JP2005168450A - Method for separation and purification of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the separation and purification of nucleic acid having excellent separation performance and good washing efficiency, enabling easy and quick treatment and using a porous membrane suitable for automation and size reduction and available in large quantities having essentially same separation performance and provide a washing liquid effective for reducing the residual liquid volume while keeping the separation performance of the nucleic acid. <P>SOLUTION: The method for the separation and purification of a nucleic acid contains (1) a step of passing a specimen solution containing the nucleic acid through a nucleic acid-adsorbing porous membrane to adsorb the nucleic acid in the porous membrane, (2) a step of washing the porous membrane holding the adsorbed nucleic acid and (3) a step of passing a recovering liquid through the porous membrane to effect the desorption of the nucleic acid from the porous membrane. The nucleic acid adsorbing porous membrane adsorbs the nucleic acid by an interaction essentially free from the participation of ionic bond and the surface tension of the washing liquid is ≥35 mPa×m. <P>COPYRIGHT: (C)2005,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 obtaining a sample solution containing a nucleic acid from a specimen in order to separate and purify the nucleic acid. More specifically, using the sample solution containing the obtained nucleic acid, the nucleic acid is purified 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. The present invention relates to a method for separating and purifying nucleic acid from a contained sample.

  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.

  In the diagnostic field, nucleic acids are used in various ways. 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. The purification of sample nucleic acids from serum, urine and bacterial cultures also adds the risk of contamination and false positive results.

  As one of widely known purification methods, there is a method in which a nucleic acid is adsorbed on a solid phase such as silicon dioxide, silica polymer, magnesium silicate, etc., and purified by subsequent operations such as washing and desorption (for example, Patent Document 1). Although this method is excellent in separation performance, it is not sufficient in terms of simplicity, speed, automation, and suitability for miniaturization, industrial mass production of adsorption media having the same performance is difficult, and handling is inconvenient. There are problems such as difficulty in processing into various shapes.

  In Patent Document 2, as one method for separating and purifying nucleic acid easily and efficiently, a solution having a nucleic acid adsorbed on a solid phase and a solution desorbing nucleic acid from the solid phase are used, respectively, and has a hydroxyl group on the surface. A method for separating and purifying a nucleic acid by adsorbing and desorbing the nucleic acid on a solid phase composed of an organic polymer has been reported.

The above-mentioned prior art relating to the invention of this application includes the following documents.
Japanese Patent Publication No. 7-51065 JP 2003-128691 A

The operation for separating and purifying nucleic acids is required to be simple and rapid, and the above-described conventional method for separating and purifying nucleic acids is an advanced method in that respect. There is a need for improved accuracy. In order to meet this demand, it is required to reduce the amount of the porous membrane occlusion solution adsorbing nucleic acid components and the amount of the adhering solution on the cartridge wall. For this purpose, the cartridge containing the porous membrane is also dried after the cleaning and before the recovery process, but this is not desirable in terms of reversing the ease of operation.
The present invention has been carried out from such a background, and its purpose is to separate and purify nucleic acid by adsorbing nucleic acid in a sample to a nucleic acid-adsorbing porous membrane and then desorbing it through washing or the like. Nucleic acid using a porous membrane that has excellent separation performance, good washing efficiency, simple, rapid, excellent in automation and downsizing, and can produce a large amount of substantially the same separation performance It is to provide a separation and purification method.
In particular, it is to provide a cleaning solution for a cartridge containing a porous membrane, which is applied to the separation and purification of nucleic acid using the porous membrane, and which achieves both reduction of the amount of residual liquid and securing of nucleic acid separation performance.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have substantially no ionic bond as the porous membrane in the method for separating and purifying nucleic acid including the process of adsorbing and desorbing the nucleic acid on the porous membrane. By using a porous membrane that adsorbs nucleic acids by interaction, and adjusting the surface tension of the washing liquid to 35 mPa · m or more, the amount of residual liquid after washing can be reduced while ensuring the separation performance of nucleic acids. I found it. The present invention has been completed based on this finding.

That is, according to the present invention, (1) a step of allowing a sample solution containing a nucleic acid to pass through a nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid in the porous membrane; A step of washing the nucleic acid-adsorbing porous membrane in a state in which the nucleic acid is adsorbed by passing through the porous membrane, and (3) passing the recovered liquid through the nucleic acid-adsorbing porous membrane In the method for separating and purifying nucleic acid comprising the step of desorbing nucleic acid from the nucleic acid, the nucleic acid-adsorbing porous membrane is a porous membrane that adsorbs nucleic acid by an interaction that does not substantially involve ionic bonds, and the washing solution Provided is a method for separating and purifying a nucleic acid, wherein the surface tension is 35 mPa · m or more.
In the method for separating and purifying nucleic acid of the present invention, in each of the above steps (1), (2) and (3), the sample solution, the washing solution or the recovery solution containing the nucleic acid is applied to the nucleic acid-adsorbing porous membrane under pressure. It is preferable to let it pass.
In each of the above steps, a sample solution, a washing solution or a recovery solution containing nucleic acid is injected into one opening of a nucleic acid separation and purification cartridge containing the nucleic acid-adsorbing porous membrane in a container having at least two openings. Preferably, the inside of the cartridge is pressurized using a pressure difference generating device coupled to the one opening of the cartridge, and the injected liquids are allowed to pass through and discharged from the other openings.

The gist of the present invention is to use a porous membrane that adsorbs nucleic acids by an interaction that does not substantially involve ionic bonds as the nucleic acid-adsorbing porous membrane, and adjust the surface tension of the cleaning liquid for cleaning the membrane to 35 mPa · m or more. It is that. The surface tension of pure water is 72.5 mPa · m when it is in contact with air at room temperature, but the cleaning solution is usually used to improve the separation between nucleic acid and degradation products such as cell membranes and cytoplasm derived from the specimen sample. Since a chaotropic substance or a water-soluble organic solvent is contained, the surface tension is generally lowered thereby. It has been found that when the surface tension is lowered, the wettability of the cleaning liquid is improved and the residual liquid on the porous membrane and the cartridge device wall is increased. When the amount of the residual liquid increases, the mixing of the residual liquid into the recovered liquid increases and the cleaning efficiency decreases. Therefore, in the present invention, priority is given to avoiding a reduction in cleaning effect due to a reduction in surface tension, and the content of chaotropic substances and water-soluble organic solvents that are inherently effective in cleaning properties is an amount that maintains a surface tension of at least 35 mPa · m. As a result, the object of the invention was achieved. A preferable surface tension is 35 to 150 mPa · m, and more preferably 35 to 100 mPa · m.
Within this surface tension range, the cleaning efficiency is high, so that the cleaning operation can be performed only once, and also in this respect, the nucleic acid separation and purification operation can be speeded up. Further, since there is no need to provide a drying step between the washing step and the recovery step, simplification and speeding up can be promoted from this point.

