JP2006238854A - Method for isolating and refining nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and inexpensive method for isolating and refining a nucleic acid quickly, excellent in isolating performance and automating suitability, high in washing efficiency, thus enabling RNA or DNA to be selectively retrieved from a mixed sample of the RNA and DNA. <P>SOLUTION: The method for isolating and refining RNA or DNA to selectively retrieve the RNA or DNA comprises (1) a step of passing a nucleic acid mixture solution containing the RNA and DNA through a nucleic acid-adsorbing porous membrane to effect adsorbing the nucleic acids, (2) a step of passing a washing liquid through the nucleic acid-adsorbing porous membrane to wash the membrane under nucleic acid adsorbed state and (3) a step of passing a retrieving liquid through the nucleic acid-adsorbing porous membrane to effect desorbing the nucleic acid off the membrane. In this method, the washing liquid is characterized by containing ≤50 mass% of a water-miscible organic solvent and containing no chaotropic salt. <P>COPYRIGHT: (C)2006,JPO&NCIPI

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 or DNA from a nucleic acid mixture containing RNA and DNA. More specifically, a method for separating and purifying RNA or DNA 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. About.

  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 washing, desorption, etc., 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, a solution consisting of an organic polymer having a hydroxyl group on the surface is used by 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 to a 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 desorption 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

However, in the conventional method for separating and purifying nucleic acid, there is no particular description of a method for efficiently separating and purifying DNA or RNA efficiently from a mixed solution containing DNA and RNA.
Accordingly, an object of the present invention is a cheaper method for separating and purifying nucleic acid by adsorbing nucleic acid in a specimen to a nucleic acid-adsorbing porous membrane and then desorbing it through washing or the like. An object of the present invention is to provide a method for separating and purifying nucleic acids using a porous membrane that is efficient, simple, rapid, excellent in automation, and capable of producing a large amount of substantially the same separation performance. In particular, it is to provide a cheaper separation and purification method for selectively recovering RNA or DNA from a mixed sample of RNA and DNA.

As a result of intensive studies to solve the above problems, the present inventors have determined that the alcohol concentration in the washing liquid is 50 mass in the method for separating and purifying nucleic acid including the process of adsorbing and desorbing the nucleic acid mixture containing RNA and DNA to and from the porous membrane. The present invention has been completed by discovering that elution of DNA and selective separation and purification of RNA or DNA can be achieved by adjusting the ratio to less than%. Furthermore, in the present invention, RNA can be separated and purified with high purity and high yield by using an aqueous solution having a salt concentration of 0.5 M or less as a recovery solution.
In the present invention, it is preferable to use a porous membrane that adsorbs nucleic acids by an interaction not involving ionic bonds as the porous membrane in order to obtain the effects of the present invention. Further, in the present invention, two openings are provided. It is preferable to use a nucleic acid separation / purification cartridge containing a porous membrane made of an organic polymer in a container.
That is, the present invention has the following configuration.

  1. (1) a step of allowing a nucleic acid mixture solution containing RNA and DNA to pass through the inside of the nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid; (2) passing a washing solution through the nucleic acid-adsorbing porous membrane to adsorb the nucleic acid. Washing the nucleic acid-adsorbing porous membrane in a state, and (3) passing the recovered liquid through the nucleic acid-adsorbing porous membrane to desorb nucleic acids from the porous membrane, A separation and purification method for selectively recovering RNA or DNA, which is a washing solution containing a solvent at a concentration of 50% by mass or less and containing no chaotropic substance.

  2. The method for separating and purifying RNA or DNA according to item 1 above, wherein the concentration of the water-soluble organic solvent in the washing solution is 5 to 40% by mass.

  3. The nucleic acid-adsorbing porous membrane is a porous membrane made of an organic polymer that adsorbs nucleic acids with a weak interaction that does not involve ionic bonds, and selectively recovers RNA or DNA according to item 1 or 2 above Separation and purification method.

  4). 4. A separation and purification method for selectively recovering RNA or DNA according to any one of Items 1 to 3, wherein the nucleic acid-adsorbing porous membrane is a porous membrane made of an organic polymer having a hydroxyl group.

  5. 5. The method for selectively separating and purifying RNA according to any one of 1 to 4 above, wherein the porous membrane is a porous membrane made of an organic material obtained by saponifying a mixture of acetylcellulose having different acetyl values.

