JP2006136285A - Method for separating and purifying nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating and purifying a nucleic acid by allowing the nucleic acid in a specimen to be adsorbed on a solid surface, by which the treatment is efficiently carried out in a short time so as not to cause contamination, and the apparatus therefor can be miniaturized. <P>SOLUTION: The method for separating and purifying the nucleic acid involving adsorption and desorption steps of the nucleic acid on the solid, using a solution for adsorbing the nucleic acid on the solid and a solution for desorbing the nucleic acid from the solid respectively, is characterized by that the solution for enabling the nucleic acid to be adsorbed on the solid contains a proteolytic enzyme and a stabilizer of the proteolytic enzyme. <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 obtaining a sample solution containing a nucleic acid from a specimen in order to separate and purify the nucleic acid. More specifically, the sample containing nucleic acid is used to contain 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 difference generator. The present invention relates to a method for separating and purifying nucleic acid from a 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.

  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 reported (Patent Document 1).

JP 2003-128691 A

  An object of the present invention is to provide a method for separating and purifying nucleic acid by adsorbing a nucleic acid in a specimen to a nucleic acid-adsorbing porous membrane and then desorbing it through washing or the like. Another object of the present invention is a porous material that has excellent separation performance, good washing efficiency, is simple, rapid, excellent in automation and downsizing, and can produce a large amount of materials having substantially the same separation performance. It is to provide a method for separating and purifying nucleic acid using a functional membrane.

  Another object of the present invention is to provide a nucleic acid separation and purification apparatus that can be processed in a short time and efficiently to prevent contamination and that can be miniaturized.

  As a result of diligent investigations to solve the above problems, the present inventors have found that in a method for separating and purifying nucleic acid including a process of adsorbing and desorbing a nucleic acid on a porous membrane, the porous membrane has an interaction that does not involve ionic bonds. It has been found that by using a porous membrane that adsorbs nucleic acid, it is possible to separate and purify nucleic acid from a sample containing nucleic acid with high yield and high purity. The present invention has been completed based on these findings.

  That is, according to the present invention, there is provided a method for separating and purifying nucleic acid, wherein the sample solution containing nucleic acid contains a proteolytic enzyme.

That is, the present invention achieves the object by the following configuration.
<1> (1) passing a sample solution containing nucleic acid through a solid phase and adsorbing the nucleic acid into the solid phase;
(2) passing the washing solution through the solid phase and washing the solid phase with the nucleic acid adsorbed; and (3) passing the recovery solution through the solid phase to desorb the nucleic acid from the solid phase. A method for separating and purifying nucleic acid, wherein the solution for adsorbing nucleic acid to a solid phase contains a proteolytic enzyme and a proteolytic enzyme stabilizer.
<2> The method for separating and purifying nucleic acid according to the above <1>, comprising a step of adding a premixed proteolytic enzyme stabilizer and proteolytic enzyme to a sample solution containing nucleic acid.
<3> Separation and purification of a nucleic acid according to <1> or <2>, wherein the proteolytic enzyme stabilizer is at least one of sugar, sugar alcohol, polyethylene glycol, and metal ion Method.
<4> The method for separating and purifying nucleic acid according to any one of <1> to <3>, wherein the proteolytic enzyme is at least one of serine protease, cysteine protease, metalloprotease, and acidic protease. .
<5> The method for separating and purifying nucleic acid according to any one of <1> to <4>, wherein the proteolytic enzyme is provided in a proteolytic enzyme solution not containing a nucleolytic enzyme.
<6> A solution for adsorbing nucleic acid on a solid phase contains at least a compound selected from proteolytic enzymes and antifoaming agents, chaotropic salts, nucleic acid stabilizers, buffers, water-soluble organic solvents, and surfactants. The method for separating and purifying nucleic acid according to any one of <1> to <5>, wherein the pretreatment liquid containing one kind is a solution obtained by adding and mixing a specimen containing cells or viruses .
<7> The nucleic acid according to any one of <1> to <6>, wherein the proteolytic enzyme is simultaneously added as one reagent together with other reagents such as a chaotropic salt and a surfactant in advance. Separation and purification method.
<8> The method for separating and purifying nucleic acid according to <6> or <7>, wherein the antifoaming agent comprises two components, a silicon-based antifoaming agent and an alcohol-based antifoaming agent.
<9> The method for separating and purifying nucleic acid according to any one of <6> to <8>, wherein the pretreatment liquid contains a nucleic acid stabilizer at a concentration of 0.1 to 20% by mass. .
<10> The method for separating and purifying nucleic acid according to <9>, wherein the nucleic acid stabilizer is a reducing agent.
<11> The method for separating and purifying nucleic acid according to <10>, wherein the reducing agent is a mercapto compound.
<12> The method for separating and purifying nucleic acid according to <9>, wherein the nucleic acid stabilizer is a chelating agent.
<13> The method for separating and purifying nucleic acid according to any one of <6> to <12>, wherein the chaotropic salt is a guanidinium salt.
<14> The method for separating and purifying nucleic acid according to any one of <6> to <13>, wherein the water-soluble organic solvent contains any of methanol, ethanol, propanol, and butanol.
<15> The method for separating and purifying nucleic acid according to any one of <1> to <14>, wherein the solid phase is a solid phase composed of silica or a derivative thereof, diatomaceous earth, or alumina.
<16> The method for separating and purifying nucleic acid according to any one of <1> to <14>, wherein the solid phase is a solid phase composed of an organic polymer.
<17> The method for separating and purifying nucleic acid according to <16>, wherein the solid phase composed of an organic polymer is a solid phase composed of an organic polymer having a polysaccharide structure.
<18> The method for separating and purifying nucleic acid according to <17>, wherein the solid phase composed of an organic polymer having a polysaccharide structure is a solid phase composed of acetylcellulose.
<19> The description <18>, wherein the solid phase composed of acetyl cellulose is a solid phase composed of an organic polymer obtained by saponifying a mixture of acetyl cellulose or acetyl cellulose having different acetyl values. The method for separating and purifying nucleic acid as described.
<20> The method for separating and purifying nucleic acid according to <19>, wherein the saponification rate of a mixture of acetylcelluloses having different acetyl values is 5% or more, comprising an organic polymer having a polysaccharide structure The method for separating and purifying nucleic acid according to <17>, wherein the solid phase is a solid phase composed of regenerated cellulose.
<22> The method for separating and purifying nucleic acid according to any one of <15> to <21>, wherein the solid phase is a porous membrane.
<23> The method for separating and purifying nucleic acid according to <22>, wherein the porous membrane is a front and back asymmetric porous membrane.
<24> The method for separating and purifying nucleic acid according to <22> or <23>, wherein the porous membrane is a porous membrane having an average pore size of 0.1 to 10.0 μm.
<25> The method for separating and purifying nucleic acid according to any one of <22> to <24>, wherein the porous membrane is a porous membrane having a thickness of 10 to 500 μm.
<26> The method for separating and purifying nucleic acid according to any one of <15> to <21>, wherein the solid phase is non-porous.
<27> The method for separating and purifying nucleic acid according to any one of <15> to <26>, wherein the solid phase is coated on beads.
<28> The method for separating and purifying nucleic acid according to <27>, wherein the beads are magnetic beads.
<29> Any one of <1> to <28>, wherein nucleic acid is adsorbed and desorbed using a nucleic acid separation and purification unit containing a solid phase in a container having at least two openings. The method for separating and purifying the nucleic acid as described.
<30> (a) a solid phase, (b) a nucleic acid separation and purification comprising a container having at least two openings for accommodating the solid phase, and (c) a pressure difference generator coupled to one opening of the container. The method for separating and purifying nucleic acid according to any one of <1> to <29>, wherein the nucleic acid is adsorbed and desorbed using a unit.
<31> The method for separating and purifying nucleic acid according to <30>, wherein the pressure difference generator is a pressurizing device.
<32> The method for separating and purifying nucleic acid according to <30>, wherein the pressure difference generator is a reduced pressure apparatus.
<33> The method for separating and purifying nucleic acid according to any one of <30> to <32>, wherein the pressure difference generator is detachably coupled to one opening of the container.
<34> The method for separating and purifying nucleic acid according to claim 30 or 31, comprising the following steps (a) to (f):
(a) preparing a sample solution containing nucleic acid using a specimen, and injecting the sample solution containing the nucleic acid into one opening of the nucleic acid separation and purification unit;
(b) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the sample solution containing the injected nucleic acid is discharged from the other opening, thereby fixing the container. A step of adsorbing nucleic acid to a solid phase by contacting the phase;
(c) injecting a washing solution into the one opening of the nucleic acid separation and purification unit;
(d) The container is pressurized by using a pressure difference generator connected to the one opening of the nucleic acid separation and purification unit, and the injected washing solution is discharged from the other opening to be brought into contact with the solid phase. A step of washing the solid phase,
(e) injecting a liquid capable of desorbing the nucleic acid adsorbed on the solid phase into the one opening of the nucleic acid separation and purification unit;
(f) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and a liquid capable of desorbing the injected nucleic acid is discharged from the other opening, A step of desorbing nucleic acid adsorbed on the solid phase and discharging it out of the container.
<35> A description <34> including the step of washing the solid phase with a washing solution after bringing the DNA-degrading enzyme solution into contact with the solid phase before the step (e) The method for separating and purifying nucleic acid according to 1.
<36> The method for separating and purifying nucleic acids according to <1> to <35>, wherein the washing solution is a solution containing 20 to 100% by mass of methanol, ethanol, isopropanol, or n-propanol.
<37> The nucleic acid according to any one of <1> to <36>, wherein the solution capable of desorbing the nucleic acid adsorbed on the solid phase is a solution having a salt concentration of 0.5 mol / L or less. Separation and purification method.
<38> A reagent kit for performing the method described in any one of <1> to <37>.
<39> Description apparatus <1>-the apparatus for performing the method described in any one of <38>.