The washing liquid preferably contains a water-soluble salt, particularly a water-soluble halide salt as another nucleic acid separation functional compound, instead of reducing or not containing the above-mentioned components having a low surface tension. Among the compounds, chloride is suitable in that it promotes dissolution of impurities while adsorbing nucleic acids and enhances the separation between nucleic acids and impurities.
On the other hand, monovalent or divalent cations are preferable as the cation component of the water-soluble salt, and potassium salt or sodium salt is particularly preferable, and sodium salt is particularly suitable.
The salt concentration is preferably 10 mmol / L or more. In particular, it is preferable that sodium chloride is contained in an amount of 20 mmol / L or more in order to exhibit the intended effect.

  The simple and rapid method for separating and purifying nucleic acid according to the present invention is such that when a porous membrane made of an organic material having a hydroxyl group is used as the nucleic acid-adsorbing porous membrane, the washing is performed quickly and there is little residual liquid. Becomes prominent. Among the porous membranes of organic materials having a hydroxyl group, porous membranes having a polysaccharide structure are preferred, and a regenerated cellulose porous membrane is particularly effective. Further details of the porous membrane will be described later.

  The nucleic acid separation and purification method of the present invention can be carried out particularly easily and quickly by using a nucleic acid separation and purification cartridge, a reagent kit and a positive automatic apparatus for nucleic acid separation and purification.

  The porous membrane uses a porous membrane that adsorbs nucleic acids through an interaction that does not involve ionic bonds, and uses a cleaning solution having a surface tension of 35 mPa · m or higher, and subsequently recovers the nucleic acid by the method of separating and purifying nucleic acid of the present invention. It is possible to reduce the amount of residual liquid in the process and ensure the separation performance of the nucleic acid, and it is possible to separate and purify the nucleic acid from the sample containing the nucleic acid in a simple, rapid and automatable manner.

The method for separating and purifying nucleic acid according to the present invention comprises (1) a step of allowing a sample solution containing nucleic acid to pass through a nucleic acid-adsorbing porous membrane, and adsorbing the nucleic acid in the porous membrane; (2) the nucleic acid-adsorbing porous It includes at least a step of washing the membrane in a state where nucleic acid is adsorbed, and (3) a step of passing the recovered liquid through the nucleic acid-adsorbing porous membrane to desorb the nucleic acid 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 is passed through the nucleic acid-adsorbing porous membrane in a pressurized state. Preferably, in each step of the above (1), (2) and (3), in one opening of the nucleic acid separation and purification cartridge containing the nucleic acid-adsorbing porous membrane in a container having at least two openings, A sample solution, a washing solution or a recovery solution containing nucleic acid is injected, the inside of the cartridge is pressurized using a pressure difference generator connected to the one opening of the cartridge, and each injected solution is allowed to pass through. It is discharged from the opening. By passing a sample solution, a washing solution or a recovery solution containing nucleic acid through the porous membrane under pressure, the apparatus can be automated in a compact manner, which is preferable. The pressurization is preferably performed at a rate of 10 to 200 kpa, more preferably 40 to 100 kpa.

  In the nucleic acid separation and purification step, the steps from injecting the sample solution containing the first nucleic acid to obtaining the nucleic acid outside the nucleic acid separation and purification cartridge are within 10 minutes, and in a suitable situation, the sample containing nucleic acid is 2 minutes. It is possible to finish within. In the nucleic acid separation and purification step, the nucleic acid can be obtained in a yield of 50% or more, and in a suitable situation, the sample containing the nucleic acid can be obtained in a yield of 90% or more.

  In the nucleic acid separation and purification step, nucleic acids having a molecular weight ranging from 1 kbp to 200 kbp, preferably from 20 kbp to 140 kbp can be recovered. That is, long-chain nucleic acids can be recovered as compared with the conventional spin column method (Qiagen) using a glass filter.

  In the nucleic acid separation and purification step, the absorbance ratio (260 nm / 280 nm) of the spectral absorbance of 260 nm and 280 nm obtained with an ultraviolet-visible spectrophotometer is 1.6 to 2.0 in the case of DNA, and in the case of RNA. Can recover a nucleic acid having a purity of 1.8 to 2.2, and can constantly obtain a high-purity nucleic acid with a small amount of impurities. Furthermore, it is possible to recover a nucleic acid having a purity of about 1.8 when the absorbance ratio (260 nm / 280 nm) in the ultraviolet-visible spectrophotometer is about DNA and about 2.0 when RNA is used.

  In the above process, examples of the pressure difference generating device include a pump capable of pressurization such as a syringe, pipetter, evaporator, or peristaltic pump, or 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.

  There are no limitations on the specimens that can be used in the present invention. For example, in the diagnostic field, whole blood collected as specimens, plasma, serum, urine, feces, semen, saliva and other body fluids, or plants (or parts thereof), Of interest are solutions prepared from biological materials such as animals (or parts thereof), bacteria, viruses, etc., or lysates and homogenates thereof.

  First, these specimens are treated with an aqueous solution (nucleic acid solubilizing reagent) containing a reagent for solubilizing nucleic acid by dissolving cell membranes and nuclear membranes. As a result, the cell membrane and the nuclear membrane are dissolved, and the nucleic acid is dispersed in the aqueous solution to obtain a sample solution containing the nucleic acid.

  In order to solubilize cell membrane and nuclear membrane and solubilize nucleic acid, for example, when the target sample is whole blood, (1) removal of red blood cells, (2) removal of various proteins, and (3) white blood cells And dissolution of the nuclear membrane is required. (1) Removal of red blood cells and (2) removal of various proteins are performed in order to prevent non-specific adsorption to the membrane and clogging of the porous membrane. (3) Lysis of leukocytes and lysis of the nuclear membrane are subject to extraction. Each is required to solubilize certain nucleic acids. In particular, (3) lysis of leukocytes and nuclear membrane lysis are important steps, and in the method of the present invention, it is necessary to solubilize nucleic acids by this step.

