JP2007068452A - Method for separating and refining nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly obtaining a nucleic acid in high yield and high purity, in the method for separating and refining the nucleic acid by adsorbing the nucleic acid in a solution containing the nucleic acid prepared from an animal tissue, an animal tissue containing a scleroprotein or a component derived from the animal tissue to the solid phase surface, passing through a washing process, etc., and then desorbing the nucleic acid from the solid phase surface by using a recovering solution. <P>SOLUTION: The method for separating and refining the nucleic acid comprises a step for adsorbing the nucleic acid to the solid phase and then desorbing the nucleic acid from the solid phase by each using a sample solution containing the nucleic acid, the solution for adsorbing the nucleic acid to the solid phase and the recovering solution. In the method, a sample solution obtained by steps for treating the tissue containing the animal tissue and the scleroprotein with an acid and an alkali, as necessary, carrying out hot water treatment when using a tissue such as bone containing the sceleroprotein and adding a tissue solution after the above treatment is used as the sample solution containing the nucleic acid and a solid phase composed of an organic polymer having a polysaccharide structure is used as the solid phase. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for separating and purifying nucleic acids in animal tissues. More specifically, the present invention relates to a solid phase used in a method for separating and purifying nucleic acid, a sample solution containing nucleic acid and a treatment solution, and a method for separating and purifying nucleic acid using these.

  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 genomic nucleic acids, nucleic acid probes, and plasmid nucleic acids.

  Also in the diagnostic field, nucleic acids are used for various purposes in various forms. For example, nucleic acid probes are routinely used for detection and diagnosis of human pathogens. Nucleic acids are also used to detect genetic disorders and food contaminants. In addition, nucleic acids are routinely used in location confirmation, identification, and isolation for a given nucleic acid for various purposes, including genetic map creation and cloning, gene expression by genetic recombination, genetic diagnosis and identification. .

  However, in many cases, the nucleic acid can be obtained only in a very small amount, and the operation of isolation and purification is complicated and requires a lot of time. This complicated process requiring time is likely to lead to loss of nucleic acid. In addition, when nucleic acid is purified from samples obtained from animal tissues, bones, teeth, blood, urine and bacterial cultures, there is a problem that contamination occurs.

As one of the methods for solving the above problems and separating and purifying nucleic acid simply and efficiently, an organic polymer having a hydroxyl group on the surface is prepared by using a solution for adsorbing nucleic acid on the solid phase and a solution for desorbing nucleic acid from the solid phase, respectively. Patent Document 1 discloses a method for separating and purifying nucleic acid by adsorbing and desorbing nucleic acid on a solid phase composed of molecules.
JP 2003-128691 A

  When carrying out the method for separating and purifying nucleic acid as described above, especially when separating and purifying nucleic acid from a solution obtained by treating animal tissue, there are many problems in the sample solution obtained from animal tissue. there were. For this reason, it has been desired to rapidly recover the target nucleic acid from minute animal tissues with high yield and high purity. Accordingly, an object of the present invention is to adsorb nucleic acid in a solution containing nucleic acid prepared from animal tissue, animal tissue containing hard protein or a component derived from animal tissue to a solid phase surface, and then desorb the nucleic acid by washing or the like. In a method for separating and purifying a nucleic acid, a method for obtaining a nucleic acid rapidly, with a high yield and with a high purity is provided.

As a result of intensive studies to solve the above-mentioned problems, the present inventors added an acid or alkaline solution to animal tissue, animal tissue containing hard protein or a component derived from animal tissue to soften the tissue, and then harden the tissue. When using a tissue such as bone containing protein, a sample solution containing nucleic acid is prepared by adding a tissue fluid solution to animal tissue that has been subjected to hydrothermal treatment. Further, a pretreatment solution is added to prepare a solution for adsorbing nucleic acids on the solid phase, and when adsorbing nucleic acids in the solution on the solid phase, a solid phase made of an organic polymer having a polysaccharide structure is used as the solid phase. Thus, it was found that the target nucleic acid can be recovered rapidly, with high yield and with high purity. Particularly in the present invention, it has been found that the yield and purity of the target nucleic acid are dramatically improved by using a solid phase composed of an organic polymer obtained by saponifying acetyl cellulose or a mixture of acetyl cellulose having different acetyl values. . The present invention has been completed based on these findings.

  That is, according to the present invention, animal tissue, animal tissue containing hard protein, or animal-derived components are treated with acid or alkali, and when using a tissue such as bone containing hard protein, hydrothermal treatment is performed. A step of adsorbing and desorbing nucleic acid on a solid phase using a solution for adsorbing nucleic acid in a solution containing nucleic acid prepared by adding a lysing solution and a solution (recovery solution) for desorbing nucleic acid from the solid phase, respectively. A solid phase composed of an organic polymer having a polysaccharide structure in the solid phase, preferably a solid phase composed of an organic polymer obtained by saponifying a mixture of acetyl cellulose or acetyl cellulose having different acetyl values. A method for separating and purifying nucleic acid is provided.