  6). 6. The method for selectively separating and purifying RNA or DNA according to any one of 1 to 5 above, wherein the porous membrane is an asymmetric porous membrane.

  7). RNA or DNA in any one of said 1-6 whose sample solution is the liquid which added the water-soluble organic solvent to the solution obtained by processing the test substance containing a cell or a virus with an RNA solubilization reagent The selective separation and purification method.

  8). 8. The method for selectively separating and purifying RNA or DNA as described in 7 above, wherein the specimen containing cells comprises cultured cells.

  9. 8. The method for selectively separating and purifying RNA or DNA according to item 7, wherein the specimen containing cells comprises animal tissue.

10. 10. The method for selectively separating and purifying RNA according to any one of 7 to 9 above, wherein the sample is homogenized before or after addition of the nucleic acid solubilizing reagent.

11. 8. The method for selectively separating and purifying RNA or DNA according to the above 7, wherein the nucleic acid solubilizing reagent contains at least one selected from chaotropic salts, nucleic acid stabilizers, surfactants, buffers and antifoaming agents.

12 12. The method for selectively separating and purifying RNA or DNA according to the above 11, wherein the chaotropic substance is at least one selected from guanidine hydrochloride or guanidine thiocyanate.

13. 13. The water-soluble organic solvent according to any one of the above items 1 to 12, wherein the water-soluble organic solvent is at least one alcohol selected from methyl alcohol, ethyl alcohol, propyl alcohol and isomers thereof, and butyl alcohol and isomers thereof. A method for selectively separating and purifying RNA or DNA.

14 Any of the above 1 to 13, wherein the cleaning liquid is a solution containing 5 to 50% by mass of at least one alcohol selected from methyl alcohol, ethyl alcohol, propyl alcohol and isomers thereof, and butanol and isomers thereof. A method for selectively separating and purifying RNA or DNA according to claim 1.

15. 15. The method for selectively separating and purifying RNA according to any one of 1 to 14 above, wherein the washing solution contains a water-soluble salt.

16. 16. The method for selectively separating and purifying RNA as described in 15 above, which comprises 10 mmol / L or more of a water-soluble salt.

17. Item 17. The method for selectively separating and purifying RNA according to Item 15 or 16, comprising a water-soluble salt in an amount of 10 mmol / L to 1 mol / L.

18. 18. The method for selectively separating and purifying RNA or DNA according to any one of 1 to 17 above, wherein the washing solution is a solution containing 10 mmol / L to 1 mol / L of chloride.

19. The selective separation of RNA or DNA according to any one of 1 to 18 above, wherein the recovery solution capable of desorbing RNA adsorbed on the nucleic acid-adsorbing porous membrane is a solution having a salt concentration of 0.5 mol / L or less. Purification method.

20. In each of the steps (1), (2) and (3), the sample solution containing the nucleic acid, the washing solution or the recovery solution is passed through the nucleic acid-adsorbing porous membrane, and the solution is put in a container having at least two openings. 20. The method for selectively separating and purifying RNA or DNA according to any one of the above items 1 to 19, which is carried out using a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane that can pass through the inside.

21. The sample solution containing the nucleic acid, the washing solution or the recovery solution is passed through the nucleic acid-adsorbing porous membrane using a pressure generator, and the pressure generator is detachably coupled to one opening of the nucleic acid separation and purification cartridge. 21. The method for selectively separating and purifying RNA or DNA according to any one of 1 to 20 above, which is a pump to be used.

22. A nucleic acid separation and purification cartridge and a reagent kit for performing the nucleic acid separation and purification step according to any one of Items 1 to 21.

23. An apparatus for automatically performing the method for separating and purifying nucleic acid according to any one of the above items 1 to 22.

  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. This is a method for separating and purifying nucleic acid used, and RNA or DNA can be selectively recovered from a mixed sample of DNA and RNA at a lower cost and easily.

  The method for separating and purifying nucleic acid according to the present invention includes (1) a step of allowing a nucleic acid mixture solution containing RNA and DNA to pass through a nucleic acid adsorbing porous membrane and adsorbing the nucleic acid, and (2) a washing solution for the nucleic acid adsorbing porous membrane. At least a step of washing the nucleic acid-adsorbing porous membrane with the nucleic acid adsorbed thereon, and (3) a step of passing the recovered liquid through the nucleic acid-adsorbing porous membrane and desorbing the nucleic acid from the porous membrane. Is included.