  The specimen containing the nucleic acid may be a specimen containing a single nucleic acid or a specimen containing a plurality of different types of nucleic acids. The type of nucleic acid to be collected is not particularly limited, such as DNA or RNA. The number of specimens may be one or more. The length of the nucleic acid to be recovered is not particularly limited, and for example, a nucleic acid having an arbitrary length of several bp to several Mbp can be used. From the viewpoint of handling, the length of the nucleic acid to be recovered is generally about several bp to several hundreds kbp. The nucleic acid separation and purification method of the present invention can be used to recover nucleic acids that are relatively longer than conventional simple nucleic acid separation and purification methods, and is preferably used to recover nucleic acids of 50 kbp or more, more preferably 70 kbp or more. I get out.

  Hereinafter, a process of dissolving a cell membrane and a nuclear membrane, solubilizing a nucleic acid, and obtaining a sample solution containing the nucleic acid from a specimen will be described. In the present invention, a nucleic acid solubilizing reagent is used to solubilize nucleic acid by dissolving the cell membrane and the nuclear membrane. Examples of the nucleic acid solubilizing reagent include a solution containing a chaotropic salt, a surfactant, and a proteolytic enzyme.

  A method for obtaining a sample solution containing a nucleic acid from a specimen by dissolving a cell membrane and a nuclear membrane and solubilizing a nucleic acid includes: (I) a step of injecting a specimen containing cells or viruses into a container; A step of adding a nucleic acid solubilizing reagent solution containing a chaotropic salt and a surfactant and mixing the specimen and the nucleic acid solubilizing reagent solution, (III) a step of incubating the mixed solution obtained above, (IV) Examples thereof include a method including a step of adding a water-soluble organic solvent to the incubated mixed solution.

  In the step of dissolving the cell membrane and the nuclear membrane, solubilizing the nucleic acid, and obtaining a sample solution containing the nucleic acid from the specimen, homogenizing the specimen improves the suitability for automation. The homogenizing treatment can be performed by, for example, ultrasonic treatment, using sharp protrusions, using high-speed stirring treatment, processing for extruding from fine voids, processing using glass beads, or the like.

  Further, in the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the amount of nucleic acid recovered can be obtained by using a nucleic acid solubilizing reagent containing a proteolytic enzyme. In addition, the recovery efficiency is improved, and it is possible to reduce the amount and speed of the specimen containing the necessary nucleic acid.

As the proteolytic enzyme, at least one proteolytic enzyme can be preferably used from serine protease, cysteine protease, metal protease and the like. As the proteolytic enzyme, a mixture of a plurality of proteolytic enzymes can be preferably used.
The serine protease is not particularly limited, and for example, protease K can be preferably used.
The cysteine protease is not particularly limited, and for example, papain and cathepsins can be preferably used.
The metal protease is not particularly limited, and for example, carboxypeptidase can be preferably used.
The proteolytic enzyme can be used at a concentration of preferably 0.001 IU to 10 IU, more preferably 0.01 IU to 1 IU per 1 ml of the total volume of the reaction system at the time of addition.

  As the proteolytic enzyme, a proteolytic enzyme that does not contain a nucleolytic enzyme can be preferably used. A proteolytic enzyme containing a stabilizer can be preferably used. As the stabilizer, metal ions can be preferably used. Specifically, magnesium ion is preferable, and it can be added in the form of, for example, magnesium chloride. Proteolytic enzyme is stable, and can be added, for example, in the form of magnesium chloride. By including a proteolytic enzyme stabilizer, the amount of proteolytic enzyme required for nucleic acid recovery can be reduced, and the cost required for nucleic acid recovery can be reduced. The proteolytic enzyme stabilizer is preferably contained at a concentration of 1 to 1000 mM, more preferably 10 to 100 mM, based on the total amount of the reaction system.

The proteolytic enzyme may be mixed with other trials such as chaotropic salts and surfactants in advance and used for nucleic acid recovery as one reagent.
The proteolytic enzyme may be provided as two or more reagents separate from other reagents such as chaotropic salts and surfactants. In the latter case, a reagent containing a proteolytic enzyme is first mixed with a sample, and then mixed with a reagent containing a chaotropic salt and a surfactant. Moreover, after mixing the reagent containing a chaotropic salt and a surfactant previously, you may mix a proteolytic enzyme.
In addition, the proteolytic enzyme can be dropped directly into a specimen or a mixed solution of the specimen and a reagent containing a chaotropic salt and a surfactant directly from the proteolytic enzyme storage container. In this case, the operation can be simplified.

It is also preferable that the nucleic acid solubilizing reagent is supplied in a dried state. Moreover, the container previously containing the proteolytic enzyme of the dried state like freeze-drying can be used. A sample solution containing nucleic acid can also be obtained by using both the nucleic acid solubilizing reagent supplied in a dried state and the container previously containing the proteolytic enzyme in a dried state.
When a sample solution containing nucleic acid is obtained by the above method, the storage stability of the nucleic acid solubilizing reagent and proteolytic enzyme is good, and the operation can be simplified without changing the nucleic acid yield.

The method for mixing the specimen with a nucleic acid solubilizing reagent solution containing a chaotropic salt and a surfactant 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.

  Increasing the yield of nucleic acid to be separated and purified by incubating a mixture of a sample and a nucleic acid solubilizing reagent solution containing a chaotropic salt and a surfactant at the optimal temperature and reaction time of the proteolytic enzyme Can do. The incubation temperature is usually 20 ° C. to 70 ° C., preferably the optimum temperature for the proteolytic enzyme, preferably 37 ° C. to 70 ° C., particularly preferably 50 ° C. to 65 ° C. The incubation time is usually 1 to 90 minutes, preferably the optimal reaction time for the proteolytic enzyme. The incubation method is not particularly limited, and can be performed by putting it in a hot water bath or a warmer.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the nucleic acid solubilizing reagent solution containing the surfactant and the chaotropic salt is preferably pH 5 to 10, More preferably, those having a pH of 6 to 9, more preferably a pH of 7 to 8 are used.