The present invention achieves the above object by the following configuration.
1.
(1) a step of preparing a solution for adsorbing nucleic acid on a solid phase;
(2) A step of bringing the solution for adsorbing nucleic acid onto the solid phase into contact with the solid phase to adsorb the nucleic acid onto the solid phase;
(3) bringing the washing solution into contact with the solid phase and washing the solid phase with the nucleic acid adsorbed; and (4) bringing the recovery solution into contact with the solid phase to desorb the nucleic acid from the solid phase. Process,
In the method for separating and purifying nucleic acid having
The solution for adsorbing nucleic acid to the solid phase contains nucleic acid obtained by adding a tissue lysate after acid or alkali treatment of animal tissue, animal tissue containing hard protein or animal tissue-derived components A method for separating and purifying nucleic acid, which is a solution obtained by further adding a pretreatment liquid to a sample solution, wherein the solid phase is a solid phase composed of an organic polymer having a polysaccharide structure.
2.
(1) a step of preparing a solution for adsorbing nucleic acid on a solid phase;
(2) A step of bringing the solution for adsorbing nucleic acid onto the solid phase into contact with the solid phase to adsorb the nucleic acid onto the solid phase;
(3) bringing the washing solution into contact with the solid phase and washing the solid phase with the nucleic acid adsorbed; and (4) bringing the recovery solution into contact with the solid phase to desorb the nucleic acid from the solid phase. Process,
In the method for separating and purifying nucleic acid having
A solution for adsorbing nucleic acid to the solid phase is obtained by adding a pretreatment solution and a proteolytic enzyme to an animal tissue, an animal tissue containing hard protein, or an ingredient derived from animal tissue after acid or alkali treatment. A method for separating and purifying nucleic acid, wherein the solid phase is a solid phase comprising an organic polymer having a polysaccharide structure.
3.
3. The method for separating and purifying nucleic acid according to 1 or 2 above, which comprises an extraction step by hydrothermal treatment after acid or alkali treatment.
4).
4. The method for separating and purifying nucleic acid according to any one of 1 to 3, wherein the animal tissue containing hard protein is selected from bone, cartilage, tooth, tendon, skin, nail and hair.
5.
1. The tissue lysate is a solution containing at least one selected from a surfactant, a buffer, a nucleic acid stabilizer, an alkali metal halide, a chaotropic salt, a proteolytic enzyme, and an antifoaming agent. 5. The method for separating and purifying nucleic acid according to 3 or 4.
6).
The nucleic acid separation and purification according to any one of 1 to 5, wherein the pretreatment liquid is a solution containing at least one selected from an antifoaming agent, a nucleic acid stabilizer, a chaotropic salt, a surfactant, and a buffer. Method.
7).
7. The method for separating and purifying nucleic acid as described in 5 or 6 above, wherein the surfactant is a nonionic surfactant. 8).
8. The method for separating and purifying nucleic acid as described in 7 above, wherein the nonionic surfactant is a polyoxyethylene surfactant.
9.
9. The method for separating and purifying nucleic acid as described in 8 above, wherein the polyoxyethylene surfactant is a polyoxyethylene sorbitan surfactant.
10.
10. The method for separating and purifying a nucleic acid according to any one of 5 to 9 above, wherein the buffer is BisTris (Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane).
11.
The method for separating and purifying nucleic acid according to any one of 1 to 10, wherein the organic polymer having a polysaccharide structure is acetylcellulose.
12
11. The method for separating and purifying nucleic acid according to any one of 1 to 10, wherein the organic polymer having a polysaccharide structure is an organic polymer obtained by saponifying acetyl cellulose or a mixture of acetyl cellulose having different acetyl values.
13.
13. The method for separating and purifying nucleic acid as described in 12 above, wherein the saponification rate of the mixture of acetylcelluloses having different acetyl values after saponification is 5% or more.
14
13. The method for separating and purifying nucleic acid as described in 12 above, wherein the saponification rate of the mixture of acetylcelluloses having different acetyl values after saponification is 10% or more.
15.
11. The method for separating and purifying nucleic acid according to any one of 1 to 10, wherein the organic polymer having a polysaccharide structure is regenerated cellulose.
16.
The method for separating and purifying nucleic acid according to any one of 1 to 15, wherein the solid phase is a porous membrane.
17.
17. The method for separating and purifying nucleic acid as described in 16 above, wherein the porous membrane is an asymmetric porous membrane.
18.
18. The method according to 16 or 17 above, wherein the porous membrane is a porous membrane having an average pore size of 0.1 to 10.0 μm.
19.
The method according to any one of 16 to 18, wherein the porous membrane is a porous membrane having a thickness of 10 to 500 µm.
20.
The method for separating and purifying nucleic acid according to any one of 1 to 19, wherein a solution for adsorbing nucleic acid to a solid phase is obtained by further adding a water-soluble organic solvent.
21.
21. The method for separating and purifying nucleic acid as described in 20 above, wherein the water-soluble organic solvent contains at least one selected from methanol, ethanol, propanol, and butanol.
22.
The method for separating and purifying nucleic acid according to any one of 1 to 21, further comprising a step of removing undissolved residual tissue components from a sample solution obtained by adding a tissue lysis solution to animal tissue.
23.
The method for separating and purifying nucleic acid according to any one of 1 to 22, 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.
24.
(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 1 to 22, wherein the nucleic acid is adsorbed and desorbed using a nucleic acid separation and purification unit comprising:
25.
25. The method for separating and purifying nucleic acid as described in 24 above, wherein the pressure difference generator is a pressurized device.
26.
25. The method for separating and purifying nucleic acid as described in 24 above, wherein the pressure difference generator is an apparatus for reducing pressure.
27.
27. The method for separating and purifying nucleic acid according to any one of 24 to 26, wherein the pressure difference generator is detachably coupled to one opening of the container.
28.
26. The method for separating and purifying nucleic acid as described in 24 or 25 above, which comprises the following steps.
(1a) a step of dissolving a tissue by adding a tissue lysis solution to an animal tissue and treating the animal tissue to prepare a sample solution containing a nucleic acid;
(1b) adding a pretreatment solution to the sample solution containing the nucleic acid to prepare a solution for adsorbing the nucleic acid on the solid phase;
(2a) injecting a solution for adsorbing nucleic acid on the solid phase into one opening of a container having at least two openings for containing the solid phase;
(2b) A pressure difference generator is connected to the one opening to bring the inside of the container into a pressurized state, and a solution for adsorbing nucleic acid to the injected solid phase is discharged from the other opening, thereby Contacting the solid phase with the nucleic acid to adsorb the nucleic acid to the solid phase,
(3a) removing the pressure difference generator from the one opening and injecting a cleaning liquid into the one opening; (3b) connecting the pressure difference generator to the one opening to bring the inside of the container into a pressurized state; A step of cleaning the solid phase by contacting the cleaning liquid with the solid phase in the container by discharging the injected cleaning liquid from the other opening,
(4a) removing the pressure difference generator from the one opening and injecting the recovered liquid into the one opening; (4b) connecting the pressure difference generator to the one opening to bring the inside of the container into a pressurized state. A step of discharging the injected recovery liquid from the other opening, bringing the recovery liquid into contact with the solid phase in the container, desorbing the nucleic acid adsorbed on the solid phase, and discharging it out of the container.
29.
Before the step (4a) above,
(3c) a step of contacting the DNA-degrading enzyme solution with the solid phase and then washing the solid phase with a washing solution;
The method for separating and purifying nucleic acid as described in 28 above, comprising:
30.
30. The method for separating and purifying nucleic acid according to any one of 1 to 29, wherein the washing solution is a solution containing 20 to 100% by mass of at least one selected from methanol, ethanol, isopropanol and n-propanol.
31.
31. The method for separating and purifying nucleic acid according to any one of 1 to 30, wherein the recovered solution is a solution having a salt concentration of 0.5 mol / L or less.
32.
Any of the above 1 to 31 comprising a (i) nucleic acid separation and purification unit, (ii) a tissue lysate or proteolytic enzyme, (iii) a pretreatment liquid, (iv) a washing liquid, and (v) a reagent for the recovery liquid A reagent kit for performing the method described in 1.
33.
An apparatus for performing the method according to any one of 1 to 32.