  Preferably, in each of the above steps (1), (2) and (3), the nucleic acid mixture solution, the washing solution or the recovery solution containing DNA and RNA is passed through the nucleic acid adsorbing porous membrane using a pressure generator. More preferably, in each of the above steps (1), (2) and (3), one of the nucleic acid separation and purification cartridges containing the nucleic acid-adsorbing porous membrane in a container having at least two openings. A sample solution containing nucleic acid, a cleaning solution, or a recovery solution is injected into the opening, and the inside of the cartridge is pressurized using the pressure difference generator coupled to the one opening of the cartridge, and passes through each injected solution. It is made to discharge from other openings. The apparatus can be automated in a compact manner by passing a nucleic acid mixture solution containing DNA and RNA, a washing solution, or a recovery solution through the porous membrane under pressure, 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 and DNA can be separated and purified in the following steps using a nucleic acid separation and purification cartridge containing the nucleic acid-adsorbing porous membrane.
That is, (a) one opening of a nucleic acid separation and purification cartridge containing a nucleic acid adsorbing porous membrane in which a solution containing a nucleic acid mixture containing DNA and RNA can be passed through a container having at least two openings. (B) a nucleic acid mixture solution containing the injected DNA and RNA, with the inside of the nucleic acid separation / purification cartridge being pressurized using the pressure difference generator coupled to the one opening of the nucleic acid separation / purification cartridge. Is passed through the nucleic acid-adsorbing porous membrane and discharged from the other opening of the nucleic acid separation / purification cartridge, whereby the nucleic acid is adsorbed in the nucleic acid-adsorption porous membrane, (c) the one opening of the nucleic acid separation / purification cartridge A step of injecting a washing solution into the cartridge, (d) the nucleic acid separation and purification cartridge is pressurized using the pressure difference generator coupled to the one opening of the nucleic acid separation and purification cartridge And (e) a nucleic acid separation and purification cartridge, wherein the injected washing liquid is passed through the nucleic acid-adsorbing porous membrane and discharged from the other openings, thereby washing the nucleic acid-adsorbing porous membrane with the nucleic acid adsorbed. (F) injecting the recovery liquid into the one opening of the above, (f) using the pressure difference generator connected to one opening of the nucleic acid separation and purification cartridge to pressurize the inside of the cartridge and injecting the recovery liquid Can be passed through the nucleic acid-adsorbing porous membrane and discharged from other openings, whereby the nucleic acid is desorbed from the nucleic acid-adsorbing porous membrane and discharged out of the cartridge for nucleic acid separation and purification.

In the above nucleic acid separation and purification process, the process from injecting 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 20 minutes, and in a suitable situation within 2 minutes. It is.

  In the nucleic acid purification step, the measurement value (260 nm / 280 nm) with an ultraviolet-visible spectrophotometer is 1.6 to 2.0 for DNA, and 1.8 to 2.2 for RNA. Can be recovered, and high-purity nucleic acid with a small amount of impurities can be steadily obtained. Furthermore, a nucleic acid having a purity of around 1.8 when DNA (260 nm / 280 nm) measured with an ultraviolet-visible spectrophotometer is used, and around 2.0 when RNA is collected can be recovered.

  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 sample that can be used in the present invention is not particularly limited as long as it contains nucleic acid. For example, in the diagnostic field, whole blood collected as a sample, plasma, serum, urine, feces, semen, saliva and other body fluids, or Of interest are 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. 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. Animal tissue is used as an animal (or part thereof) specimen. All tissues constituting an individual such as liver, kidney, spleen, brain, heart, lung and thymus that can be collected when the animal is dissected can be used.

  These specimens 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 specimen will be described. The step is usually (I) a step of injecting a specimen containing cells or viruses into a container, (II) any one of chaotropic salts, nucleic acid stabilizers, surfactants, buffers and antifoaming agents in the container. A step of adding a nucleic acid solubilizing reagent solution containing two or more, mixing the specimen and the nucleic acid solubilizing reagent solution, and (III) adding a water-soluble organic solvent to the mixed solution obtained above.

