to obtain a certain level of nucleic acid regardless of the skill of the operator.

  The following is an automatic process for automatically separating and purifying nucleic acid from a sample containing nucleic acid using a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane in a container having at least two openings and a pressure generator. Although the example of the automatic device to perform is shown, an automatic device is not limited to this.

  The automatic device uses a nucleic acid separation / purification cartridge containing a nucleic acid-adsorbing porous membrane through which the solution can pass, and injects and pressurizes a sample liquid containing nucleic acid into the nucleic acid separation / purification cartridge. Is adsorbed on the nucleic acid-adsorbing porous membrane, and then the washing solution is dispensed into the nucleic acid separation and purification cartridge and pressurized to remove impurities, and then the recovered solution is dispensed into the nucleic acid separation and purification cartridge. A nucleic acid separation and purification device for automatically separating and purifying a nucleic acid adsorbed on a membrane and recovering it together with a recovery liquid, wherein the waste liquid container contains the nucleic acid separation and purification cartridge, the sample liquid, and the drainage of the cleaning liquid And a mounting mechanism that holds a recovery container that stores a recovery liquid containing the nucleic acid, a pressurized air supply mechanism that introduces pressurized air into the nucleic acid separation and purification cartridge, and the nucleic acid separation In manufacturing the cartridge is characterized in by comprising a dispensing mechanism for dispensing a washing liquid and recovered.

(1) Production of nucleic acid purification cartridge The nucleic acid separation / purification cartridge (hereinafter also simply referred to as a nucleic acid purification cartridge) having an inner diameter of 7 mm and a portion accommodating a nucleic acid-adsorbing porous membrane is produced.

(2) A porous membrane obtained by saponifying a porous membrane of triacetyl cellulose is used as the nucleic acid-adsorbing porous membrane, and the membrane is accommodated in the nucleic acid-adsorbing porous membrane housing part of the nucleic acid purification cartridge prepared in (1) above.

[Example 1]
(3) Preparation of Nucleic Acid Solubilizing Reagent Solution and Washing Solution A nucleic acid solubilizing reagent solution and a washing solution having the following formulation are prepared.

(Nucleic acid solubilizing reagent solution-1)
Guanidine thiocyanate (Wako Pure Chemical Industries, Ltd.) 3.5 mol / L
BisTris (Dojin Chemical Co., Ltd.) 0.25 mol / L
Adjust to pH 6.5 using hydrochloric acid (add 1.0% by volume 2-mercaptoethanol before use.)

(Nucleic acid solubilizing reagent-2)
Tween 20 (Wako Pure Chemical Industries) 15wt / vol%
BisTris (Dojin Chemical Co., Ltd.) 0.1 mol / L
Adjust to pH 6.0 using hydrochloric acid

(Cleaning solution)
Tris-HCl (pH 7.5) 10 mmol / L
Sodium chloride 100mmol / L
70% ethanol by volume

(Recovered liquid)
Tris-HCl (pH 6.5) 1 mmol / L

(4) Preparation of Sample Solution Containing Nucleic Acid A 5 mmφ zirconia ball is put in a 2.0 ml tube for a bead mill homogenizer (manufactured by Tommy Seiko, MicroSmash). The mouse liver rapidly frozen with liquid nitrogen is made into small pieces with scissors, and 30 mg is weighed into the 2.0 ml tube. 350, 385, 420, 455 μl of nucleic acid solubilizing reagent-1 is added, and homogenized at 4200 rpm for 120 seconds with a bead mill homogenizer (Tomy Seiko, MicroSmash). Centrifuge at 15000 rpm for 3 minutes at room temperature and transfer the supernatant to a new 1.5 ml tube so as not to remove tissue debris. 175 μl of nucleic acid solubilizing reagent-2 is added thereto, and the mixture is stirred for 15 seconds with a vortex mixer. Further, 175, 140, 105, and 70 μl each of 99.5% or more special grade ethanol are added and stirred for 1 minute with a vortex mixer (the final ethanol concentrations in the sample solution are 25, 20, 15, and 10%, respectively).