  By the method of the present invention, high purity nucleic acid can be efficiently separated from a sample solution containing nucleic acid prepared from animal cells with high yield.

The nucleic acid separation and purification method according to the present invention will be specifically described.
The nucleic acid separation and purification method of the present invention comprises:
(1) Step of subjecting tissue, tissue containing hard protein or tissue-derived component to alkali or acid treatment (2) Step of performing hydrothermal treatment only when using tissue such as bone containing hard protein (3) Nucleic acid in solid phase A step of preparing a solution for adsorption (hereinafter also referred to as “nucleic acid adsorption solution”) (hereinafter also referred to as “solution preparation step”),
(4) A step of bringing the solution for adsorbing nucleic acid onto the solid phase (nucleic acid adsorption solution) into contact with the solid phase and adsorbing the nucleic acid on the solid phase (hereinafter also referred to as “adsorption step”),
(5) a step of bringing the washing solution into contact with the solid phase and washing the solid phase in a state where the nucleic acid is adsorbed (hereinafter also referred to as “washing step”); and (6) bringing the recovered solution into contact with the solid phase. A step of desorbing nucleic acid from the solid phase (hereinafter also referred to as “recovery step”),
Is included at least.
That is, in the present invention, a pretreatment solution is added to a sample solution containing nucleic acid prepared from animal tissue to prepare a solution for adsorbing nucleic acid on a solid phase (nucleic acid adsorption solution). By bringing the sample solution into contact with the solid phase, the nucleic acid in the sample solution is adsorbed on the solid phase, washed with a washing solution, and then the nucleic acid adsorbed on the solid phase is desorbed from the solid phase using the recovery solution. Preferably, the sample solution containing a nucleic acid prepared from animal tissue is at least one selected from a surfactant, a buffer, a nucleic acid stabilizer, an alkali metal halide proteolytic enzyme, and an antifoaming agent. Obtained by adding a tissue lysate containing The nucleic acid adsorption solution is prepared by adding a pretreatment liquid containing at least one selected from an antifoaming agent, a nucleic acid stabilizer, a chaotropic salt, a surfactant, and a buffer to the sample solution containing the obtained nucleic acid. The added solution is preferred.

<Solution preparation process>
[Sample solution containing nucleic acid]
The “sample” in the sample solution means a sample obtained from any animal tissue 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.

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

[Animal tissue and its dissolution]
There is no limitation on animal tissue that can be used in the present invention, but it is intended for animal organs such as kidney, liver, pancreas, heart, lung, and brain, and parts thereof, or parts of the body such as animal tails and ears. .
In addition, cells separated from tissues, and liquid / solid components obtained from animals themselves or tissues can be targeted. Cultured cells, cells that can proliferate independently, and particles that can grow within cells can also be targeted. Moreover, the thing extract | collected by rubbing animal tissue can also be made into object.
The collected sample may be used as it is or frozen. Moreover, the thing frozen and preserve | saved may be used, and you may thaw | decompress and use as it is. Furthermore, the thing preserve | saved in the preservation | save solution, the thing preserve | saved in the shape of a flake, the thing embedded by resin etc. can be used. The sample may be stored using a tissue lysis solution described later as a storage solution.