  According to the method for separating and purifying nucleic acid of the present invention, it is possible to separate and purify nucleic acid from a sample containing nucleic acid with good separation performance, in a simple, rapid, and automated manner.

  The method for separating and purifying nucleic acid according to the present invention will be specifically described. In the present invention, the nucleic acid in the sample solution is adsorbed to the solid phase by bringing the sample solution containing the nucleic acid into contact with the solid phase, and then the nucleic acid adsorbed to the solid phase is used with a suitable solution described below. Desorb from the solid phase. Preferably, the sample solution containing nucleic acid comprises at least one compound selected from the group consisting of proteolytic enzymes, nucleic acid stabilizers, chaotropic salts, surfactants, buffers and antifoaming agents. Is a solution obtained by further adding a water-soluble organic solvent to a solution obtained by processing using a pretreatment liquid containing a nucleic acid solubilizing reagent.

  The sample solution containing nucleic acid that can be used in the present invention is not limited, but in the diagnostic field, for example, whole blood collected as a specimen, plasma, serum, urine, stool, semen, saliva and other body fluids, cells, bacteria, Of interest are solutions prepared from biological materials such as plants (or parts thereof), animals (or parts thereof), etc., or lysates and homogenates thereof.

  First, these specimens are treated with an aqueous solution containing a reagent that dissolves the cell membrane and solubilizes the nucleic acid. As a result, the cell membrane and the nuclear membrane are dissolved, and the nucleic acid is dispersed in the aqueous solution.

  In the present invention, the “nucleic acid” may be single-stranded or double-stranded DNA or RNA, and there is no molecular weight limitation.

  A sample means any sample containing nucleic acid. The type of nucleic acid in the sample may be one type or two or more types. The length of each nucleic acid 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 is generally about several bp to several hundreds kbp.

  For cell membrane lysis and nucleic acid solubilization, for example, when the sample of interest is whole blood, E. Removal of red blood cells Removal of various proteins, and C.I. Leukocyte lysis and nuclear membrane lysis are required. A. E. Removal of red blood cells and B. The removal of various proteins is performed in order to prevent non-specific adsorption to the solid phase and clogging of the porous membrane. Leukocyte lysis and nuclear envelope lysis are required to solubilize the nucleic acid to be extracted. For example, said A, B, and C can be achieved simultaneously by incubating at 60 degreeC for 10 minutes in the state which added the guanidine hydrochloride, TritonX-100, and protease K (made by SIGMA).

The pretreatment liquid contains a proteolytic enzyme from the viewpoint of improving the recovery amount and recovery efficiency of the nucleic acid, reducing the amount of the sample containing the necessary nucleic acid, and speeding up the sample.
As the proteolytic enzyme, at least one proteolytic enzyme of serine protease, cysteine protease, and metalloprotease can be 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 concentration of the proteolytic enzyme in the pretreatment liquid is preferably 0.001 IU to 10 IU, more preferably 0.01 IU to 1 IU per 1 mL of the total volume at the time of addition.
As the proteolytic enzyme, a proteolytic enzyme that does not contain a nucleolytic enzyme can be preferably used.

The proteolytic enzyme stabilizer may be added to the proteolytic enzyme in advance and used in a mixed state, or may be added separately to the sample solution before and after using the proteolytic enzyme.
As the proteolytic enzyme stabilizer, saccharides, sugar alcohols, polyethylene glycols, and metal ions can be preferably used. Specifically, among saccharides, glucose, maltose, sucrose, lactose, and trehalose are preferable. Among sugar alcohols, sorbitol, mannitol, and myo-inositol are preferable. In polyethylene glycol, it can be preferably used that the average molecular weight is in the range of 200 to 10,000. Examples of the metal ions include divalent metal ions such as magnesium and calcium. Magnesium ions are preferable, and for example, magnesium ions can be added in the form of magnesium chloride.
By including a stabilizer in the proteolytic enzyme, the amount of proteolytic enzyme necessary for nucleic acid recovery can be reduced, and the cost required for nucleic acid recovery can be reduced.
The concentration of the protease stabilizer in the pretreatment liquid is preferably 1 to 1000 mmol / L, more preferably 10 to 100 mmol / L.

The proteolytic enzyme may be preliminarily mixed with other reagents such as an antifoaming agent, chaotropic salt, surfactant, etc. and used for recovery of nucleic acid as a pretreatment liquid (hereinafter referred to as pretreatment liquid A). good.
In addition, the proteolytic enzyme includes two or more reagents that are separate from the pretreatment liquid (hereinafter referred to as pretreatment liquid B) containing other reagents such as an antifoaming agent, chaotropic salt, and surfactant. May be provided. In the latter case, the reagent containing the proteolytic enzyme is first mixed with the sample, and then mixed with the pretreatment liquid B. Alternatively, the pretreatment liquid B may be mixed with the specimen first, and then the proteolytic enzyme may be mixed.
Alternatively, the proteolytic enzyme can be dropped directly into the specimen or a mixed liquid of the specimen and the pretreatment liquid B in the form of eye drops directly from the proteolytic enzyme storage container. In this case, the operation can be simplified.

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

  Specific examples of the antifoaming agent used in the present invention include a silicon-based antifoaming agent (eg, silicone oil, dimethylpolysiloxane, silicone emulsion, modified polysiloxane, silicone compound, etc.), an alcohol-based antifoaming agent (eg, acetylene glycol). , Heptanol, ethylhexanol, higher alcohol, polyoxyalkylene glycol, etc.), ether-based antifoaming agents (for example, heptylcellosolve, nonylcellosolv-3-heptylcorbitol, etc.), oil-based antifoaming agents (for example, animal and vegetable oils, etc.) ), Fatty acid 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 ), Phosphate ester antifoaming agents (for example, octyl sodium phosphate), amine-based antifoaming agents (for example, diamylamine), amide-based antifoaming agents (for example, stearamide), and other antifoaming agents 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.

  Examples of the nucleic acid stabilizer include those having an action of inactivating nuclease activity. Depending on the sample, a nuclease or the like that degrades the nucleic acid may be contained. When the nucleic acid is homogenized, the nuclease acts on the nucleic acid, and the yield may be drastically reduced. The nucleic acid stabilizer is preferable because it can cause the nucleic acid in the specimen to exist stably.

As the nucleic acid stabilizer having an action of inactivating the nuclease activity, compounds generally used as a reducing agent can be used. 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 acids such as formic acid and oxalic acid, mercapto compounds and the like. Of these, mercapto compounds are preferred. Examples of mercapto compounds include N-acetylcysteine, mercaptoethanol, alkyl mercaptan, and the like. In particular, β-mercaptoethanol is preferable. Mercapto compounds may be used alone or in combination.
The concentration of the nucleic acid stabilizer in the pretreatment solution is preferably 0.1 to 20% by mass, and more preferably 0.3 to 15% by mass. The concentration of the mercapto compound in the pretreatment liquid is preferably 0.1 to 10% by mass, and more preferably 0.5 to 5% by mass.

  Moreover, a chelating agent can be used as a nucleic acid stabilizer having an action of inactivating the nuclease activity. Examples of chelating agents include EDTA, NTA, EGTA and the like. Chelating agents may be used alone or in combination.

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. Of these, guanidine hydrochloride is preferable. These salts may be used alone or in combination. The chaotropic salt concentration in the pretreatment liquid is preferably 0.5 mol / L or more, more preferably 0.5 to 4 mol / L, and still more preferably 1 to 3 mol / L.
Instead of the chaotropic salt, urea can also be used as the chaotropic substance.

Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants.
In the present invention, nonionic surfactants and cationic surfactants can be preferably used.
Examples of nonionic surfactants include polyoxyethylene alkylphenyl ether surfactants, polyoxyethylene alkyl ether surfactants, and fatty acid alkanolamides, with polyoxyethylene alkyl ether surfactants being preferred. Among the polyoxyethylene alkyl ether surfactants, POE decyl ether, POE lauryl ether, POE tridecyl ether, POE alkylene decyl ether, POE sorbitan monolaurate, POE sorbitan monooleate POE sorbitan monostearate, polyoxyethylene sorbite tetraoleate, POE alkylamine, and POE acetylene glycol are more preferable.