  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 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 comprising 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.

  A process for obtaining a sample solution containing nucleic acid from a specimen by dissolving a cell membrane / nuclear membrane and solubilizing nucleic acid will be described. In the process of dissolving the cell membrane / nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the specimen is homogenized before or after the nucleic acid solubilizing reagent is added, thereby improving the appropriateness of the automated processing. To do. 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 specimen, a water-soluble organic solvent is then added to the mixture. 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 methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol and isomers thereof can be preferably used. The final concentration of the sample solution containing nucleic acids of these water-soluble organic solvents is preferably 5 to 90% by mass. 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, in the next step, the sample solution is easily removed after contacting the sample solution with the nucleic acid-adsorbing porous membrane.

The nucleic acid-adsorbing porous membrane used in the present invention and the adsorption process will be described below.
The nucleic acid-adsorbing 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.

  Moreover, the nucleic acid-adsorbing porous membrane of the present invention is preferably 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 bonding with the 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, and if it is too long, it is not preferable from the viewpoint of operability. 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.
According to the present invention, by passing the cleaning solution of the present invention through the nucleic acid-adsorbing porous membrane, DNA can be eluted, only RNA can be adsorbed, and the amount of the sample containing the necessary nucleic acid can be made very small. That is, it is possible to separate the DNA from the liquid in which the washing liquid is flowed.

  The cleaning liquid of the present invention is an aqueous solution containing a water-soluble organic solvent at a concentration of 50% by mass or less, preferably an aqueous solution containing a water-soluble organic solvent at a concentration of 1% by mass or more and 50% by mass or less. 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 in which the nucleic acid is not desorbed from the nucleic acid-adsorbing porous membrane but the impurities are desorbed.

  If the ratio of the water-soluble organic solvent contained in the washing solution is too large, DNA cannot be eluted in the washing step. Therefore, the contamination of DNA increases in the RNA solution recovered by the recovery solution. If the amount is too small, not only DNA but also RNA is desorbed from the porous membrane in the washing step, and the amount of RNA in the recovered solution is reduced. Therefore, the ratio of the water-soluble organic solvent contained in the cleaning liquid is 50% by mass or less, preferably 5 to 40% by mass.

  As the water-soluble organic solvent contained in the cleaning liquid, alcohols such as methanol, ethanol, isopropanol, n-isopropanol, and butanol can be used, and ethanol is particularly preferable.

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 liquid, the concentration 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. Is more preferable, and 0.1 mol / L or less is more preferable. More preferably, the water-soluble salt is sodium chloride, and further sodium chloride is contained at 20 mmol / L or more.

Furthermore, the cleaning liquid is characterized by not containing 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, a highly pure nucleic acid having a measurement value (260 nm / 230 nm) of 1.5 or more can be recovered with an ultraviolet-visible spectrophotometer. In a suitable situation, it is possible to recover high-purity nucleic acid whose measured value (260 nm / 230 nm) with an ultraviolet-visible spectrophotometer is around 2.0.
Here, the chaotropic substance is urea, guanidine isothiocyanate, guanidine thiocyanate, sodium isothiocyanate, sodium iodide, potassium iodide, or the like as described above.

  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 washing residual liquid should not remain in the cartridge so that the liquid used for adsorption and washing, particularly the washing liquid, does not affect the next step. Is important.

Therefore, the surface tension of the cleaning liquid should be less than 0.035 J / m ^{2 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. Is preferred. 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.

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 relative 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 liquid, preferably, purified distilled water, 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. The recovered liquid preferably has an ionic strength of 500 mmol / L or less, and more preferably has a salt concentration of 50 to 0.01 mmol / L. 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, thereby obtaining a recovery solution containing the nucleic acid of a desired concentration. A recovery solution containing nucleic acid at a concentration suitable for PCR and the like 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, by desorbing the nucleic acid from the porous membrane at a temperature of the recovered liquid of 0 to 10 ° C., the function of the nucleolytic enzyme can be suppressed without adding any reagent or special operation to prevent degradation by the enzyme. The nucleic acid solution can be easily and efficiently obtained by preventing the degradation of the nucleic acid.