Since animal tissue is generally solid, it needs to be dissolved in a treatment solution or physically destroyed. In the present invention, acid or alkali is first added to soften these animal tissues, animal tissues containing hard proteins, or components derived from animal tissues. When nucleic acid is extracted from a tissue such as bone containing hard protein after the softening treatment, it is dissolved by a process of adding a tissue lysis solution after hot water treatment. At this time, it is preferable to cut animal tissues into small pieces in advance by cutting or freeze-grinding. For example, the acid treatment is immersed in an acid solution of 1 to 10%, preferably 1 to 5%, more preferably 1 to 3% for a short time of 1 to 48 hours, preferably 1 to 24 hours, more preferably 1 to 12 hours. The alkali treatment is immersed in an alkaline solution of 1 to 10%, preferably 1 to 5%, more preferably 1 to 3%, for 1 to 100 days, preferably 10 to 50 days, more preferably 15 to 30 days. The hydrothermal treatment is performed at 30 to 100 ° C., preferably 40 to 80 ° C., more preferably 45 to 60 ° C. for 1 to 10 hours. After acid or alkali treatment and hot water treatment, a tissue lysis solution is added to the animal tissue as described above, and the temperature of the solution is 30 ° C to 75 ° C, preferably 40 ° C to 65 ° C, more preferably 50 ° C to 60 ° C. For example, it can be carried out by allowing the solution to stand with stirring using a shaker or the like for several hours to overnight or for several days while maintaining at 55 ° C. You may stand still and sometimes stir. Depending on the dissolved state of the tissue, the composition concentration of the tissue lysate can be changed, the treatment temperature, the number of stirrings, and the treatment time can be changed, or it can be immersed in the tissue lysate and left to stand. . Further, when using a calcified tissue such as bone, it can be decalcified. As yet another method, a step of fragmenting animal tissue using physical means such as a mill or a homogenizer can be used.
As a guide, 1 to 2000 μl of tissue lysate can be used per 0.1 mg to 200 mg of tissue, and can be increased or decreased from the above range depending on the state of the tissue. In the case of a liquid separated from a tissue, 1 to 2000 μl can be used per 1 μl to 2 ml, and can be increased or decreased from the above range depending on the state of the liquid. In the case of cells and particles, 1 to 2000 μl can be used per 10 to 1 × 10 ⁸ , and can be increased or decreased from the above range depending on the state of the cells and particles.
The pH can be used in the range of 4 to 12, preferably 6 to 11, and more preferably 7.5 to 9.5. These increase / decrease ranges can be increased or decreased by a volume suitable for the extraction operation.
The composition of the tissue lysis solution will be described later in [Composition of tissue lysis solution and pretreatment solution].

  In the present invention, as described above, the sample solution obtained in the step of adding the acid or alkali solution and the tissue lysis solution to the animal tissue is used. Also, calcified tissue such as bone can be subjected to hot water treatment. When undissolved residual tissue components remain in these liquids, it is preferable to further perform a step of removing the undissolved residual tissue components by centrifugation or filter filtration.

In addition, when the target nucleic acid to be collected is DNA, RNA may be degraded in advance by adding an RNA-degrading enzyme solution to the sample solution obtained in the step of adding the tissue lysis solution to the animal tissue. it can. In addition, when the target nucleic acid is RNA, DNA can be previously degraded by adding a DNA degrading enzyme solution to the sample solution obtained in the step of adding the tissue lysate to the animal tissue.
The enzyme solution may contain a buffer, may contain a monovalent or divalent alkali metal salt, and the alkali metal may be a combination of monovalent and divalent alkali metals. Polyhydric alcohols such as glycerol may be contained.
For example, 10 to 100 mg / ml RNase can be added in an amount of 0.1 to 100 μl per 200 μl of the tissue lysate, and can be increased or decreased from the above range depending on the state of the sample. Preferably, 1-50 μl of 100 mg / ml RNase can be added per 200 μl of tissue lysate.
These enzymes may be natural products, recombinants, or a mixture of two or more.

[Nucleic acid adsorption solution]
The sample solution obtained in the step of adding the tissue lysis solution to the animal tissue is treated with a pretreatment solution in order to solubilize the nucleic acid in the solution. Thereby, the cell membrane and the nuclear membrane are further dissolved, and the nucleic acid is dispersed in the sample solution to form a nucleic acid adsorption solution.
As a standard, the pretreatment liquid can be used in an amount of 1 to 2000 μl per 0.1 mg to 200 mg of mass used for dissolving the dissolved tissue, and can be increased or decreased from the above range depending on the amount of the dissolved tissue. In the case of a liquid separated from a tissue, 1 to 2000 μl can be used per 1 μl to 2 ml, and can be increased or decreased from the above range depending on the state of the liquid. In the case of cells and particles, 1 to 2000 μl can be used per 10 to 1 × 10 ⁸ , and can be increased or decreased from the above range depending on the state of the cells and particles. These increase / decrease ranges can be increased or decreased by a volume suitable for the extraction operation.
The pH can be used in the range of 3-12, preferably 4-8, more preferably 5-7.
When the sample solution obtained in the step of adding the tissue lysis solution to the animal tissue and the pretreatment solution are mixed, the sample solution can be stirred at 30 to 3000 rpm for 1 second to 30 minutes. Moreover, inversion mixing can be performed once to 30 times. Further, the pipetting operation can be performed once to 50 times. Further, the tapping operation can be performed once to 50 times. One of these may be performed as the mixing method, or these may be used in combination. It is preferable to mix well.
You may heat after mixing. For example, you may heat at 30 to 95 degreeC for 1 minute-60 minutes. Preferably, it can be heated to 50 to 75 ° C. for 1 to 15 minutes.