Examples of the cationic surfactant include cetyltrimethylammonium promide, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, and cetylpyridinium chloride.
These surfactants may be used alone or in combination. The concentration of the surfactant in the pretreatment liquid is preferably 0.1 to 20% by mass.

Examples of the buffer 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] ethanesulfonicasid), PIPES (Piperaxine-1,4-bis (2-ethanesulfonic acid)), ACES (N- (2-Acetamino) -2-aminoethanesulfonic acid), CAPS (N-Cyclohexyl-3-aminopropanesulfonic acid), TES (N-Tris (hydroxyl -methyl) methyl-2-aminoethanesulfonic acid).
These buffering agents preferably have a concentration in the pretreatment liquid of 1 to 300 mmol / L.

  A water-soluble organic solvent may be added to dissolve a defoamer, nucleic acid stabilizer, chaotropic salt, surfactant, buffer, etc. and mix to obtain a sample solution. Examples of the water-soluble organic solvent include alcohols, acetone, acetonitrile, dimethylformamide and the like. Among these, alcohols are preferable because the solubility of the compound contained in the sample solution can be increased. As alcohols, any of primary alcohol, secondary alcohol, and tertiary alcohol may be used. Among them, methanol, ethanol, propanol and its isomer, butanol and its isomer, and the like are mentioned, and ethanol is more preferable. These water-soluble organic solvents may be used alone or in combination. The concentration of these water-soluble organic solvents in the sample solution is preferably 1 to 20% by mass.

  Next, a water-soluble organic solvent is added to the aqueous solution in which the nucleic acid is dispersed, and brought into contact with the solid phase. By this operation, the nucleic acid in the sample solution is adsorbed to the solid phase. In order to adsorb the nucleic acid solubilized by the operation described above in this specification to the solid phase, a water-soluble organic solvent is mixed with the solubilized nucleic acid mixture, and a salt is contained in the obtained nucleic acid mixture. Must exist.

  That is, by destroying the hydration structure of water molecules present around the nucleic acid, the nucleic acid is solubilized in an unstable state. When nucleic acid in this state is brought into contact with a solid phase composed of an organic polymer having a hydroxyl group on the surface, the polar group on the nucleic acid surface interacts with the polar group on the solid phase surface, and the nucleic acid is adsorbed on the solid surface. It is thought to do. In the method of the present invention, the nucleic acid can be brought into an unstable state by mixing a water-soluble organic solvent with the solubilized nucleic acid mixture and the presence of a salt in the obtained nucleic acid mixture.

  Examples of the water-soluble organic solvent include alcohols, acetone, acetonitrile, dimethylformamide and the like. Among these, alcohols are preferable. As alcohols, any of primary alcohol, secondary alcohol, and tertiary alcohol may be used. Among them, methanol, ethanol, propanol and its isomer, butanol and its isomer, and the like are mentioned, and ethanol is more preferable.

  Moreover, it is preferable that the final concentration in the sample solution containing the nucleic acid of these water-soluble organic solvents is 5-90 mass%. More preferably, it is 20 mass%-60 mass%. The concentration of ethanol added is particularly preferably as high as possible without causing pseudo-collects.

  Salts present in the resulting nucleic acid mixture include various chaotropic substances (guanidinium salts, sodium iodide, sodium perchlorate), sodium chloride, potassium chloride, ammonium chloride, sodium bromide, potassium bromide, bromide. Calcium, ammonium bromide and the like are preferred. In particular, a guanidinium salt is particularly preferable because it has the effects of cell membrane lysis and nucleic acid solubilization.

  The sample solution preferably has a pH of 3 to 10, more preferably a pH of 4 to 9, and even more preferably a pH of 5 to 8.

In addition, the sample solution containing the obtained nucleic acid preferably has a surface tension of 0.05 J / m ² or less, a viscosity of 1 to 10,000 mPa, and a specific gravity of 0.8 to 1.2. By making the solution in this range, in the next step, the sample solution containing the nucleic acid is passed through the solid phase, and the solution remaining after adsorbing the nucleic acid is easily removed.

  The type of the solid phase is not particularly limited. For example, a solid phase composed of an organic polymer having a hydroxyl group on the surface, a solid phase composed of silicon dioxide, silica polymer or magnesium silicate, silica or a derivative thereof, diatomaceous earth, or alumina. A solid phase consisting of can be used. Preferably, a solid phase composed of an organic polymer having a polysaccharide structure can be used. More preferably, it is a solid phase on which a nucleic acid is adsorbed by an interaction that does not substantially involve ionic bonds. This means that it is not “ionized” under the use conditions on the solid phase side, and it is presumed that the nucleic acid and the solid phase become attracted 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. This is a solid phase having a hydrophilic group, and it is presumed that by changing the polarity of the environment, acetic acid and the hydrophilic group of the solid phase are attracted to each other.

  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. As the hydrophilic group, one having a moderate interaction with water is good (see Chemical Encyclopedia, Kyoritsu Shuppan Co., Ltd., “Hydrophilic group”, “Group that is 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.

  Here, the solid phase having a hydrophilic group means that the material itself forming the solid phase has a hydrophilic group, or introducing a hydrophilic group by treating or coating the material forming the solid phase. . When the material forming the solid phase is treated or coated, the material forming the solid phase may be either organic or inorganic. For example, a solid phase in which the material forming the solid phase itself is an organic material having a hydrophilic group, a solid phase in which a solid phase of an organic material having no hydrophilic group is processed to introduce a hydrophilic group, or an organic material having no hydrophilic group A solid phase in which a hydrophilic group is introduced by coating with a material having a hydrophilic group on the solid phase, a solid phase in which the solid phase forming material itself is an inorganic material having a hydrophilic group, and a solid phase of an inorganic material having no hydrophilic group. A solid phase in which a hydrophilic group is introduced by treating a phase, a solid phase in which a hydrophilic group is introduced by coating with a material having a hydrophilic group on a solid phase of an inorganic material having no 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 solid phase.

  Examples of the solid phase of the material having a hydrophilic group include a solid phase of an organic material having a hydroxyl group. As the solid phase of the organic material having a hydroxyl group, polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, an organic material having a polysaccharide structure, etc. Can be mentioned.

  Organic materials having a polysaccharide structure include cellulose, hemicellulose, dextran, agarose, dextrin, amylose, amylopectin, starch, glycogen, pullulan, mannan, glucomannan, lichenan, isolikenan, laminaran, carrageenan, xylan, fructan, alginic acid, hyaluronic acid Chondroitin, chitin, chitosan and the like can be preferably used, but the polysaccharide materials and derivatives thereof are not limited to the materials mentioned above. Moreover, it can use preferably also about the ester derivative of one of the said polysaccharide structures. Further, saponified products of ester derivatives having any of the above polysaccharide structures can be more preferably used.

  The ester of the polysaccharide structure ester derivative may be selected from any one or more of carboxylic acid ester, nitrate ester, sulfate ester, sulfonate ester, phosphate ester, phosphonate ester, and pyrophosphate ester. preferable. Further, saponified products of any of the above-mentioned polysaccharide structures, such as carboxylic acid esters, nitrate esters, sulfate esters, sulfonate esters, phosphate esters, phosphonate esters, and pyrophosphate esters, can be further preferably used.

  The carboxylic acid ester having any polysaccharide structure is preferably selected from any one or more of alkyl carbonyl ester, alkenyl carbonyl ester, aromatic carbonyl ester, and aromatic alkyl carbonyl ester. Furthermore, saponified products of any of the above-mentioned polysaccharide-structured alkylcarbonyl esters, alkenylcarbonyl esters, aromatic carbonyl esters, and aromatic alkylcarbonyl esters can be further preferably used.

  Examples of the ester group of the alkylcarbonyl ester having any polysaccharide structure include acetyl group, propionyl group, butyroyl group, barrel group, peptanoyl group, octanoyl group, decanoyl group, dodecanoyl group, tridecanoyl group, hexadecanoyl group, octadecanoyl group, octadecanoyl group, and octadecanoyl group. It is preferably selected from any one or more of noyl groups. Further, an ester group selected from any one or more of the acetyl group, propionyl group, butyroyl group, barrel group, peptanoyl group, octanoyl group, decanoyl group, dodecanoyl group, tridecanoyl group, hexadecanoyl group and octadecanoyl group Any of the polysaccharide saponified products having the above can also be preferably used.

  It is preferable that the ester group of the alkenylcarbonyl ester having any polysaccharide structure is selected from one or more of an acryl group and a methacryl group. Moreover, the saponified product of any one of the polysaccharide structures having an ester group selected from any one or more of the acryl group and the methacryl group can be further preferably used.