  In addition, when the temperature of the recovered solution is 10 to 35 ° C., the nucleic acid can be recovered at a general room temperature, and the nucleic acid can be desorbed and separated and purified 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 desorbed from the porous membrane easily and at a high recovery rate without complicated operations.

  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.

  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 apparatus to perform is shown, an automatic apparatus 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 collected liquid, wherein the waste liquid container accommodates the nucleic acid separation and purification cartridge, the sample liquid and the washing liquid discharge liquid. And a mounting mechanism for holding a recovery container for storing a recovery liquid containing the nucleic acid, a pressurized air supply mechanism for introducing 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 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)
Guanidine hydrochloride (Life Technology) 382g
Tris (Life Technology) 12.1g
TritonX-100 (ICN) 10g
Add to 1000ml of distilled water
(1.0% by volume 2-mercaptoethanol is added before use.)

(Cleaning solution)
10 mM Tris-HCl 20-50% ethanol

(4) Nucleic acid purification procedure Prepare human promyelocytic leukemia cell (HL60) culture solution. The cell number is adjusted to 1 × 10 ⁶ and the cells are washed with Ca ²⁺ and Mg ²⁺ free PBS. After centrifuging with a swing rotor at 4 ° C, 300 g for 5 minutes, pellet the floating cells, remove the supernatant, and resuspend the cells by tapping. Add 350 μl of the nucleic acid solubilizing reagent solution and stir with a vortex mixer for 1 minute. Thereafter, 350 μl of 70% ethanol is added and stirred for 5 seconds with a vortex mixer. After stirring, the mixture is injected into one opening of the nucleic acid purification cartridge having the nucleic acid adsorbing porous membrane prepared in the above (1) and (2), and then a pressure difference generator is connected to the one opening to separate and purify the nucleic acid. The inside of the cartridge is pressurized, and the sample solution containing the injected nucleic acid is passed through the nucleic acid-adsorbing porous membrane to contact the nucleic acid-adsorbing porous membrane and discharged from the other opening of the nucleic acid separation and purification cartridge. Subsequently, 500 μl of a washing solution containing ethanol at a concentration of 20 to 50% is injected into the one opening of the nucleic acid separation and purification cartridge, and a pressure difference generator is coupled to the one opening, and the inside of the nucleic acid separation and purification cartridge is Under pressure, the injected cleaning solution is passed through the nucleic acid-adsorbing porous membrane and discharged from the other opening. Repeat the same operation 3 times. Subsequently, 100 μl of the recovery solution is injected into the one opening of the nucleic acid separation and purification cartridge, and a pressure difference generator is connected to the one opening of the nucleic acid separation and purification cartridge so that the inside of the nucleic acid separation and purification cartridge is pressurized. The injected recovery liquid is passed through the nucleic acid-adsorptive porous membrane, discharged from the other openings, and this liquid is recovered.

(Examples and Comparative Examples)
Each operation was performed under the same conditions as described above except that the cleaning liquid having the ethanol concentration (Tris-HCl concentration is constant) shown in Tables 1 and 2 was used as the cleaning liquid in the above example.

(5) Agarose gel electrophoresis of recovered nucleic acid In each of the above samples, the amount of nucleic acid in the recovered washing solution and recovered solution is determined from the electrophoresis of 1% agarose gel, and the fluorescence intensity is determined in advance. A calibration curve of RNA was prepared and its concentration was determined. Table 1 shows the DNA and RNA concentrations of the recovered solution, and Table 2 shows the DNA and RNA concentrations of the washing solution.

As is clear from the results in Tables 1 and 2, when the ethanol concentration in the washing step is 50% by mass or less, the amount of DNA contamination is small. From this result, it can be seen that by using the method of the present invention, the amount of DNA mixed in the nucleic acid recovery solution can be reduced, and RNA can be selectively recovered.
On the other hand, when the ethanol concentration in the washing solution is higher than 50% by mass, it can be seen that DNA contamination increases.

As is clear from the results in Tables 1 and 2, when the ethanol concentration in the washing step is 50% by mass or less, the amount of DNA contamination is small. From this result, it can be seen that by using the method of the present invention, the amount of DNA mixed in the nucleic acid recovery solution can be reduced, and RNA can be selectively recovered.
On the other hand, when the ethanol concentration in the washing solution is higher than 50% by mass, it can be seen that DNA contamination increases.