[Composition of solution for softening tissue]
The acid or alkali solution used in the present invention is a 1 to 10% (% by weight) solution.
The acid solution is preferably a solution containing at least one selected from inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid and organic acids such as acetic acid and citric acid. The alkaline solution is preferably a solution containing at least one selected from a metal hydroxide and an amino group-containing compound.

[Composition of tissue lysate and pretreatment solution]
The tissue lysis solution used in the present invention contains at least one selected from a surfactant, a buffer, a nucleic acid stabilizer, an alkali metal halide, a chaotropic salt, a proteolytic enzyme, and an antifoaming agent. It is preferable that The pretreatment liquid is preferably a solution containing at least one selected from an antifoaming agent, a nucleic acid stabilizer, a chaotropic salt, a surfactant and a buffer.

(Surfactant)
Specific examples of the surfactant used in the present invention include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants, and anionic surfactants and nonionic surfactants are preferably used.

  Examples of anionic surfactants include sulfate ester surfactants, sulfonate surfactants, carboxylate surfactants, phosphate ester surfactants, alkyl sulfate ester surfactants Agents are preferred. In particular, sodium dodecyl sulfate is preferable. Preferably, it is contained in a tissue lysis solution and can act on tissue dissolution.

Examples of nonionic surfactants include polyoxyethylene surfactants and fatty acid alkanolamides, with polyoxyethylene surfactants being preferred. Examples of the polyoxyethylene surfactant include polyoxyethylene alkylphenyl ether surfactants and polyoxyethylene alkyl ether surfactants, and polyoxyethylene alkyl ether surfactants are preferable. Among polyoxyethylene (POE) 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 Further preferred are rate, polyoxyethylene sorbite tetraoleate, POE alkylamine, and POE acetylene glycol. Among these, polyoxyethylene sorbitan surfactants are particularly preferable. It is particularly effective and preferable for adsorbing nucleic acids to the membrane.

These surfactants can be used alone or in combination. The density | concentration which can use surfactant can be selected in the range used as 0-100 mass% in a pretreatment liquid. The concentration of the surfactant in the pretreatment liquid is preferably 0.1 to 20% by mass. The working concentration is preferably in the range of 0.05 to 10% by mass.
In the case of an anionic surfactant, it can be added to the pretreatment liquid so as to be 0.01 to 50% by mass. Preferably, it can select in the range of 0.1-20 mass%. The working concentration is preferably in the range of 0.05 to 10% by mass.
In the case of a nonionic surfactant, it can be added to the pretreatment liquid so as to be 0.01 to 50% by mass. Preferably, it can select in the range of 0.1-20 mass%. The working concentration is preferably in the range of 0.05 to 10% by mass.
Also when used in combination, preferably, the anionic surfactant is in the range of 0.1 to 20% by mass in the pretreatment liquid, and the nonionic surfactant is in the range of 0.1 to 20% by mass in the pretreatment liquid. The range can be selected within the range of%. The working concentration of the anionic surfactant is preferably in the range of 0.05 to 10% by mass. The working concentration of the nonionic surfactant is preferably in the range of 0.05 to 10% by mass.
The concentration of the surfactant in the tissue lysate can be selected in the range of 0 to 100% by mass, which is 0.1 to 20% by mass, when the tissue is dissolved and adsorbed to the membrane. It is preferable. The working concentration is preferably in the range of 0.05 to 10% by mass.
In the case of an anionic surfactant, it can be added so as to be 0.01 to 50% by mass. Preferably, it can select in the range of 0.1-20 mass%. The working concentration is preferably in the range of 0.05 to 10% by mass.
In the case of a nonionic surfactant, it can be added so as to be 0.01 to 50% by mass. Preferably, it can select in the range of 0.1-20 mass%. The working concentration is preferably in the range of 0.05 to 10% by mass.
Also when used in combination, preferably, the anionic surfactant is in the range of 0.1 to 20% by mass in the pretreatment liquid, and the nonionic surfactant is in the range of 0.1 to 20% by mass in the pretreatment liquid. The range can be selected within the range of%. The working concentration of the anionic surfactant is preferably in the range of 0.05 to 10% by mass. The working concentration of the nonionic surfactant is preferably in the range of 0.05 to 10% by mass.

(Buffering agent)
Specific examples of the buffer used in the present invention include a commonly used pH buffer (buffer). Preferably, the pH buffer agent normally used for a biochemical test is mentioned. Examples of such a buffer include a buffer consisting of citrate, phosphate or acetate, Tris-HCl, TE (Tris-HCl / EDTA), TBE (Tris-Borate / EDTA), TAE (Tris-Acetate). / EDTA), Good buffer. Good buffering agents include MES (2-Morpholinoethanesulfonic acid), Bis-Tris {Bis (2-hydoroxyethyl) iminotris (hydroxymethyl) methane}, HEPES {2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic asid }, PIPES {Piperaxine-1,4-bis (2-ethanesulfonic acid)}, ACES {N- (2-Acetamino) -2-aminoethanesulfonic acid}, CAPS {N-Cyclohexyl-3-aminopropane-sulfonic acid}, TES {N-Tris (hydroxymethyl) methyl-2-aminoethane-sulfonic acid}.
The concentration of these buffering agents in the tissue lysis solution and the pretreatment solution is preferably 1 to 300 mmol / L. More preferably, it can select in the range of 20-150 mmol / L.

(Nucleic acid stabilizer)
Specific examples of the nucleic acid stabilizer used in the present invention include those having an action of inactivating nuclease activity. Depending on the animal tissue, 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 stably cause nucleic acids in animal tissues to exist.