In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the concentration of the chaotropic salt in the nucleic acid solubilizing reagent solution is 0.5 mol / L or more. It is preferably 0.5 mol / L to 7 mol / L, and more preferably 2 mol / L to 6 mol / L. As the chaotropic salt, guanidine hydrochloride is preferable, but other chaotropic salts (guanidine isothiocyanate, guanidine thiocyanate, urea, sodium isothiocyanate, potassium iodide, sodium iodide) can also be used. These salts may be used alone or in combination.

The nucleic acid solubilizing reagent solution may contain a water-soluble organic solvent. As this water-soluble organic solvent, alcohol is 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 preferably used. 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%.

In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the surfactant mixed with the chaotropic salt and the proteolytic enzyme in the specimen is, for example, nonionic A surfactant, a cationic surfactant, an anionic surfactant, and 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, Polyoxyethylene alkyl ether selected from POE sorbitan monooleate, POE sorbitan monostearate, polyoxyethylene sorbitol 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.

When recovering nucleic acids other than RNA, such as DNA, in the step of dissolving the cell membrane and nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, an RNase is added to the nucleic acid solubilizing reagent solution. It is preferable to add. In this case, interference by RNA coexisting with the recovered nucleic acid can be reduced. It is also preferable to add a DNA-degrading enzyme inhibitor.
On the other hand, when recovering nucleic acids other than DNA such as RNA, it is preferable to add a DNA degrading enzyme to the nucleic acid solubilizing reagent solution. In this case, interference by DNA coexisting with the recovered nucleic acid can be reduced. It is also preferable to add an RNase inhibitor. As the RNase inhibitor, those that specifically inhibit RNase are preferable.
The RNA degrading enzyme is not particularly limited, and for example, an RNA-specific degrading enzyme such as ribonuclease H (RNase H) can be preferably used.
The DNA degrading enzyme is not particularly limited, and for example, a DNA-specific degrading enzyme such as DNase I can be preferably used.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, an alcohol is preferably used as the water-soluble organic solvent to be added to the incubated mixture. Can do. 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 these water-soluble organic solvents in the nucleic acid solubilizing reagent solution is preferably 5 to 90%.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain the sample solution containing the nucleic acid from the specimen, the sample solution containing the nucleic acid 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.), oil-based antifoaming agents (eg, animal and vegetable oils, etc.), fatty acid type Antifoaming agents (eg, stearic acid, oleic acid, palmitic acid, etc.), metal soap 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.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain the sample solution containing the nucleic acid from the specimen, the sample solution containing the nucleic acid has a surface tension of 50 mPa · m or less. The viscosity is preferably 1 to 10,000 mPa · s, and the specific gravity is preferably 0.8 to 1.2.

  Hereinafter, the nucleic acid-adsorbing porous membrane used in the present invention and the adsorption process 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.

  The nucleic acid-adsorbing porous membrane of the present invention is characterized in that it is a porous membrane that adsorbs nucleic acids through an interaction that does not involve ionic bonds, and thereby, isolation and purification of nucleic acids with excellent separation performance and high washing efficiency can do. In particular, when the washing solution contains a water-soluble salt, particularly when it contains a water-soluble salt and a water-soluble organic solvent, the washing effect is excellent and nucleic acid can be recovered with high purity. More preferably, the nucleic acid-adsorptive porous membrane is a porous membrane having a hydrophilic group, and the material forming the porous membrane itself treats the porous membrane having the hydrophilic group or the material forming the porous membrane. Or the porous membrane which introduce | transduced the hydrophilic group by coating is meant. 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 hydrophilic group is introduced by treating a porous film of an organic material having no hydrophilic group, A porous film in which a hydrophilic group is introduced by coating a porous film made of an organic material that does not have a hydrophilic group, a porous film in which the material forming the porous film itself is an inorganic material having a hydrophilic group, hydrophilic Porous membranes with hydrophilic groups introduced by treating porous membranes of inorganic materials that do not have groups, and inorganic groups without inorganic groups coated with materials having hydrophilic groups to introduce hydrophilic groups A porous film or the like can be used, but an organic material such as an organic polymer is preferably used as a material for forming the porous film from the viewpoint of ease of processing.

  The hydrophilic group refers to a polar group (atomic group) capable of interacting with water, and all groups (atomic groups) involved in nucleic acid adsorption are applicable. The hydrophilic group preferably has a moderate interaction strength with water, and examples thereof include a hydroxyl group, a carboxyl group, a cyano group, and an oxyethylene group, with a hydroxyl group being preferred.

Examples of the porous film having a hydrophilic group include a porous film made of an organic material having a hydroxyl group. Examples of the organic material having a hydroxyl group include surface saponified products of acetylcellulose described in JP-A No. 2003-128691. The acetyl cellulose may be any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose, but triacetyl cellulose is particularly preferable. In this case, the amount (density) of hydroxyl groups on the solid surface can be controlled by the degree of saponification treatment (saponification degree). 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 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, the porous membrane of acetylcellulose is saponified.
The amount (density) of spatial hydroxyl groups can be controlled by a combination of the degree of saponification treatment (saponification degree) and the pore diameter of the porous membrane. In this case, the porous membrane may be a front-back symmetrical porous membrane, but a back-asymmetric porous membrane can be preferably used.

  As porous membranes of organic materials having hydroxyl groups, polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, acetylcellulose, different acetyl values Examples of the porous film formed include a mixture of acetylcellulose, and a porous film of an organic material having a polysaccharide structure can be preferably used.

In particular, as a porous film of an organic material having a hydroxyl group, an organic polymer porous film made of a mixture of acetylcelluloses having different acetyl values can be 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 (molar ratio) of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99. Furthermore, the mixing ratio of triacetyl cellulose and diacetyl cellulose is preferably 90:10 to 50:50.

Moreover, as the porous film of the organic material having a hydroxyl group, a porous film made of an organic material obtained by saponifying a mixture of acetyl cellulose having different acetyl values can be preferably used. Organic materials obtained by saponifying a mixture of acetyl celluloses having different acetyl values include saponified products of a mixture of triacetyl cellulose and diacetyl cellulose, saponified products of a mixture of triacetyl cellulose and monoacetyl cellulose, triacetyl cellulose, diacetyl cellulose and mono A saponified product of a mixture of acetylcellulose and a saponified product of a mixture of diacetylcellulose and monoacetylcellulose can also be preferably used. In particular, a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose can be preferably used.
The mixing ratio of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99. Furthermore, the mixing ratio of triacetyl cellulose and diacetyl cellulose is preferably 90:10 to 50:50.