  It is preferable that the ester group of the aromatic carbonyl ester having any one of the polysaccharide structures is selected from at least one of a benzoyl group and a naphthaloyl group. Further, any of the polysaccharide structure saponified products having an ester group selected from at least one of the benzoyl group and naphthaloyl group can be used more preferably.

Nitric esters of any of the above polysaccharide structures include nitrocellulose, nitrohemicellulose, nitrodextran, nitroagarose, nitrodextrin, nitroamylose, nitroamylopectin, nitroglycogen, nitropullulan, nitromannan, nitroglucomannan, nitrolicenan, Sorikenan, nitrolaminaran, nitrocarrageenan, nitroxylan, nitrofructan, nitroalginic acid, nitrohyaluronic acid, nitrochondroitin, nitrochitin, nitrochitosan and the like can be preferably used.
In addition, nitrocellulose, nitrohemicellulose, nitrodextran, nitroagarose, nitrodextrin, nitroamylose, nitroamylopectin, nitroglycogen, nitropullulan, nitromannan, nitroglucomannan, nitroligenanan, nitroisoliquenan, nitrolaminaran, nitrocarrageenan Further, saponified products such as nitroxylan, nitrofructan, nitroalginic acid, nitrohyaluronic acid, nitrochondroitin, nitrochitin and nitrochitosan can be used more preferably.

  Examples of the sulfate ester having a polysaccharide structure include cellulose sulfate, hemicellulose sulfate, dextran sulfate, agarose sulfate, dextrin sulfate, amylose sulfate, amylopectin sulfate, glycogen sulfate, pullulan sulfate, mannan sulfate, glucomannan sulfate, lichenan sulfate, and isolikenan. Sulfuric acid, laminaran sulfate, carrageenan sulfate, xylan sulfate, fructan sulfate, alginate sulfate, hyaluronic acid sulfate, chondroitin sulfate, chitin sulfate, chitosan sulfate and the like can be preferably used. In addition, cellulose sulfate, hemicellulose sulfate, dextran sulfate, agarose sulfate, dextrin sulfate, amylose sulfate, amylopectin sulfate, glycogen sulfate, pullulan sulfate, mannan sulfate, glucomannan sulfate, lichenan sulfate, isolikenane sulfate, laminaran sulfate, carrageenan sulfate, Saponified products such as xylan sulfate, fructan sulfate, alginic acid sulfuric acid, hyaluronic acid sulfuric acid, chondroitin sulfuric acid, chitin sulfuric acid, chitosan sulfuric acid and the like can be further preferably used.

  The polysaccharide structure sulfonic acid ester is preferably selected from any one or more of alkyl sulfonic acid esters, alkenyl sulfonic acid esters, aromatic sulfonic acid esters, and aromatic alkyl sulfonic acid esters. Furthermore, saponified products of any of the above-described polysaccharide-structured alkyl sulfonates, alkenyl sulfonates, aromatic sulfonates, and aromatic alkyl sulfonates can be more preferably used.

  Examples of the phosphate ester having any polysaccharide structure include cellulose phosphate, hemicellulose phosphate, dextran phosphate, agarose phosphate, dextrin phosphate, amylose phosphate, amylopectin phosphate, glycogen phosphate, pullulan phosphate, mannan phosphate. Acids, glucomannan phosphates, lichenan phosphates, isolikenan phosphates, laminaran phosphates, carrageenan phosphates, xylan phosphates, fructan phosphates, alginate phosphates, hyaluronic acid phosphates, chondroitin phosphates, chitin phosphates, chitosan phosphates, etc. Can be used. In addition, cellulose phosphate, hemicellulose phosphate, dextran phosphate, agarose phosphate, dextrin phosphate, amylose phosphate, amylopectin phosphate, glycogen phosphate, pullulan phosphate, mannan phosphate, glucomannan phosphate, lichenan phosphate Further, saponified products such as isolikenan phosphate, laminaran phosphate, carrageenan phosphate, xylan phosphate, fructan phosphate, alginate phosphate, hyaluronic acid phosphate, chondroitin phosphate, chitin phosphate, chitosan phosphate can be used more preferably. .

  Examples of the phosphonic acid ester having any polysaccharide structure include cellulose phosphonic acid, hemicellulose phosphonic acid, dextran phosphonic acid, agarose phosphonic acid, dextrin phosphonic acid, amylose phosphonic acid, amylopectin phosphonic acid, glycogen phosphonic acid, pullulan phosphonic acid, and mannan. Phosphonic acid, glucomannan phosphonic acid, lichenan phosphonic acid, isolikenane phosphonic acid, laminaran phosphonic acid, carrageenan phosphonic acid, xylan phosphonic acid, fructan phosphonic acid, alginic acid phosphonic acid, hyaluronic acid phosphonic acid, chondroitin phosphonic acid, chitin phosphonic acid, Chitosan phosphonic acid and the like can be preferably used. In addition, cellulose phosphonic acid, hemicellulose phosphonic acid, dextran phosphonic acid, agarose phosphonic acid, dextrin phosphonic acid, amylose phosphonic acid, amylopectin phosphonic acid, glycogen phosphonic acid, pullulan phosphonic acid, mannan phosphonic acid, glucomannan phosphonic acid, lichenane phosphonic acid Saponification products such as acid, isollikenan phosphonic acid, laminaran phosphonic acid, carrageenan phosphonic acid, xylan phosphonic acid, fructan phosphonic acid, alginic acid phosphonic acid, hyaluronic acid phosphonic acid, chondroitin phosphonic acid, chitin phosphonic acid, chitosan phosphonic acid Furthermore, it can be preferably used.

  Examples of the above-described pyrophosphoric acid ester having a polysaccharide structure include cellulose pyrophosphate, hemicellulose pyrophosphate, dextran pyrophosphate, agarose pyrophosphate, dextrin pyrophosphate, amylose pyrophosphate, amylopectin pyrophosphate, glycogen pyrophosphate, pullulan pyrophosphate, mannan Pyrophosphoric acid, glucomannan pyrophosphate, lichenane pyrophosphate, isollichenane pyrophosphate, laminaran pyrophosphate, carrageenan pyrophosphate, xylan pyrophosphate, fructan pyrophosphate, alginate pyrophosphate, hyaluronic acid pyrophosphate, chondroitin pyrophosphate, chitin pyrolin Acid, chitosan pyrophosphate and the like can be preferably used. In addition, cellulose pyrophosphate, hemicellulose pyrophosphate, dextran pyrophosphate, agarose pyrophosphate, dextrin pyrophosphate, amylose pyrophosphate, amylopectin pyrophosphate, glycogen pyrophosphate, pullulan pyrophosphate, mannan pyrophosphate, glucomannan pyrophosphate, Saponified products such as Kenan pyrophosphate, Isolicenan pyrophosphate, Laminaran pyrophosphate, Carrageenan pyrophosphate, Xylan pyrophosphate, Fructan pyrophosphate, Alginate pyrophosphate, Hyaluronic acid pyrophosphate, Chondroitin pyrophosphate, Chitin pyrophosphate, Chitosan pyrophosphate Further, it can be preferably used.

  Examples of the ether derivative having any polysaccharide structure include methylcellulose, ethylcellulose, carboxymethylcellulose, carboxyethylcellulose, carboxyethyl-carbamoylethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, cyanoethylcellulose, carbamoyl. Although ethyl cellulose etc. can be used, it is not limited to these. Preferably, hydroxymethyl cellulose and hydroxyethyl cellulose can be used.

  Any of the above-mentioned polysaccharides having a hydroxyl group halogenated with an arbitrary degree of substitution can be preferably used.

  Preferred examples of the solid phase comprising an organic polymer having a polysaccharide structure include acetyl cellulose, and an organic polymer solid phase comprising a mixture of acetyl celluloses having different acetyl values can be used. As acetylcellulose mixtures having different acetyl values, triacetylcellulose and diacetylcellulose mixtures, triacetylcellulose and monoacetylcellulose mixtures, triacetylcellulose and diacetylcellulose and monoacetylcellulose mixtures, diacetylcellulose and monoacetylcellulose mixtures are preferably used. Can do. 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 particularly preferred solid phase composed of acetyl cellulose is a surface saponified product of acetyl cellulose described in JP-A No. 2003-128691. The surface saponified product of acetylcellulose is a saponified acetylcellulose mixture with different acetyl values, saponified product of triacetylcellulose and diacetylcellulose mixture, saponified product of triacetylcellulose and monoacetylcellulose mixture, triacetylcellulose 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. The saponification rate (surface saponification rate) of the solid phase obtained by the saponification treatment is preferably 5% or more and 100% or less, and more preferably 10% or more and 100% or less. In order to increase the surface area of the solid phase having a hydroxyl group, it is preferable to saponify the solid phase of acetylcellulose. The solid phase may be a front and back symmetric porous membrane, but a front and back asymmetric porous membrane can be preferably used.