(5) RNA separation and purification operation The sample solution prepared in (4) above is injected into one opening of a nucleic acid purification cartridge having a nucleic acid-adsorbing porous membrane prepared in (1) and (2) above, and then the above-mentioned one A pressure difference generator is coupled to the opening of the cartridge, the inside of the cartridge for separating and purifying nucleic acid is pressurized, and the sample solution containing the injected nucleic acid is passed through the nucleic acid-adsorbing porous membrane, thereby contacting the nucleic acid-adsorbing porous membrane. The nucleic acid separation and purification cartridge is discharged from the other opening. Subsequently, 750 μl of a washing solution is injected into the one opening of the nucleic acid separation and purification cartridge, a pressure difference generator is connected to the one opening, the inside of the nucleic acid separation and purification cartridge is pressurized, and the injected washing solution is added to the nucleic acid. Pass through the adsorptive porous membrane and discharge through other openings. Repeat the same operation 3 times. Subsequently, 100 μl of the recovered solution is injected into the one opening of the nucleic acid separation and purification cartridge, and incubated at room temperature for 120 seconds. A pressure difference generator is connected to the one opening of the cartridge for nucleic acid separation and purification so that the inside of the cartridge for separation and purification of the nucleic acid is pressurized. The injected recovery liquid is passed through the porous nucleic acid adsorbing porous membrane and discharged from the other opening. This liquid is collected.

(6) Washing process passage time and amount of collected nucleic acid recovered The porous membrane passage time (washing process passage time) of the washing liquid in the washing step in the above operation (5) and the concentration of the collected liquid containing the collected nucleic acid Was measured. The measurement method used was a method using absorbance at 260 nm. The results are shown in Table 1.

As is clear from Table 1, it can be seen that a high yield of highly pure RNA was obtained when the ethanol concentration in the sample solution was 10 to 25%. Further, when the ethanol concentration is in the range of 10 to 25%, the cleaning process passage time is 1 minute or less, and clogging does not occur.
From the above, according to the present invention, it is possible to increase the amount of specimen that can be processed and, as a result, increase the amount of RNA recovered.

Claims (6)

(1) A step of preparing a sample solution by adding a nucleic acid solubilizing reagent to a biomaterial, and further adding a water-soluble organic solvent or a solution containing a water-soluble organic solvent to the mixture,
(2) passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane to adsorb the nucleic acid to the porous membrane;
(3) passing the washing solution through the nucleic acid-adsorbing porous membrane and washing the porous membrane with the RNA adsorbed; and (4) passing the recovered solution through the nucleic acid-adsorbing porous membrane and removing the porous membrane from the porous membrane. In an RNA separation and purification method comprising a step of desorbing RNA,
The nucleic acid-adsorbing porous membrane is a porous membrane obtained by saponifying a porous membrane of triacetyl cellulose and adsorbing nucleic acids by an interaction not involving ionic bonds,
Wherein the water-soluble organic solvent is at least one alcohol methanol, ethanol, is whether we selected propanol及beauty blanking Tano Le,
And the RNA isolation | separation purification method whose water-soluble organic-solvent density | concentration in the said sample solution is 10-25 volume%.   The method for separating and purifying RNA according to claim 1, wherein the alcohol is ethanol.   The method for separating and purifying RNA according to claim 1 or 2, wherein the biomaterial is a specimen containing cells or viruses.   The method for separating and purifying RNA according to claim 3, wherein the specimen containing cells is animal tissue.   In each of the steps (1), (2), (3) and (4), a container having at least two openings for allowing a sample solution, a washing solution or a recovery solution containing a nucleic acid to pass through the nucleic acid-adsorbing porous membrane. A nucleic acid separation / purification cartridge and a pressure generator containing a nucleic acid-adsorbing porous membrane through which the solution can pass, and the pressure generator can be attached to and detached from one opening of the nucleic acid separation / purification cartridge. The method for separating and purifying RNA according to any one of claims 1 to 4, which is a pump to be bound.   A nucleic acid separation and purification cartridge and a reagent kit for performing the RNA separation and purification method according to any one of claims 1 to 5.