[Example 2]
(6) 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-B1)
Guanidine thiocyanate (Wako Pure Chemical Industries) 3.5M
BisTris (Dojin Chemical Co., Ltd.) 0.25M
Adjust to pH 6.5 using hydrochloric acid (add 1.0% by volume 2-mercaptoethanol before use.)

(Nucleic acid solubilizing reagent-B2)
Tween 20 (Wako Pure Chemical Industries) 15%
BisTris (manufactured by Dojin Chemical) 0.1M
Adjust to pH 6.0 using hydrochloric acid

(Cleaning liquid-B)
Tris-HCl (pH 7.5) 10 mM
Sodium chloride 0.5M
Ethanol 5-12.5%

(Recovered liquid-B)
Tris-HCl (pH 6.5) 1 mM

(7) Preparation of animal tissue nucleic acid mixture solution Mouse liver rapidly frozen with liquid nitrogen is cut into small pieces with scissors, and 5 mg is weighed into a 1.5 ml tube. Add 350 μl of the nucleic acid solubilizing reagent-B1 and homogenize it with a Rotor-Stator homogenizer (Polytron, KINEMATICA) until it is uniform. Centrifuge at 8000 xg 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-B2 is added thereto and stirred for 15 seconds with a vortex mixer. Further, add 175 μl of 99.5% or more special grade ethanol and stir for 1 minute with a vortex mixer.

(8) RNA separation and purification operation: Inject into one opening of a nucleic acid purification cartridge having a nucleic acid-adsorbing porous membrane prepared in (1) and (2) above, and then connect a pressure difference generator to the one opening. The nucleic acid separation and purification cartridge is pressurized, and the sample solution containing the injected nucleic acid is passed through the nucleic acid adsorption porous membrane to contact the nucleic acid adsorption porous membrane. Discharge. Subsequently, 750 μl of a cleaning solution-B containing ethanol at a concentration of 5 to 12.5% is injected into the one opening of the nucleic acid separation and purification cartridge, and a pressure difference generator is connected to the one opening to separate the nucleic acid. The inside of the purification cartridge is pressurized, the injected cleaning solution is passed through the nucleic acid-adsorbing porous membrane, and discharged from the other opening. Repeat the same operation 3 times. Subsequently, 100 μl of the recovered liquid-B is injected into the one opening of the nucleic acid separation and purification cartridge, and a pressure difference generator is 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. Then, the injected recovery liquid is passed through the nucleic acid-adsorbing porous membrane and discharged from the other openings, and this liquid is recovered.

[Example 3]
Each operation was performed in the same manner as (8) except that the ethanol concentration of the cleaning liquid-B was changed to 30, 20, and 0%.

(9) Agarose gel electrophoresis of recovered nucleic acids The nucleic acids purified in Example 2 and Comparative Example 2 were subjected to 1% agarose gel electrophoresis. The results are shown in FIG.

(10) Amount of recovered nucleic acid The concentration of the recovered liquid containing the nucleic acid recovered in Example 2 and Example 3 was measured. The measurement method used was a method using absorbance at 260 nm. The results are shown in Table 3.

As apparent from FIG. 1, when the ethanol concentration in the washing step of Example 2 is 5 to 12.5%, almost no DNA contamination is observed. On the other hand, when the ethanol concentration in the washing process of Comparative Example 2 is 20 to 30%, it can be seen that DNA is mixed. Furthermore, when the ethanol concentration in Comparative Example 2 is 0%, the amount of nucleic acid recovered is reduced although there is no DNA contamination.
From the above, it can be seen that according to the present invention, the amount of DNA contamination in the nucleic acid recovery solution can be considerably reduced, and RNA can be selectively recovered at a high yield.

It is a figure which shows the result of having carried out 1% agarose gel electrophoresis of the nucleic acid refine | purified in Example 2 and Comparative Example 2. FIG.