Here, 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 portion of acetylcellulose in contact with the saponification solution becomes regenerated cellulose and hydroxyl groups are introduced. The regenerated cellulose thus prepared is different from the original cellulose in terms of crystal state and the like. In the present invention, it is particularly preferable to use a regenerated cellulose solid phase as the solid phase.
In order to change the saponification rate, the saponification treatment may be performed by changing the concentration of sodium hydroxide. The saponification rate can be easily measured by NMR (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 solid phase 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 solid phase.
As a method for bonding a graft polymer chain to a solid phase of an organic material, a method in which a solid phase and a graft polymer chain are chemically bonded, or a compound having a double bond capable of polymerization starting from a solid phase is polymerized to graft polymer chain. There are two methods.

First, in the method of chemically bonding the solid phase and the graft polymer chain, a polymer having a functional group that reacts with the solid phase at the end or side chain of the polymer is used, and the functional group and the functional group of the solid phase are chemically treated. It can be grafted by reacting. The functional group that reacts with the solid phase is not particularly limited as long as it can react with the functional group on the solid phase. For example, a silane coupling group such as alkoxysilane, isocyanate group, amino group, hydroxyl group, carboxyl 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 terminal, and a polymer having an isocyanate group at the polymer terminal. 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 starting from a solid phase to form a graft polymer chain is generally called surface graft polymerization. The surface graft polymerization method is a method in which an active species is given on the surface of a substrate by a method such as plasma irradiation, light irradiation, or heating, and a compound having a polymerizable double bond disposed so as to contact a solid phase is solidified by polymerization. Refers to the method of combining with the phase.
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 of polymers, oligomers, and monomers having a hydrophilic group can be used as long as they have 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 monomers having a hydroxyl group, such as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and glycerol monomethacrylate. 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 the solid phase 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 acetylcellulose mixtures, and polymers having a polysaccharide structure are preferred.

  In addition, after coating a solid phase of an organic material having no hydrophilic group with acetyl cellulose or an acetyl cellulose mixture having a different acetyl value, the coated acetyl cellulose or an acetyl cellulose mixture having a different acetyl value can be saponified. In this case, the saponification rate is preferably 5% or more and 100% or less. Furthermore, the saponification rate is preferably 10% or more and 100% or less.

  Examples of the solid phase that is an inorganic material having a hydrophilic group include a solid phase containing a silica compound. A glass filter can be mentioned as a solid phase 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 of introducing a hydrophilic group into the solid phase of an inorganic material having no hydrophilic group, there are a method of chemically bonding the solid phase and a graft polymer chain having a hydrophilic group, and a hydrophilic property having a double bond in the molecule. There are two methods of polymerizing a graft polymer chain starting from a solid phase using a monomer having a group.
When chemically bonding a solid phase and a graft polymer chain having a hydrophilic group, a functional group that reacts with a functional group at the end of the graft polymer chain is introduced into the inorganic material, and the graft polymer is chemically bonded thereto. In addition, when using a monomer having a hydrophilic group having a double bond in the molecule and polymerizing a graft polymer chain starting from a solid phase, the starting point when polymerizing a compound having a double bond Is introduced into the inorganic material.

  The graft polymer having a hydrophilic group and the monomer having a hydrophilic group having a double bond in the molecule are described in the above-described method for introducing a hydrophilic group into the solid phase of an organic material having no hydrophilic group. A graft polymer having a hydrophilic group and a monomer 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 solid phase 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. An acetylcellulose mixture etc. can be mentioned.

  In addition, after coating a solid phase of an inorganic material having no hydrophilic group with acetyl cellulose or an acetyl cellulose mixture having a different acetyl value, the coated acetyl cellulose or an acetyl cellulose mixture having a different acetyl value can be saponified. . In this case, the saponification rate is preferably 5% or more and 100% or less. Furthermore, the saponification rate is preferably 10% or more and 100% or less.

  As a solid phase of an inorganic material having no hydrophilic group, a solid phase produced by processing a metal such as aluminum, ceramics such as glass, cement, ceramics, new ceramics, silicon, activated carbon, or the like can be given.

  The solid phase is preferably used in the form of a filter or a membrane because the solution can pass through the inside. In this case, the thickness is preferably 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 solid phase through which the solution can pass is preferably 0.1 μm to 10 μm in average pore size. More preferably, the average pore diameter is 1 μm to 5 μm. As a result, a sufficient surface area for adsorbing nucleic acids is obtained and clogging is difficult. The average pore size of the solid phase through which this solution can pass can be determined using the bubble point method (according to ASTM 316-86, JIS 3832).

  The solid phase through which the solution can pass is preferably an asymmetric porous membrane. Here, the front and back asymmetry indicates a property that the physical property or chemical property of the membrane is changed from one surface of the porous membrane to the other surface. An example of the physical properties of the membrane is the average pore size. The chemical properties of the membrane include the degree of saponification. When using a porous membrane having an asymmetric average pore size in the present invention, it is preferable to change the average pore size from large to small in the direction in which the liquid passes. Here, 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. More preferably, the ratio of the maximum pore diameter to the minimum pore diameter is 5 or more. As a result, a sufficient surface area for adsorbing nucleic acids is obtained and clogging is difficult.

The solid phase through which the solution can pass is preferably 50 to 95% in porosity. 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, a bubble point is 0.2-4 kgf / cm < ² >.

  The solid phase through which the solution can pass is preferably 0.1 to 100 kPa in pressure loss. 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 solid phase through which the solution can pass is preferably 1 to 5000 mL per minute per 1 cm ^{2 of} membrane when water is passed at 25 ° C. and 1 kg / cm ² of pressure. . 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 minute per 1 cm ^{2 of} membrane.

  The solid phase through which the solution can pass is preferably 0.1 μg or more of nucleic acid adsorbed per 1 mg of the porous membrane. More preferably, the amount of nucleic acid adsorbed per 1 mg of the porous membrane is 0.9 μg or more.

The flow rate when the sample solution containing nucleic acid is passed through the solid phase is preferably ² to 1500 μL / sec per cm ^{2 of the} solid phase in order to obtain an appropriate contact time of the liquid with the solid phase. . If the contact time of the liquid with the solid phase 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} solid phase area.

  Moreover, when the solution to be used can pass through the inside of the solid phase, one kind may be used, but a plurality may be used. The plurality of solid phases may be the same material or different.

  After adsorbing the nucleic acid to the solid phase. By washing the solid phase, the recovery amount and purity of the nucleic acid can be improved, and the amount of the sample 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 step may be completed only once for speeding up, and it is preferable to repeat the cleaning multiple times when purity is more important.

  The cleaning liquid is preferably a solution containing a water-soluble organic solvent and / or a water-soluble salt. Moreover, you may contain a buffering agent and surfactant as needed. The washing liquid needs to have a function of washing out impurities in the sample solution adsorbed on the solid phase together with the nucleic acid. For that purpose, it is necessary to have a composition that does not desorb nucleic acids from the solid phase but desorbs impurities. For this purpose, since the nucleic acid is hardly soluble in water-soluble organic solvents such as alcohol, it is suitable for desorbing components other than the 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 impurities and unnecessary components is improved.

  As the water-soluble organic solvent contained in the cleaning liquid, alcohol, acetone or the like can be used, and alcohol is preferable. Alcohols include methanol, ethanol, propanol, and butanol. The propanol may be either isopropanol or n-propanol, and butanol may be linear or branched. A plurality of these alcohols can be used. Among these, it is preferable to use ethanol. The amount of the water-soluble organic solvent contained in the cleaning liquid is preferably 20 to 100% by mass, and more preferably 40 to 80% by mass.

On the other hand, the water-soluble salt contained in the cleaning liquid is preferably a halide salt, and more preferably chloride. The water-soluble salt is preferably a monovalent or divalent cation, particularly preferably an alkali metal salt or an alkaline earth metal salt, particularly preferably a sodium salt or potassium salt, and most preferably a sodium salt.
When the 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 preferable, and 0.1 mol / L or less is more preferable. More preferably, the water-soluble salt is sodium chloride, and it is particularly preferable that sodium chloride is contained in an amount of 20 mmol / L or more.

  Examples of the buffering agent and the surfactant include the above-described buffering agent and surfactant. Among these, a solution containing ethanol, Tris and Triton X-100 is preferable. The preferable concentrations of Tris and Triton X-100 are 10 to 100 mmol / L and 0.1 to 10% by mass, respectively.

  It is preferable that the cleaning liquid does not contain a chaotropic substance. Thereby, the possibility that the chaotropic substance is mixed in the recovery process subsequent to the cleaning process can be reduced. When a chaotropic substance is mixed during the recovery step, an enzyme reaction such as a PCR reaction is often inhibited. Therefore, it is ideal that the washing liquid 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. Here, the chaotropic substance is urea, guanidine salt, sodium isothiocyanate, sodium iodide, potassium iodide or the like.