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, magnesium, calcium, aluminum, zinc and other highly electropositive metals, or those 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 tissue lysis solution and the pretreatment solution is preferably 0.1 to 20% by mass, more preferably 0.3 to 15% by mass.

  Moreover, a chelating agent can be used as a nucleic acid stabilizer having an action of inactivating the nuclease activity. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and EGTA. Chelating agents can be used alone or in combination. For example, EDTA can be used in the working concentration range of 1 to 300 mmol / L. Preferably, the inclusion in a tissue lysate can act to inactivate endogenous nuclease activity. Moreover, it is preferable that the density | concentration in the said tissue solution and pretreatment liquid of a chelating agent is 1-1000 mmol / L, More preferably, it is 5-100 mmol / L.

(Alkali metal halide)
Specific examples of the alkali metal halide used in the present invention are sodium, potassium and lithium halides, preferably chloride. The alkali metal halide can be used at a concentration of 1 to 200 mM in the tissue lysate.

(Proteolytic enzyme)
Specific examples of the proteolytic enzyme used in the present invention include serine protease, cysteine protease, and metalloprotease, and at least one proteolytic enzyme can be preferably used. As the proteolytic enzyme, a mixture of a plurality of proteolytic enzymes can be preferably used.
The serine protease is not particularly limited, and for example, protease K can be preferably used. The cysteine protease is not particularly limited, and for example, papain and cathepsins can be preferably used. The metal protease is not particularly limited, and for example, carboxypeptidase can be preferably used.

The concentration of the proteolytic enzyme in the tissue lysate is preferably 0.001 to 10 IU, more preferably 0.01 to 1 IU per 1 mL of the total volume at the time of addition. In addition, it can be used at a working concentration of 0.05 to 20 mg / mL. These enzymes may be natural products, recombinants, or a mixture thereof. In order to stably maintain the action of the proteolytic enzyme, a buffer may be added to the tissue lysate. For example, Tris-HCl can be contained at 1 to 200 mmol / L.

  As the proteolytic enzyme, a proteolytic enzyme that does not contain a nucleolytic enzyme can be preferably used. A proteolytic enzyme containing a stabilizer can be preferably used. As the stabilizer, metal ions can be preferably used. Specifically, magnesium ions and calcium ions are preferable, and they can be added in the form of, for example, magnesium chloride or calcium acetate. By including a proteolytic enzyme stabilizer, the amount of proteolytic enzyme required for nucleic acid recovery can be reduced, and the cost required for nucleic acid recovery can be reduced. A buffer solution can be contained in the proteolytic enzyme solution, or a polyhydric alcohol can be added. For example, Tris-HCl can be contained in an amount of 0.1 to 200 mmol / L as a buffer solution, or glycerol can be contained in an amount of 1 to 70% by volume as a polyhydric alcohol. These can be used alone or in combination.

(Defoamer)
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, 2-ethylhexanol, higher alcohol, polyoxyalkylene glycol, etc.), ether-based antifoaming agents (for example, heptylcellosolve, nonylcellosolv-3-heptylsorbitol, 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) , Tributylphos Phosphate ester-based 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 Examples include foaming agents (eg, ferric sulfate, bauxite, etc.). Preferably, they are a silicon type antifoamer and an alcohol type antifoamer. These antifoaming agents may be used alone or in combination. Particularly preferably, the antifoaming agent is a combination of two components, a silicon antifoaming agent and an alcohol antifoaming agent. Moreover, as an alcohol type | system | group antifoamer, it is preferable to use an acetylene glycol type | system | group field antifoamer.

The antifoaming agent may be added directly to the obtained nucleic acid adsorption solution, or may be contained in the tissue lysis solution and / or the pretreatment solution. When the antifoaming agent is not contained in the pretreatment liquid, the timing of adding the antifoaming agent may be before or after using the pretreatment liquid. Moreover, you may add before using a tissue solution.
The concentration of the antifoaming agent in the tissue dissolving solution and / or the pretreatment solution can be selected in the range of 0 to 10% by mass. The concentration in the nucleic acid adsorption solution is preferably 0.1 to 10% by mass, more preferably 0.1 to 2% by mass.

(Chaotropic salt)
Specific examples of chaotropic salts used in the present invention include guanidine salts, sodium isothiocyanate, sodium iodide, potassium iodide and the like. 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 can be used alone or in combination. The concentration of chaotropic salt in the tissue lysate or pretreatment solution and the nucleic acid purification solution obtained by the method for separating and purifying nucleic acid of the present invention is preferably 0.5 mol / L or more, more preferably 0.8. 5-4 mol / L, more preferably 1-3 mol / L.
Instead of the chaotropic salt, urea can also be used as the chaotropic substance. Urea may be selected in the range of 0 to 10 mol / L.

  The solution for adsorbing nucleic acid on the solid phase (nucleic acid adsorption solution) is preferably a solution obtained by further adding a water-soluble organic solvent. That is, it is preferable to add a pretreatment solution to the sample solution obtained in the step of adding the tissue lysis solution to the animal tissue, and further add a water-soluble organic solvent to obtain a nucleic acid adsorption solution. By adding a water-soluble organic solvent to the nucleic acid adsorption solution in which the nucleic acid is solubilized and dispersed, the nucleic acid in the nucleic acid adsorption solution is effectively adsorbed to the solid phase when brought into contact with the solid phase. Therefore, it is preferable. Furthermore, the presence of a salt in the obtained nucleic acid adsorption solution is preferable because the solubilized nucleic acid can be more effectively adsorbed to the solid phase.