The saponification treatment refers to bringing acetyl cellulose into contact with a saponification treatment solution (for example, sodium hydroxide aqueous solution). Thereby, it becomes a regenerated cellulose and a hydroxyl group is introduced into the portion of acetylcellulose that has come into contact with the saponification solution.
In order to increase the nucleic acid separation efficiency, it is preferable that the number of introduced hydroxyl groups is larger. For example, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.
In order to change the saponification rate, the saponification treatment may be performed by changing the concentration of sodium hydroxide. The surface saponification rate can be determined by NMR or IR.

  Moreover, as a porous film of an organic material having a hydroxyl group, a porous film of cellulose can be preferably used. As the porous membrane of cellulose, a porous membrane of regenerated cellulose can be preferably used. Regenerated cellulose is obtained by saponifying the solid surface or the whole of acetyl cellulose by saponification treatment, and is different from the original cellulose in terms of crystal state and the like.

As a method for introducing a hydrophilic group into a porous film made 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 compound having a double bond that can be polymerized starting from the porous film are polymerized. There are two ways to make the graft polymer chain.

  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, and this functional group and the porous It can be grafted by chemically reacting with a functional group of the membrane. The functional group that reacts with the porous membrane is not particularly limited as long as it can react with the functional group of the porous membrane. For example, a silane coupling group such as alkoxysilane, an isocyanate group, an amino group, and a hydroxyl group. And carboxyl group, sulfonic acid group, phosphoric acid group, epoxy group, allyl group, methacryloyl group, acryloyl group 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 based on a porous membrane to form a graft polymer chain is generally called surface graft polymerization. The surface graft polymerization method is a method of polymerizing a compound having a polymerizable double bond disposed so as to be in contact with the porous film by giving active species on the surface of the substrate by a method such as plasma irradiation, light irradiation, and heating. It refers to the method of bonding with a porous 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 made of an organic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for 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 ease of 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 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 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 membrane of an inorganic material having no hydrophilic group, a method of chemically bonding the porous membrane and a graft polymer chain, and a hydrophilic group having a double bond in the molecule are used. There are two methods for polymerizing a graft polymer chain using a monomer having a porous membrane as a starting point.
When the porous membrane 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 a graft polymer chain is polymerized starting from a porous membrane using a monomer having a hydrophilic group having a double bond in the molecule, a compound having a double bond is polymerized. The starting functional group 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 porous film of the organic material having no hydrophilic group is chemically bonded to the graft polymer chain. In the method, the described graft polymers having hydrophilic groups 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 of an inorganic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for 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 ease of 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 acetyl cellulose or a mixture of acetyl celluloses having different acetyl values on a porous film of an inorganic material having no hydrophilic group, the coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values is coated. Can also be saponified. 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. .

As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a thickness of 10 μm to 500 μm can be used. More preferably, a porous film having a thickness of 50 μm to 250 μm can be used.

  In addition, as the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a minimum pore diameter of 0.22 μm or more can be used. More preferably, a porous membrane having a minimum pore diameter of 0.5 μm or more can be used. As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a ratio of the maximum pore diameter to the minimum pore diameter of 2 or more can be used. More preferably, a porous membrane having a ratio of the maximum pore diameter to the minimum pore diameter of 5 or more can be used.

Moreover, as the nucleic acid-adsorptive porous membrane through which the solution can pass, a porous membrane having a porosity of 50 to 95% can be used. More preferably, a porous film having a porosity of 65 to 80% can be used. As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a bubble point of 0.1 to 10 kgf / cm ² can be used. More preferably, a porous film having a bubble point of 0.2 to 4 kgf / cm ² can be used.

  As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a pressure loss of 0.1 to 100 kPa can be used. More preferably, a porous film having a pressure loss of 0.5 to 50 kPa can be used. Here, the pressure loss is the minimum pressure required to pass water per 100 μm thickness of the membrane.

Moreover, as a nucleic acid-adsorptive porous membrane through which the solution can pass, the water permeation amount when passing water at a pressure of 1 kg / cm ² at 25 ° C. is 1 to 1 per 1 cm ^{2 of} the membrane. A porous membrane that is 5000 mL can be used. More preferably, a porous membrane having a water permeability of 5 to 1000 mL per minute per 1 cm ^{2 of} membrane when water is passed at 25 ° C. and a pressure of 1 kg / cm ² can be used.

  Further, as the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane in which the nucleic acid adsorption amount per 1 mg of the porous membrane is 0.1 μg or more can be preferably used. More preferably, a porous membrane having a nucleic acid adsorption amount of 0.9 μg or more per 1 mg of the porous membrane can be used.

  Moreover, as a nucleic acid-adsorbing porous membrane through which the solution can pass, when a square porous membrane with a side of 5 mm is immersed in 5 mL of trifluoroacetic acid, it does not dissolve within 1 hour but dissolves within 48 hours. A porous membrane made of a cellulose derivative can be preferably used. Further, a porous membrane made of a cellulose derivative that dissolves within 1 hour when immersed in 5 mL of trifluoroacetic acid, but does not dissolve within 24 hours when immersed in 5 mL of dichloromethane is preferable. Can be used.

  When passing a sample solution containing nucleic acid through the nucleic acid-adsorbing porous membrane, it is preferable to pass the sample solution from one surface to the other surface. When passing a sample solution containing nucleic acid through the nucleic acid-adsorbing porous membrane, it is preferable to pass the sample solution from the side having the larger pore diameter of the nucleic acid-adsorbing porous membrane to the smaller side.

The flow rate when the sample solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane is preferably ² to 1500 μL / sec per cm ^{2 of} the membrane area. Furthermore, it is preferable that it is 5-700 microliters / sec per area cm < ² > of a film | membrane.

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

  A cartridge for separating and purifying nucleic acid containing a nucleic acid-adsorbing porous membrane through which the above solution can pass inside 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.

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

  It is preferable that the nucleic acid separation and 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 the material of the container, plastics such as polypropylene, polystyrene, polycarbonate, polyvinyl chloride, polymethacrylic ester, polyethylene, polyester, and nylon can be used. Biodegradable materials can also be preferably used. In addition, the container may be transparent or colored.

  The nucleic acid-adsorbing porous membrane accommodated in the cartridge may have any shape such as a circle, a square, a rectangle and an ellipse, and a circle is particularly preferable.