  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 is preferably 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. 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.

However, 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 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.

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. However, it is preferable that the amount be 200 μL / mm ² or less because operability can be maintained and sample outflow can be suppressed.

In the washing step, the flow rate when the washing solution is passed through the solid phase is preferably 2 to 1500 μL / sec, more preferably 5 to 700 μL / sec per unit area (cm ² ) of the membrane. If the passage speed is lowered and time is taken, the washing is sufficiently performed. However, the above range is preferable because the operation of separating and purifying nucleic acid can be speeded up without reducing the washing efficiency.

  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. Further, in the washing step, mechanical vibration or ultrasonic stirring can be applied to the nucleic acid separation and purification cartridge simultaneously with the washing step. Alternatively, it can be washed by centrifugation.

  Next, the washed solid phase is brought into contact with a solution capable of desorbing the nucleic acid adsorbed on the solid phase. Since the target nucleic acid is contained in this solution, it is recovered and provided for subsequent operation, for example, amplification of the nucleic acid by PCR (polymerase chain reaction).

  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 recovered liquid that is often used is several tens to several hundreds of μL. However, when the amount of the sample is extremely small, or conversely when a large amount of nucleic acid is to be separated and purified, the amount of the recovered liquid is 1 to several tens of mL You can change the range.

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

  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 solution may be injected several times, such as when collecting a large amount of nucleic acid.

  In the recovery step, a stabilizer for preventing degradation of the recovered nucleic acid can be added to the nucleic acid recovery solution. As the stabilizer, antibacterial agents, antifungal agents, nucleic acid degradation inhibitors and the like can be added. Examples of the nucleic acid degradation inhibitor include nucleolytic enzyme inhibitors, and specific examples include EDTA. As another embodiment, a stabilizer may be added to the collection container in advance.

  Further, in the nucleic acid fraction purification step, nucleic acids having a molecular weight ranging from 1 kbp to 200 kbp, particularly 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 using a glass filter.

  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 about 1.8 when the measurement value (260 nm / 280 nm) with an ultraviolet-visible spectrophotometer is DNA and about 2.0 when RNA is RNA can be recovered.

  The nucleic acid separation and purification unit used in the present invention is coupled to (a) a solid phase, (b) a container having at least two openings containing the solid phase, and (c) one opening of the container. A pressure difference generator is preferably included. Hereinafter, the nucleic acid separation and purification unit will be described.

  There is no particular limitation on the material of the container, and it is sufficient that the solid phase can be accommodated and at least two openings can be provided. However, plastic is preferable from the viewpoint of ease of manufacture. For example, it is preferable to use a transparent or opaque resin such as polystyrene, polymethacrylic ester, polyethylene, polypropylene, polyester, nylon, and polycarbonate.

  The container has a solid phase container, the solid phase can be stored in the container, and the solid phase does not come out of the container when the sample liquid or the like is sucked and discharged, and a pressure difference generating device such as What is necessary is just to join a syringe. For this purpose, it is preferred that the container is initially divided into two parts and can be integrated after the solid phase is accommodated. Further, in order to prevent the solid phase from going out of the accommodating portion, a mesh made of a material that does not contaminate the nucleic acid can be placed above and below the solid phase.

  There is no particular limitation on the shape of the solid phase contained in the container, and in the case of a circle, square, rectangle, ellipse, membrane, a cylinder, a scroll, or a bead coated with an organic polymer having a hydroxyl group on the surface However, from the viewpoint of production suitability, highly symmetric shapes such as circles, squares, cylinders, and scrolls and beads are preferable.

  The container is usually produced in a form divided into a main body for accommodating the solid phase and a lid, and each is provided with at least one opening. The opening is used as an inlet and an outlet for a sample solution containing nucleic acid, a washing solution (nucleic acid washing buffer) and a solution capable of desorbing nucleic acid adsorbed on a solid phase (hereinafter referred to as “sample solution etc.”). It is connected to a pressure difference generator that can cause the inside of the container to be depressurized or pressurized. The shape of the main body is not particularly limited, but it is preferable to make the cross section circular in order to facilitate the manufacture and to facilitate the diffusion of the sample solution and the like over the entire surface of the solid phase. It is also preferable to make the cross section square in order not to generate solid-state cutting waste.

  The lid needs to be joined to the main body so that the inside of the container can be depressurized and pressurized by a pressure difference generator, but if this state can be achieved, the joining method can be arbitrarily selected. For example, use of an adhesive, screwing, fitting, screwing, fusion by ultrasonic heating and the like can be mentioned.

  The internal volume of the container is determined only by the amount of the sample solution to be processed, but is usually represented by the volume of the solid phase accommodated. That is, it is preferable that the thickness is about 1 mm or less (for example, about 50 to 500 μm) and the size is such that about 1 to 6 solid phases having a diameter of about 2 mm to 20 mm can be accommodated.

  The end face of the solid phase is preferably in close contact with the inner wall surface of the container to the extent that the sample solution or the like does not pass through.

  Under the solid phase facing the opening used for the inlet of the sample solution or the like, a space is provided without being in close contact with the inner wall of the container so that the sample solution or the like diffuses as evenly as possible over the entire surface of the solid phase.

  It is preferable to provide a member having a hole substantially in the center on the solid phase facing the opening coupled to the pressure difference generator. This member suppresses the solid phase and has the effect of efficiently discharging the sample solution and the like, and has a funnel-shaped or bowl-shaped inclined surface so that the liquid collects in the central hole. preferable. The size of the hole, the angle of the inclined surface, and the thickness of the member can be appropriately determined by those skilled in the art in consideration of the amount of the sample solution to be processed, the size of the container for storing the solid phase, and the like. It is preferable to provide a space between the member and the opening for storing the overflowed sample solution or the like and preventing the sample solution from being sucked into the pressure difference generator. The size of this space can also be appropriately selected by those skilled in the art. In order to efficiently collect nucleic acids, it is preferable to aspirate a sample solution containing an amount of nucleic acid larger than the entire solid phase.

  In addition, in order to prevent the sample solution and the like from concentrating only in the portion directly under the opening being sucked and to allow the sample solution and the like to pass through the solid phase relatively uniformly, the space between the solid phase and this member It is preferable to provide a space. For this purpose, it is preferable to provide a plurality of protrusions from the member toward the solid phase. The size and number of the protrusions can be appropriately selected by those skilled in the art, but it is preferable to keep the solid phase opening area as large as possible while maintaining the space.

  Needless to say, if the container is provided with three or more openings, it is necessary to temporarily block the extra openings in order to allow suction and discharge of the liquid accompanying decompression and pressurization operations. Absent.

  The pressure difference generator first sucks a sample solution containing nucleic acid by reducing the pressure in the container containing the solid phase. Examples of the pressure difference generating device include a pump capable of suction and pressurization such as a syringe, a pipetter, or a peristaltic pump. 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 container.

  Next, a method for purifying nucleic acid using the above-described nucleic acid separation and purification unit will be described.

  In the method for separating and purifying nucleic acid of the present invention, preferably, nucleic acid can be adsorbed and desorbed using a nucleic acid separation and purification unit in which the solid phase is accommodated in a container having at least two openings.

  More preferably, a nucleus including (a) the solid phase, (b) a container having at least two openings for containing the solid phase, and (c) a pressure difference generator coupled to one opening of the container. Nucleic acid can be adsorbed and desorbed using an acid separation and purification unit.

In this case, the first embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(a) preparing a sample solution containing nucleic acid using a specimen, and inserting one opening of the nucleic acid separation and purification unit into the sample solution;
(b) using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit to draw a sample solution containing nucleic acid under reduced pressure in the container and bring it into contact with the solid phase;
(c) A step of bringing the container into a pressurized state using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit, and discharging the sample solution containing the aspirated nucleic acid out of the container;
(d) inserting one opening of the nucleic acid separation and purification unit into a washing solution (nucleic acid washing buffer);
(e) a step of bringing the container into a reduced pressure state using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit, sucking the washing liquid, and bringing it into contact with the solid phase;
(f) a step of pressurizing the inside of the container using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit, and discharging the sucked washing liquid out of the container;
(g) inserting one opening of the nucleic acid separation and purification unit into a liquid capable of desorbing the nucleic acid adsorbed on the solid phase;
(h) Using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit, the container is depressurized, and a solution capable of desorbing nucleic acid adsorbed on the solid phase is sucked and contacted with the solid phase. And a step of causing
(i) A step in which the inside of the container is pressurized using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit, and a liquid capable of desorbing the nucleic acid adsorbed on the solid phase is discharged out of the container.
In (b), (e), and (h), it is preferable to suck an amount of the solution that comes into contact with almost the entire solid phase. However, if the solution is sucked into the pressure difference generator, the device is contaminated. adjust. After sucking an appropriate amount of solution, the inside of the unit container is pressurized using a pressure difference generator, and the sucked liquid is discharged. It is not necessary to leave an interval before this operation, and it may be discharged immediately after suction.