  The presence of the water-soluble organic solvent and salt destroys the hydration structure of water molecules present around the nucleic acid, so that the nucleic acid is solubilized in an unstable state. When a nucleic acid in this state is brought into contact with a solid phase consisting of a polysaccharide-structured organic polymer having a hydroxyl group on the surface, the nucleic acid interacts between the polar group on the nucleic acid surface and the polar group on the solid phase surface, and the nucleic acid It is thought that it adsorbs on top. In the method of the present invention, as described above, mixing a water-soluble organic solvent with a solubilized solution for adsorbing nucleic acid and the presence of a salt in the resulting solution makes the nucleic acid unstable. Is preferable.

(Water-soluble organic solvent)
Examples of such water-soluble organic solvents 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. Of these, methanol, ethanol, propanol and its isomer, butanol and its isomer can be preferably used. More preferred is ethanol. These water-soluble organic solvents may be used alone or in combination.

The final concentration of these water-soluble organic solvents in the nucleic acid adsorption solution is preferably 5 to 90% by mass. More preferably, it is 20 mass%-60 mass%. When ethanol is used, it is particularly preferable that the addition concentration be as high as possible within this range and within the range where no pseudo-collects are generated.
As a standard, the water-soluble organic solvent can be used in an amount of 1 to 2000 μl per mass of 0.1 mg to 200 mg used when dissolved in the dissolved tissue, and can be increased or decreased from the above range depending on the amount of the dissolved tissue. In the case of a liquid separated from a tissue, 1 to 2000 μl can be used per 1 μl to 2 ml, and can be increased or decreased from the above range depending on the state of the liquid. In the case of cells and particles, 1 to 2000 μl can be used per 10 to 1 × 10 ⁸ , and can be increased or decreased from the above range depending on the state of the cells and particles. These increase / decrease ranges can be increased or decreased by a volume suitable for the extraction operation.
As described above, the water-soluble organic solvent may be added alone after adding the pretreatment liquid, or may be obtained in the step of adding the tissue solution to the animal tissue after mixing with the treatment liquid. It may be added to the sample solution. Furthermore, it may be added to the sample solution obtained in the step of adding the tissue lysis solution to the animal tissue after mixing with the treatment solution, and then a water-soluble organic solvent may be further added alone.
When mixing after adding a water-soluble organic solvent, it can stir at 30-3000 rpm with a stirring apparatus for 1 second to 30 minutes. Moreover, inversion mixing can be performed once to 30 times. Further, the pipetting operation can be performed once to 50 times. Further, the tapping operation can be performed once to 50 times. One of these may be performed, or these may be used in combination. It is preferable to mix well.

(Salt in nucleic acid adsorption solution)
Salts preferably present in the nucleic acid adsorption solution include various chaotropic substances (guanidinium salt, sodium iodide, sodium perchlorate), sodium chloride, potassium chloride, ammonium chloride, sodium bromide, potassium bromide, odor Calcium guanide, ammonium bromide and the like, and guanidinium salt is particularly preferable since it has both the effect of solubilizing the cell membrane and solubilizing the nucleic acid.

The pH of the obtained nucleic acid adsorption solution is preferably 3 to 10, more preferably 4 to 9, and still more preferably 5 to 8.
The temperature may be 5 ° C to 50 ° C, preferably 10 ° C to 40 ° C, more preferably 15 ° C to 35 ° C.

Further, the obtained nucleic acid adsorption solution preferably has a surface tension of 0.05 J / m ² or less, a viscosity of preferably 1 to 10,000 mPa, and a specific gravity in the range of 0.8 to 1.2. Preferably there is. By making the solution in this range, the nucleic acid adsorption solution is brought into contact with the solid phase in the adsorption step, and the solution remaining after adsorbing the nucleic acid is easily removed in the washing step.

<Adsorption process>
Next, a solution (nucleic acid adsorption solution) in which the nucleic acid is solubilized and dispersed in the liquid is adsorbed onto the solid phase, and the solution for adsorbing the nucleic acid on the solid phase is brought into contact with the solid phase.

[Solid phase]
In the method of the present invention, a solid phase composed of an organic polymer having a polysaccharide structure is used. An organic polymer having a polysaccharide structure can adsorb a nucleic acid by an interaction that does not substantially involve an ionic bond. “Ion binding is not substantially involved” means that it is not “ionized” under the use conditions on the solid phase side, and it is assumed that the nucleic acid and the solid phase will attract each other by changing the polarity of the environment. Is done. As a result, the nucleic acid can be isolated and purified with excellent separation performance and good washing efficiency. Since the organic polymer having a polysaccharide structure has a hydrophilic group, it is presumed that the hydrophilic group of the nucleic acid and the solid phase are attracted to each other by changing the polarity of the environment.

  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 hydrophilic groups, those with moderate interaction with water are good (see Chemical Encyclopedia, published by Kyoritsu Shuppan Co., Ltd., “Groups that are not very hydrophilic” in the section “Hydrophilic groups”), Examples thereof include a hydroxyl group, a carboxyl group, a cyano group, and an oxyethylene group. Preferably it is a hydroxyl group.

(Organic polymer with polysaccharide structure)
Organic polymers 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, hyaluron Acid, chondroitin, chitin, chitosan and the like can be preferably used. These polysaccharide structure derivatives may be used. For example, those in which the hydroxyl group of the polysaccharide structure is esterified, etherified, or halogenated with an arbitrary degree of substitution. Any of the polysaccharide structures and derivatives thereof can be used without being limited to the materials listed above. These derivatives can be produced by a conventionally known method. These derivatives can be produced by a conventionally known method. In particular, ester derivatives can be preferably used. Further, a saponified product of an ester derivative is also preferable.