  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 capable of easily taking out the nucleic acid-adsorbing porous membrane from a container having at least two openings can be used.

  Using the nucleic acid separation / purification cartridge containing the nucleic acid-adsorbing porous membrane through which each solution can pass, the nucleic acid can be separated and purified in the following steps. That is, (a) a sample solution containing nucleic acid is injected into one opening of a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane that allows the solution to pass through a container having at least two openings. (B) Depressurizing the inside of the nucleic acid separation / purification cartridge using a pressure difference generator connected to the other opening of the nucleic acid separation / purification cartridge, and the sample solution containing the injected nucleic acid is converted into a nucleic acid-adsorbing porous material. A step of adsorbing nucleic acid in the nucleic acid adsorbing porous membrane by passing through the membrane and discharging from the other opening of the nucleic acid separation and purification cartridge; (c) injecting a washing solution into the one opening of the nucleic acid separation and purification cartridge (D) Using a pressure difference generator connected to the other opening of the nucleic acid separation / purification cartridge, the inside of the nucleic acid separation / purification cartridge is depressurized, and the injected washing solution is subjected to nucleic acid adsorption. A step of washing the nucleic acid-adsorbing porous membrane with the nucleic acid adsorbed by passing through the porous membrane and discharging from the other opening; (F) The nucleic acid separation / purification cartridge is depressurized using a pressure difference generator coupled to the other opening of the nucleic acid separation / purification cartridge, or a centrifugal force is applied to the nucleic acid separation / purification cartridge, Passing the injected recovery liquid through the nucleic acid-adsorbing porous membrane and discharging it from the other opening, thereby desorbing the nucleic acid from the nucleic acid-adsorbing porous membrane and discharging it out of the cartridge for nucleic acid separation and purification; Can be done.

  As another nucleic acid separation and purification step, (a) a nucleic acid containing a nucleic acid-adsorbing porous membrane in which a solution containing a nucleic acid can be passed through a sample solution containing the nucleic acid in a container having at least two openings. A step of injecting into one opening of the separation and purification cartridge; (b) applying a centrifugal force to the nucleic acid separation and purification cartridge, passing the sample solution containing the injected nucleic acid through the nucleic acid-adsorbing porous membrane, and A step of adsorbing nucleic acid in the nucleic acid-adsorbing porous membrane by discharging from another opening, (c) a step of injecting a washing solution into the one opening of the nucleic acid separation and purification cartridge, and (d) centrifugation into the nucleic acid separation and purification cartridge. A step of washing the nucleic acid-adsorbing porous membrane in a state in which the nucleic acid is adsorbed by allowing the force to act and passing the injected cleaning liquid through the nucleic acid-adsorbing porous membrane and discharging from the other openings (e Nuclear Step of injecting the recovered liquid into the one opening of the separation / purification cartridge (f) Centrifugal force is applied to the nucleic acid separation / purification cartridge, and the injected recovery liquid is passed through the nucleic acid-adsorbing porous membrane and discharged from the other opening. Thus, a step of desorbing the nucleic acid from the nucleic acid-adsorbing porous membrane and discharging it out of the cartridge for nucleic acid separation and purification can be performed.

  Hereinafter, the cleaning process will be described. By performing the washing, the amount and purity of the nucleic acid recovered can be improved, and the amount of the specimen containing the necessary nucleic acid can be made very small. Further, by automating the cleaning and recovery operation, the operation can be performed easily and quickly. The cleaning process can be completed only once for speeding up. If the purity is more important, the washing may be 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 allowed to pass through the porous nucleic acid adsorbing porous membrane and discharged from an opening different from the 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.
In a particularly preferred embodiment, a cleaning solution is also supplied from one opening into which the sample solution is injected, passed through the nucleic acid-adsorbing porous membrane, and discharged from an opening different from the one opening, and one opening into which the sample solution is injected. In this method, a cleaning solution is supplied from different openings, passed through the nucleic acid-adsorbing porous membrane, and discharged from one opening.
The amount of the cleaning liquid in the cleaning process is preferably ² μl / mm ² or more. If the amount of the cleaning liquid 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, preferably 5 to 700 μL / sec, per unit area (cm ² ) of the membrane. More preferred. 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 by applying mechanical vibration or ultrasonic stirring or by centrifugation.

  In the washing step, the washing solution generally does not contain an enzyme such as a nucleolytic enzyme, but can contain an enzyme that degrades contaminants such as proteins. In some cases, a DNA degrading enzyme, an RNA degrading enzyme, or the like can be included. A washing solution containing a DNA-degrading enzyme is preferably used when only RNA in a specimen is selectively recovered. Conversely, a washing solution containing an RNase is preferably used when only DNA in a specimen is selectively recovered.

  In the washing step, a solution containing a water-soluble organic solvent and / or a water-soluble salt can be used as the washing liquid. 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 this purpose, it is necessary to have a composition in which nucleic acids are not desorbed from the nucleic acid-adsorbing porous membrane but impurities are desorbed. For this purpose, since a water-soluble organic solvent such as alcohol is hardly soluble in nucleic acid, it is suitable for desorbing components other than nucleic acid while retaining the nucleic acid. In addition, by adding a water-soluble salt, the effect of adsorbing nucleic acids is enhanced, so that the selective removal of unnecessary components is improved.

  As the water-soluble organic solvent contained in the cleaning liquid, methanol, ethanol, isopropanol, n-isopropanol, butanol, acetone and the like can be used, and ethanol is particularly preferable. The amount of the water-soluble organic solvent contained in the cleaning liquid is preferably 2 to 50% by mass, more preferably 2 to 40% by mass, and further preferably 3 to 30% by mass. However, the content of the water-soluble organic solvent is restricted by the lower limit of the specified range of the surface tension of the present invention described below.

As described above, the method for separating and purifying nucleic acid according to the present invention is characterized in that the nucleic acid-adsorbing porous membrane is a porous membrane that adsorbs nucleic acids by an interaction that does not substantially involve ionic bonds, and at the same time, has a surface tension. It is characterized by being 35 mPa · m or more.
The washing solution usually contains chaotropic substances and water-soluble organic solvents to improve the separation between nucleic acids and cell membranes and cytoplasm-derived degradation products derived from specimen samples. have. When the surface tension decreases, the wettability of the cleaning liquid improves, and conversely, the residual liquid on the porous membrane and the cartridge device wall increases, and when the residual liquid volume increases, the cleaning liquid is mixed into the recovery process following the cleaning process. This causes a decrease in the purity of the nucleic acid and a decrease in reactivity in the next step. Therefore, when nucleic acid is adsorbed and desorbed using a container such as a cartridge, washing residual liquid remains in the cartridge so that the liquid used for adsorption and washing, particularly the washing liquid, does not affect the next step. It is important not to.