The second embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(a) preparing a sample solution containing nucleic acid using a specimen, and injecting the sample solution containing the nucleic acid into one opening of the nucleic acid separation and purification unit;
(b) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the sample solution containing the injected nucleic acid is discharged from the other opening, thereby fixing the container. Contacting the phase;
(c) injecting a washing solution into the one opening of the nucleic acid separation and purification unit;
(d) The container is pressurized by using a pressure difference generator connected to the one opening of the nucleic acid separation and purification unit, and the injected washing solution is discharged from the other opening to be brought into contact with the solid phase. Process,
(e) injecting a liquid capable of desorbing the nucleic acid adsorbed on the solid phase into the one opening of the nucleic acid separation and purification unit;
(f) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and a liquid capable of desorbing the injected nucleic acid is discharged from the other opening, A step of desorbing the nucleic acid adsorbed on the solid phase and discharging it out of the container.
First, one opening of the nucleic acid separation and purification unit is inserted into a sample solution containing nucleic acid.

In the above step, the sample solution is added to the container without limitation, but it is preferable to use a laboratory instrument such as a pipette or a dropper. More preferably, these instruments are nuclease-free and pyrogen-free.
The method for mixing the specimen is not particularly limited. For example, when mixing, it is preferable to mix at 1 to 3 minutes at 30 to 3000 rpm 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 performing inversion mixing 5-30 times. Moreover, it can mix also by performing pipetting operation 10-50 times.
The following is an automatic method for automatically performing a process of separating and purifying nucleic acid from a sample containing nucleic acid using a nucleic acid separation and purification cartridge containing a solid phase in a container having at least two openings and a pressure generator. Although the example of an apparatus is shown, an automatic apparatus is not limited to this.

  The automatic device uses a nucleic acid separation / purification cartridge containing a solid phase that adsorbs nucleic acid, which allows the solution to pass through the inside, and injects and pressurizes a sample liquid containing nucleic acid into the nucleic acid separation / purification cartridge. Nucleic acid adsorbed on the solid phase after the nucleic acid has been adsorbed on the solid phase, the washing solution is dispensed onto the nucleic acid separation and purification cartridge and pressurized to remove impurities, and then the recovered solution is dispensed onto the nucleic acid separation and purification cartridge Is a nucleic acid separation / purification device that automatically performs separation / purification operation, and collects the nucleic acid separation / purification cartridge, a waste liquid container containing the sample liquid and the washing liquid discharge liquid, and the nucleic acid. A mounting mechanism for holding a recovery container for storing a recovery liquid, a pressurized air supply mechanism for introducing pressurized air into the nucleic acid separation and purification cartridge, and a washing in the nucleic acid separation and purification cartridge And it is characterized in that the recovery liquid comprising a dispensing dispensing mechanism.

  The mounting mechanism includes a stand mounted on the main body of the apparatus, a cartridge holder supported on the stand so as to be movable up and down, and holding the nucleic acid separation and purification cartridge, and the position relative to the nucleic acid separation and purification cartridge is exchanged below the cartridge holder. What comprises the said waste-liquid container and the container holder holding the said collection | recovery container as possible is suitable.

  The pressurized air supply mechanism includes an air nozzle that ejects pressurized air from a lower end portion, and a pressure that moves the air nozzle up and down relative to the nucleic acid separation and purification cartridge supported by the air nozzle and held in the cartridge holder. A head provided with a head and positioning means for positioning the nucleic acid separation / purification cartridge in the rack of the mounting mechanism is suitable.

  The dispensing mechanism holds a cleaning liquid dispensing nozzle that dispenses the cleaning liquid, a recovered liquid dispensing nozzle that dispenses the recovered liquid, the cleaning liquid dispensing nozzle, and the recovered liquid dispensing nozzle. A nozzle moving table that can move in sequence on the cartridge for nucleic acid separation and purification held in the mounting mechanism, a cleaning liquid supply pump that draws the cleaning liquid from the cleaning liquid bottle that stores the cleaning liquid and supplies the cleaning liquid to the cleaning liquid dispensing nozzle, and a recovery liquid It is preferable to include a recovery liquid supply pump that sucks the recovery liquid from the recovery liquid bottle and supplies it to the recovery liquid dispensing nozzle.

  According to the automatic apparatus as described above, the mounting mechanism for holding the nucleic acid separation and purification cartridge, the waste liquid container and the recovery container, the pressurized air supply mechanism for introducing pressurized air into the nucleic acid separation and purification cartridge, and the nucleic acid separation and purification cartridge A dispensing mechanism for dispensing the washing solution and the recovery solution, and injecting and pressurizing the sample solution containing the nucleic acid into the nucleic acid separation / purification cartridge equipped with the solid phase member to adsorb the nucleic acid to the solid phase member, After dispensing and washing out impurities, the sample solution is efficiently dispensed in a short time by automatically performing a nucleic acid separation and purification step of dispensing the collected solution and separating and collecting the nucleic acid adsorbed on the solid phase membrane member. The mechanism capable of automatically separating and purifying the nucleic acid can be made compact.

  When the mounting mechanism includes a stand, a vertically movable cartridge holder that holds the nucleic acid separation and purification cartridge, and a container holder that holds the waste liquid container and the recovery container in an exchangeable manner, the nucleic acid separation and purification cartridge and The set of both containers and the exchange of the waste liquid container and the collection container can be easily performed.

  In addition, when the pressurized air supply mechanism includes an air nozzle, a pressure head for moving the air nozzle up and down, and a positioning means for positioning the nucleic acid separation / purification cartridge, the pressurized air can be reliably secured with a simple mechanism. Can be supplied.

  In addition, the dispensing mechanism includes a washing liquid dispensing nozzle, a recovery liquid dispensing nozzle, a nozzle moving table that can move in sequence on the nucleic acid separation and purification cartridge, and a washing liquid drawn from the washing liquid bottle and supplied to the washing liquid dispensing nozzle. If the cleaning liquid supply pump and the recovery liquid supply pump that sucks the recovery liquid from the recovery liquid bottle and supplies it to the recovery liquid dispensing nozzle are provided, the cleaning liquid and the recovery liquid can be sequentially dispensed by a simple mechanism.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1
(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-adsorptive porous membrane was produced from high impact polystyrene.

(2) As a nucleic acid-adsorbing porous membrane, a porous membrane (pore size 2.5 μm, diameter 7 mm, thickness 100 μm, saponification rate 95%) obtained by saponifying a porous membrane of triacetyl cellulose is used. ) In the nucleic acid-adsorbing porous membrane housing part of the nucleic acid purification cartridge produced in (1).

(3) Materials and Reagents The nucleic acid purification cartridge prepared in (2) was used. A pretreatment liquid (adsorption buffer solution for nucleic acid purification) and a washing liquid (nucleic acid washing buffer) were prepared as follows.

Pretreatment solution (adsorption buffer solution for nucleic acid purification)
Guanidine hydrochloride (Life Technology) 4.95M
Rheodor TW-S120V (Kao nonionic surfactant) 3.0%
Ethanol 7.3%
Cetyltrimethylammonium bromide 2%
AK-02 (Defoam made by Shin-Etsu Chemical) 0.6%
Distilled water 1000mL

Washing solution (nucleic acid washing buffer)
Tris-HCl 20 mM
Sodium chloride 200 mM
Distilled water

(4) Nucleic acid purification operation 200 μL of human whole blood sample, 250 μL of pretreatment solution (nucleic acid purification adsorption buffer solution), 30 μL of protease solution (protease N Amano G; neutral protease manufactured by Amano Enzyme), stabilizer (glucose or sorbitol) ) 30 μL of 10% (W / V) aqueous solution was added (control was 30 μL of pure water) and incubated at 56 ° C. for 2 minutes. After incubation, 250 μL of ethanol was added and stirred. After stirring, the mixture is injected into one opening of the nucleic acid purification cartridge having the nucleic acid-adsorptive porous membrane prepared in (2), and then a pressure difference generator (tubing pump) is coupled to the one opening. The separation / purification cartridge is pressurized (80 kpa), 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. It discharged from the opening. Subsequently, a washing liquid is injected into the one opening of the nucleic acid separation and purification cartridge, a tubing pump is coupled to the one opening, the inside of the nucleic acid separation and purification cartridge is brought into a pressurized state (80 kpa), and the injected washing liquid is It passed through the nucleic acid-adsorptive porous membrane and discharged from the other openings. Subsequently, the recovery liquid is injected into the one opening of the nucleic acid separation and purification cartridge, and a tubing pump is connected to the one opening of the nucleic acid separation and purification cartridge to bring the inside of the nucleic acid separation and purification cartridge into a pressurized state (80 kpa). The injected recovery liquid was passed through the nucleic acid-adsorbing porous membrane, discharged from the other openings, and this liquid was recovered. The time required for the nucleic acid separation and purification operation (from the time when the sample solution containing the nucleic acid was injected into the cartridge to the time of recovery) was 2 minutes.