Claims (22)

  (1) a step of allowing a nucleic acid mixture solution containing RNA and DNA to pass through the inside of the nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid; (2) passing a washing solution through the nucleic acid-adsorbing porous membrane to adsorb the nucleic acid. Washing the nucleic acid-adsorbing porous membrane in a state, and (3) passing the recovered liquid through the nucleic acid-adsorbing porous membrane to desorb nucleic acids from the porous membrane, A separation and purification method for selectively recovering RNA or DNA, which is a washing solution containing a solvent at a concentration of 50% by mass or less and containing no chaotropic substance.   The separation and purification method for selectively recovering RNA or DNA according to claim 1, wherein the concentration of the water-soluble organic solvent in the washing solution is 5 to 40% by mass.   Separation and purification for selectively recovering RNA or DNA according to claim 1 or 2, wherein the nucleic acid-adsorbing porous membrane is a porous membrane made of an organic polymer that adsorbs nucleic acids with a weak interaction not involving ionic bonds. Method.   The separation and purification method for selectively recovering RNA or DNA according to any one of claims 1 to 3, wherein the nucleic acid-adsorbing porous membrane is a porous membrane made of an organic polymer having a hydroxyl group.   The method for selectively separating and purifying RNA or DNA according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane made of an organic material obtained by saponifying a mixture of acetylcelluloses having different acetyl values.   The method for selectively separating and purifying RNA or DNA according to any one of claims 1 to 5, wherein the porous membrane is an asymmetric porous membrane.   The RNA or DNA of any one of claims 1 to 6, wherein the sample solution is a solution obtained by adding a water-soluble organic solvent to a solution obtained by treating a specimen containing cells or viruses with a nucleic acid solubilizing reagent. Selective separation and purification method.   The method for selectively separating and purifying RNA or DNA according to claim 7, wherein the specimen containing cells comprises cultured cells.   The method for selectively separating and purifying RNA or DNA according to claim 7, wherein the specimen containing cells comprises animal tissue.   The method for selectively separating and purifying RNA according to any one of claims 7 to 9, wherein the sample is homogenized before or after addition of the nucleic acid solubilizing reagent.   The method for selectively separating and purifying RNA or DNA according to claim 7, wherein the nucleic acid solubilizing reagent contains at least one selected from chaotropic salts, nucleic acid stabilizers, surfactants, buffers and antifoaming agents.   The method for selectively separating and purifying RNA or DNA according to claim 11, wherein the chaotropic substance is at least one selected from guanidine hydrochloride or guanidine thiocyanate.   The RNA according to any one of claims 1 to 12, wherein the water-soluble organic solvent is at least one alcohol selected from methyl alcohol, ethyl alcohol, propyl alcohol and isomers thereof, and butyl alcohol and isomers thereof. Alternatively, a method for selective separation and purification of DNA.   The cleaning liquid according to any one of claims 1 to 13, wherein the cleaning liquid is a solution containing 5 to 50% by mass of at least one alcohol selected from methyl alcohol, ethyl alcohol, propyl alcohol and isomers thereof, and butanol and isomers thereof. The method for selectively separating and purifying RNA or DNA according to 1.   The method for selectively separating and purifying RNA according to any one of claims 1 to 14, wherein the washing solution contains a water-soluble salt.   The method for selectively separating and purifying RNA according to claim 15, comprising 10 mmol / L or more of a water-soluble salt.   The method for selectively separating and purifying RNA according to claim 15 or 16, comprising a water-soluble salt in an amount of 10 mmol / L to 1 mol / L.   The method for selectively separating and purifying RNA or DNA according to any one of claims 1 to 17, wherein the washing solution is a solution containing 10 mmol / L to 1 mol / L of chloride.   The selective separation and purification of RNA or DNA according to any one of claims 1 to 18, wherein the recovery solution capable of desorbing RNA adsorbed on the nucleic acid-adsorbing porous membrane is a solution having a salt concentration of 0.5 mol / L or less. Method.   In each of the steps (1), (2) and (3), the sample solution containing the nucleic acid, the washing solution or the recovery solution is passed through the nucleic acid-adsorbing porous membrane, and the solution is put in a container having at least two openings. A pump that uses a nucleic acid separation and purification cartridge and a pressure generation device that contain a nucleic acid-adsorbing porous membrane that can pass through the inside, and that is detachably coupled to one opening of the nucleic acid separation and purification cartridge The method for selectively separating and purifying RNA or DNA according to any one of claims 1 to 19.   A nucleic acid separation and purification cartridge and a reagent kit for performing the nucleic acid separation and purification step according to any one of claims 1 to 20.   An apparatus for automatically performing the method for separating and purifying nucleic acid according to any one of claims 1 to 20.