Therefore, in the present invention, priority is given to avoiding a reduction in cleaning effect due to a reduction in surface tension, and the content of chaotropic substances and water-soluble organic solvents that are inherently effective in cleaning properties is an amount that maintains a surface tension of at least 35 mPa · m. Adjust to.
A preferable surface tension is 35 to 150 mPa · m, and more preferably 35 to 100 mPa · m. The upper limit of the surface tension of the cleaning liquid need not be specified, but even if it is 150 mPa · m or more, no further effect can be obtained.
Since the cleaning efficiency is increased by adjusting the surface tension, the cleaning process can be performed only once, and in this respect also, the nucleic acid separation and purification operation can be speeded up. Further, since there is no need to provide a drying step between the washing step and the recovery step, simplification and speeding up can be promoted from this point.

In the present invention, instead of adjusting the content of the organic solvent or chaotropic substance to a level at which the surface tension does not exceed the appropriate range, another means for improving the separation of nucleic acids and impurities such as proteins and maintaining the detergency is provided. It is preferable to adopt such a means, and it is effective that the ionic strength of the cleaning liquid is selected in the range of 5 to 500 mmol / L and that the cleaning liquid contains a water-soluble salt in the range of 10 to 1000 mmol / L. It is.
The ionic strength can be adjusted by adjusting the kind and concentration of salts and ionic surfactants in the cleaning liquid.

As the water-soluble salt, a halide salt is preferable, and a chloride is particularly suitable. Moreover, in order to increase the ionic strength with a small amount, sulfate or phosphate may be used.
On the other hand, monovalent or divalent cations are preferable as the cation component of the water-soluble salt, and potassium salt or sodium salt is particularly preferable, and sodium salt is particularly suitable. In order to increase the ionic strength in a small amount, a magnesium salt may be used. The concentration of the salt is preferably 10 mmol / L or more. In particular, it is preferable that sodium chloride is contained in an amount of 20 mmol / L or more to exhibit the intended effect, but even if 500 mmol / L or more is added, the effect is not further increased.
These water-soluble salts and ionic strength increasing compounds tend to increase the surface tension of the cleaning solution somewhat.

  Conventionally, in the nucleic acid separation and purification process, there is a problem that contamination of the sample occurs when the washing liquid is often scattered during the washing process and adheres to others. 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.

  The process of desorbing and recovering nucleic acid from the nucleic acid adsorbing polyporous membrane is shown below. 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 recovery 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 recovery 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 the 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 in which the nucleic acid separation and purification cartridge is supplied from one opening, passed through the nucleic acid-adsorbing porous membrane, and discharged 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 amount of the recovered liquid is several tens to several hundreds of μl. However, when the amount of the sample is extremely small, or when it is desired to separate and purify a large amount of nucleic acid, the amount of the recovered liquid is 1 μl to several tens of ml. Can be changed in range.

  The pH of the recovery 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, more preferably a salt concentration of 0.5 mol / L or less, and more preferably a salt concentration of 90 mmol / L or less. By doing so, the recovery rate of nucleic acids can be improved, and more nucleic acids can be recovered. However, in the present invention, it is not prohibited to simply use water (distilled water, pure water, etc.) as the recovered liquid. The recovered nucleic acid may be single-stranded or double-stranded.

  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, (collected liquid volume) :( sample solution volume) = 1: 100 to 99: 100, more preferably (collected 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 nucleic acids from the porous membrane at a temperature of the recovered solution of 0 to 10 ° C., the function of the nucleolytic enzyme is suppressed without adding any reagent or special operation to prevent degradation by the enzyme. Thus, nucleic acid solution can be easily and efficiently obtained by preventing nucleic acid degradation.

  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 desorption of nucleic acids from the porous membrane can be easily performed 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. Examples of the nucleolytic enzyme inhibitor include EDTA. As another embodiment, a stabilizer may be added to the collection container in advance.

  The collection container used in the collection process 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. A material that does not absorb at 260 nm is, for example, quartz glass, but is not limited thereto.

  In the nucleic acid separation and purification step, the steps from injection of the nucleic acid-containing sample solution to obtaining the nucleic acid outside the cartridge for nucleic acid separation and purification can be completed in 2 to 10 minutes. In the nucleic acid separation and purification step, the nucleic acid can be obtained in a yield of 50% to 90%.

[Example 1]
(1) Production of Nucleic Acid Purification Cartridge A nucleic acid separation and purification cartridge container having an inner diameter of 7 mm and a portion for accommodating a nucleic acid-adsorbing porous membrane was produced from high impact polystyrene.
(2) As a nucleic acid-adsorbing porous membrane, a porous membrane (saponification rate 20%) obtained by saponifying a porous membrane of triacetyl cellulose (film thickness = 70 μm, average pore size = 1.2 μm) is used. It accommodated in the part which accommodates the nucleic acid adsorptive porous membrane of the container for nucleic acid separation and purification cartridges.

(3) Preparation of Nucleic Acid Solubilizing Reagent and Washing Solution RNA solubilizing reagent solution and washing solution having the formulation shown in Table 1 were prepared. The surface tension of the cleaning liquid was 42 mPa · m at 25 ° C.