(5) Quantification of recovery amount of nucleic acid The recovery amount of DNA purified by the operation of (4) was measured by measuring absorbance at a wavelength of 260 nm. The amount of DNA recovered when 30 μL of pure water was added instead of the stabilizer aqueous solution was taken as 100%, and the results are shown in Table 1 below. Moreover, the result when using glucose and sorbitol as an example of a stabilizer was shown. From the results in Table 1, it can be seen that the amount of recovered DNA can be improved by the present invention.

Example 2
(1) Addition of Stabilizer to Protease Solution Protease activity was measured using an EnzCheck protease assay kit from Molecular Probes. Glucose, sucrose, trehalose, and sorbitol stabilizers were added to a 200 mg / mL protease solution (protease N Amano G; neutral protease manufactured by Amano Enzyme) at 10% (W / V). Was stored at each temperature of 4, 25, 45, and 60 ° C. for 20 hours, and then the activity was measured. For comparison, the same measurement was performed on a sample to which no stabilizer was added.

(2) Measurement of protease activity Table 2 shows the protease activity measured by the operation of (1) as 100 when stored at 4 ° C. From this result, it can be seen that various proteolytic enzyme stabilizers used in the present invention have a stabilizing action.

Claims (39)

(1) passing a sample solution containing nucleic acid through a solid phase and adsorbing the nucleic acid into the solid phase;
(2) passing the washing solution through the solid phase and washing the solid phase with the nucleic acid adsorbed; and (3) passing the recovery solution through the solid phase to desorb the nucleic acid from the solid phase. A method for separating and purifying nucleic acid, wherein the solution for adsorbing nucleic acid to a solid phase contains a proteolytic enzyme and a proteolytic enzyme stabilizer.   2. The method for separating and purifying nucleic acid according to claim 1, comprising a step of adding a premixed proteolytic enzyme stabilizer and proteolytic enzyme to a sample solution containing the nucleic acid.   The method for separating and purifying nucleic acid according to claim 1 or 2, wherein the proteolytic enzyme stabilizer is at least one of saccharide, sugar alcohol, polyethylene glycol, and metal ion.   The method for separating and purifying nucleic acid according to any one of claims 1 to 3, wherein the proteolytic enzyme is at least one of serine protease, cysteine protease, metalloprotease, and acidic protease.   The method for separating and purifying nucleic acid according to any one of claims 1 to 4, wherein the proteolytic enzyme is provided in a proteolytic enzyme solution containing no nucleolytic enzyme.   The solution for adsorbing nucleic acid on the solid phase contains a proteolytic enzyme and at least one compound selected from an antifoaming agent, a chaotropic salt, a nucleic acid stabilizer, a buffer, a water-soluble organic solvent, and a surfactant. The method for separating and purifying nucleic acid according to any one of claims 1 to 5, which is a solution obtained by adding and mixing a pretreatment liquid to a specimen containing cells or viruses.   The method for separating and purifying nucleic acid according to any one of claims 1 to 6, wherein the proteolytic enzyme is simultaneously added as one reagent together with other reagents such as a chaotropic salt and a surfactant in advance.   The method for separating and purifying nucleic acid according to claim 6 or 7, wherein the antifoaming agent comprises two components, a silicon antifoaming agent and an alcohol defoaming agent.   The method for separating and purifying nucleic acid according to any one of claims 6 to 8, wherein the pretreatment liquid contains a nucleic acid stabilizer at a concentration of 0.1 to 20% by mass.   The method for separating and purifying nucleic acid according to claim 9, wherein the nucleic acid stabilizer is a reducing agent.   The method for separating and purifying nucleic acid according to claim 10, wherein the reducing agent is a mercapto compound.   10. The method for separating and purifying nucleic acid according to claim 9, wherein the nucleic acid stabilizer is a chelating agent.   The method for separating and purifying nucleic acid according to any one of claims 6 to 12, wherein the chaotropic salt is a guanidinium salt.   The method for separating and purifying nucleic acid according to any one of claims 6 to 13, wherein the water-soluble organic solvent contains any one of methanol, ethanol, propanol, and butanol.   The method for separating and purifying nucleic acid according to any one of claims 1 to 14, wherein the solid phase is a solid phase composed of silica or a derivative thereof, diatomaceous earth, or alumina.   The method for separating and purifying nucleic acid according to claim 1, wherein the solid phase is a solid phase composed of an organic polymer.   The method for separating and purifying nucleic acid according to claim 16, wherein the solid phase comprising an organic polymer is a solid phase comprising an organic polymer having a polysaccharide structure.   18. The method for separating and purifying nucleic acid according to claim 17, wherein the solid phase comprising an organic polymer having a polysaccharide structure is a solid phase comprising acetylcellulose.   19. The nucleic acid separation and purification according to claim 18, wherein the solid phase comprising acetyl cellulose is a solid phase comprising an organic polymer obtained by saponifying a mixture of acetyl cellulose or acetyl cellulose having different acetyl values. Method.   The method for separating and purifying nucleic acid according to claim 19, wherein the saponification rate of a mixture of acetylcelluloses having different acetyl values is 5% or more.   18. The method for separating and purifying nucleic acid according to claim 17, wherein the solid phase comprising an organic polymer having a polysaccharide structure is a solid phase comprising regenerated cellulose.   The method for separating and purifying nucleic acid according to any one of claims 15 to 21, wherein the solid phase is a porous membrane.   The method for separating and purifying nucleic acid according to claim 22, wherein the porous membrane is an asymmetric porous membrane.   The method for separating and purifying nucleic acid according to claim 22 or 23, wherein the porous membrane is a porous membrane having an average pore size of 0.1 to 10.0 µm.   The method for separating and purifying nucleic acid according to any one of claims 22 to 24, wherein the porous membrane is a porous membrane having a thickness of 10 to 500 µm.   The method for separating and purifying nucleic acid according to any one of claims 15 to 21, wherein the solid phase is non-porous.   27. The method for separating and purifying nucleic acid according to any one of claims 15 to 26, wherein the solid phase is coated on beads.   The method for separating and purifying nucleic acid according to claim 27, wherein the beads are magnetic beads.   The method for separating and purifying nucleic acid according to any one of claims 1 to 28, wherein the nucleic acid is adsorbed and desorbed using a nucleic acid separation and purification unit containing a solid phase in a container having at least two openings. .   a nucleic acid separation and purification unit comprising: (a) a solid phase; (b) a container having at least two openings for accommodating the solid phase; and (c) a pressure difference generator coupled to one opening of the container. The method for separating and purifying nucleic acid according to any one of claims 1 to 29, wherein the nucleic acid is adsorbed and desorbed.   The method for separating and purifying nucleic acid according to claim 30, wherein the pressure difference generating device is a pressurizing device.   The method for separating and purifying nucleic acid according to claim 30, wherein the pressure difference generator is a depressurizing apparatus.   The method for separating and purifying nucleic acid according to any one of claims 30 to 32, wherein the pressure difference generator is detachably coupled to one opening of the container. The method for separating and purifying nucleic acid according to claim 30 or 31, comprising the following steps (a) to (f).
(a) preparing a sample solution containing nucleic acid using a specimen, and injecting the sample solution containing the nucleic acid into one opening of the nucleic acid separation and purification unit;
(b) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the sample solution containing the injected nucleic acid is discharged from the other opening, thereby fixing the container. A step of adsorbing nucleic acid to a solid phase by contacting the phase;
(c) injecting a washing solution into the one opening of the nucleic acid separation and purification unit;
(d) The container is pressurized by using a pressure difference generator connected to the one opening of the nucleic acid separation and purification unit, and the injected washing solution is discharged from the other opening to be brought into contact with the solid phase. A step of washing the solid phase,
(e) injecting a liquid capable of desorbing the nucleic acid adsorbed on the solid phase into the one opening of the nucleic acid separation and purification unit;
(f) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and a liquid capable of desorbing the injected nucleic acid is discharged from the other opening, A step of desorbing nucleic acid adsorbed on the solid phase and discharging it out of the container.   35. The nucleic acid according to claim 34, comprising the step of bringing the DNA degrading enzyme solution into contact with the solid phase and then washing the solid phase with a washing solution before the step (e). Separation and purification method.   The method for separating and purifying nucleic acid according to claim 1 to 35, wherein the washing solution is a solution containing 20 to 100% by mass of methanol, ethanol, isopropanol or n-propanol.   37. The method for separating and purifying nucleic acid according to claim 1, wherein the solution capable of desorbing the nucleic acid adsorbed on the solid phase is a solution having a salt concentration of 0.5 mol / L or less.   A reagent kit for performing the method according to any one of claims 1 to 37.   Apparatus for performing the method according to any of claims 1-38.