(4) Nucleic Acid Separation / Purification Operation Separation / purification operation of nucleic acid in cells was performed from the culture solution of cancerous human bone marrow cells (HL60) by the following procedure. That is, 200 μl of a nucleic acid solubilizing reagent solution and 20 μl of protease K (manufactured by SIGMA) solution were added to 200 μl of this culture solution and incubated at 60 ° C. for 10 minutes. After the incubation, 200 μl of ethanol was added and stirred. After stirring, the cell culture sample was injected into one opening of the nucleic acid separation / purification cartridge equipped with the nucleic acid-adsorbing porous membrane, and then the one opening. A pressure generator (power pipette) is coupled to the pressure sensor, the inside of the nucleic acid separation / purification cartridge is pressurized, and the sample solution containing the injected nucleic acid is passed through the nucleic acid-adsorptive porous membrane. And then discharged from the other opening of the nucleic acid separation and purification cartridge. Subsequently, 500 μL of the washing solution is injected into the one opening of the nucleic acid separation and purification cartridge, a pressure generator is connected to the one opening of the nucleic acid separation and purification cartridge, and the inside of the nucleic acid separation and purification cartridge is pressurized and injected. The washed liquid was passed through the nucleic acid-adsorptive porous membrane and discharged from another opening. Subsequently, 200 μL of water as a recovery solution is injected into the one opening of the nucleic acid separation and purification cartridge, and a pressure 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. The collected recovery liquid was put into a state and allowed to pass through the nucleic acid-adsorptive porous membrane, discharged from the other openings, and this liquid was recovered.

Comparative Example 1
A liquid containing the separated nucleic acid was recovered under the same conditions and operations as in Example 1 except that the cleaning liquid having the composition shown in Table 2 was used. The surface tension of the cleaning liquid was 24 mPa · m at 25 ° C.

(5) Result of electrophoresis of extracted nucleic acid FIG. 1 is a diagram obtained by electrophoresis of a nucleic acid sample collected in this example and a size marker λHINDIII (conditions: Life Technologies, 1 mass% agarose, 100 V). , 30 minutes). As can be seen from the results in FIG. 1, nucleic acids can be separated and purified efficiently from a nucleic acid-containing specimen sample by the method of the present invention.

  Further, the purity of the nucleic acids obtained in Example 1 and Comparative Example 1 was determined as the ratio of the spectral absorbance at 260 nm and 280 nm (referred to as A260 / A280). The results are shown in Table 3.

As is clear from the results of FIG. 1 and Table 3, the recovery amount is comparable to that of the comparative example by using the method for the present invention in which the surface tension of the cleaning liquid is adjusted to 35 mPa · m or more. Has been shown to be excellent in research. That is, Table 3 specifically shows the effect of the present invention in which the amount of residual liquid is reduced by adjusting the surface tension, thereby improving the purity of the nucleic acid.
The separation and purification of the nucleic acid could be performed with a simple operation and apparatus in a short time of about 5 minutes.

  In addition, when the operation of Example 1 was repeated using a cleaning liquid having a surface tension of 45 mPa · m obtained by adding 20 mmol / L of NaCl to the cleaning liquid of Example 1, the nucleic acid was recovered with the same efficiency as that of Example 1 and the purification efficiency and purification. Was recovered.

It is the figure obtained by electrophoresis of the nucleic acid sample collect | recovered in the Example and size marker (lambda) HINDIII.

Claims (15)

(1) passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid in the porous membrane;
(2) passing a washing solution through the nucleic acid-adsorptive porous membrane to wash the nucleic acid-adsorptive porous membrane with the nucleic acid adsorbed thereon; and
(3) In the method for separating and purifying nucleic acid, comprising the step of passing the recovered liquid through the nucleic acid-adsorbing porous membrane and desorbing the nucleic acid from the porous membrane, the nucleic acid-adsorbing porous membrane has an ionic bond. A method for separating and purifying nucleic acid, characterized in that it is a porous membrane that adsorbs nucleic acid by an interaction that is not substantially involved, and the surface tension of the washing liquid is 35 mPa · m or more.   2. In each of the steps (1), (2) and (3), a sample solution, a washing solution or a recovery solution containing a nucleic acid is passed through a nucleic acid-adsorbing porous membrane under pressure. The method for separating and purifying the nucleic acid as described.   In each of the above steps (1), (2) and (3), the nucleic acid is contained in one opening of the nucleic acid separation / purification cartridge containing the nucleic acid-adsorbing porous membrane in a container having at least two openings. A sample solution, a cleaning solution or a recovery solution is injected, and the inside of the cartridge is pressurized using a pressure difference generating device coupled to the one opening of the cartridge, and each injected solution is allowed to pass through the other opening. The method for separating and purifying nucleic acid according to claim 1, wherein the nucleic acid is discharged.   The method according to any one of claims 1 to 3, wherein the cleaning liquid is an aqueous solution containing 2 to 50% by mass of methanol, ethanol, isopropanol or n-propanol or a mixture thereof.   The method according to claim 1, wherein the cleaning liquid is a solution containing 5-1000 mmol / L of a monovalent or divalent metal water-soluble salt.   The number of times of washing the nucleic acid-adsorbing porous membrane by passing the washing solution through the nucleic acid-adsorbing porous membrane to which the nucleic acid has been adsorbed is one. A method for separating and purifying nucleic acids.   The method for separating and purifying nucleic acid according to any one of claims 1 to 6, wherein a step of drying the porous membrane adsorbing the nucleic acid is not included between the washing step and the recovery step.   The method for separating and purifying nucleic acid according to any one of claims 1 to 7, wherein the nucleic acid-adsorbing porous membrane is a porous membrane of an organic polymer material having a hydroxyl group.   The method for separating and purifying nucleic acid according to claim 8, wherein the nucleic acid-adsorbing porous membrane is a porous membrane having a polysaccharide structure.   The method for separating and purifying nucleic acid according to claim 8 or 9, wherein the nucleic acid-adsorbing porous membrane is a porous membrane of regenerated cellulose.   The method for separating and purifying nucleic acid according to any one of claims 1 to 3, wherein the sample solution containing the nucleic acid is an aqueous solution of nucleic acid or a solution obtained by adding a water-soluble organic solvent to a buffer solution of nucleic acid.   The sample solution containing a nucleic acid is a solution obtained by treating a specimen containing cells or viruses with a nucleic acid solubilizing reagent and adding a water-soluble organic solvent to the sample. A method for separating and purifying a nucleic acid according to any one of the above.   The method for separating and purifying nucleic acid according to claim 11 or 12, wherein the water-soluble organic solvent is methanol, ethanol, isopropanol, n-propanol or a mixture thereof.   The method for separating and purifying nucleic acid according to claim 12, wherein the nucleic acid solubilizing reagent is a solution containing a chaotropic salt, a surfactant and a proteolytic enzyme.   The method for separating and purifying nucleic acid according to any one of claims 1 to 3, wherein the recovered solution is a solution having a salt concentration of 0.5 mmol / L or less.

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