Examples of the ester in the ester derivative having a polysaccharide structure include carboxylic acid ester, nitrate ester, sulfate ester, sulfonate ester, phosphate ester, phosphonate ester, and pyrophosphate ester, and the ester derivative is at least selected from these esters. One is preferred. Further, saponified products of these ester derivatives are also suitable.

  Examples of the carboxylic acid ester include alkyl carbonyl esters, alkenyl carbonyl esters, aromatic carbonyl esters, and aromatic alkyl carbonyl esters. When a carboxylic acid ester is used as the ester, at least one selected from these carboxylic acid esters. It is preferable that In addition, saponified products of these carboxylic acid esters are also preferred.

  Examples of the alkylcarbonyl group of the above alkylcarbonyl ester include acetyl group, propionyl group, butyroyl group, barrel group, peptanoyl group, octanoyl group, decanoyl group, dodecanoyl group, tridecanoyl group, hexadecanoyl group, and octadecanoyl group. When an alkylcarbonyl ester is used as the carboxylic acid ester, it preferably has at least one selected from these alkylcarbonyl groups. Further, saponified products of these alkyl carbonyl esters are also suitable.

  Examples of the alkenylcarbonyl group of the alkenylcarbonyl ester include an acryl group and a methacryl group. When an alkenylcarbonyl ester is used as the carboxylic acid ester, it preferably has at least one selected from these alkenylcarbonyl groups. Further, saponified products of these alkenyl carbonyl esters are also preferred.

  Examples of the aromatic carbonyl group of the aromatic carbonyl ester include a benzoyl group and a naphthaloyl group. When an aromatic carbonyl ester is used as the carboxylic acid ester, the aromatic carbonyl group has at least one selected from these aromatic carbonyl groups. It is preferable. Further, saponified products of these aromatic carbonyl esters are also preferred.

  Examples of the nitrate ester include nitrocellulose, nitrohemicellulose, nitrodextran, nitroagarose, nitrodextrin, nitroamylose, nitroamylopectin, nitroglycogen, nitropullulan, nitromannan, nitroglucomannan, nitrolikenan, nitroisoliquenan, nitrolaminaran, Examples thereof include nitrocarrageenan, nitroxylan, nitrofructan, nitroalginic acid, nitrohyaluronic acid, nitrochondroitin, nitrochitin, and nitrochitosan. Further, saponified products of these nitrates are also suitable.

  Examples of the sulfate ester 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, isolikenane sulfate, laminaran sulfate, carrageenan Examples include sulfuric acid, xylan sulfate, fructan sulfate, alginic acid sulfate, hyaluronic acid sulfuric acid, chondroitin sulfate, chitin sulfate, and chitosan sulfate. In addition, a saponified product of these sulfates is also preferable.

  Examples of the sulfonic acid ester include alkyl sulfonic acid esters, alkenyl sulfonic acid esters, aromatic sulfonic acid esters, and aromatic alkyl sulfonic acid esters. When sulfonic acid esters are used as the esters, from these sulfonic acid esters, It is preferable that at least one selected. In addition, saponified products of these sulfonic acid esters are also preferable.

  Examples of the phosphate ester include cellulose phosphate, hemicellulose phosphate, dextran phosphate, agarose phosphate, dextrin phosphate, amylose phosphate, amylopectin phosphate, glycogen phosphate, pullulan phosphate, mannan phosphate, glucomannan phosphate , Lichenan phosphoric acid, isoricenan phosphoric acid, laminaran phosphoric acid, carrageenan phosphoric acid, xylan phosphoric acid, fructan phosphoric acid, alginic acid phosphoric acid, hyaluronic acid phosphoric acid, chondroitin phosphoric acid, chitin phosphoric acid, chitosan phosphoric acid. In addition, saponified products of these phosphate esters are also preferable.

  Examples of the phosphonic acid ester 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, mannan phosphonic acid, and glucomannan phosphonic acid. Examples include acids, lichenan phosphonic acid, isolichenane 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, etc. It is done. In addition, saponified products of these phosphonic acid esters are also preferable.

  Examples of the pyrophosphate ester include cellulose pyrophosphate, hemicellulose pyrophosphate, dextran pyrophosphate, agarose pyrophosphate, dextrin pyrophosphate, amylose pyrophosphate, amylopectin pyrophosphate, glycogen pyrophosphate, pullulan pyrophosphate, mannan pyrophosphate, glucomannan pyrophosphate. Acid, lichenane pyrophosphate, isolichenane pyrophosphate, laminaran pyrophosphate, carrageenan pyrophosphate, xylan pyrophosphate, fructan pyrophosphate, alginate pyrophosphate, hyaluronic acid pyrophosphate, chondroitin pyrophosphate, chitin pyrophosphate, chitosan pyrophosphate, etc. Is mentioned. Further, saponified products of these pyrophosphate esters are also suitable.

  Examples of the ether in the above-described polysaccharide-structured ether derivative include, but are not limited to, the types of polysaccharide structures and the types of ethers. Examples of these ethers include methylcellulose, ethylcellulose, carboxymethylcellulose, carboxyethylcellulose, carboxyethyl-carbamoylethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, cyanoethylcellulose, carbamoylethylcellulose and the like. . Preferred are hydroxymethyl cellulose and hydroxyethyl cellulose.

  It can also be preferably used for those in which the hydroxyl group of the polysaccharide structure is halogenated with an arbitrary degree of substitution.

  Preferred examples of the organic polymer having a polysaccharide structure include acetylcellulose, and acetylcellulose mixtures having different acetyl values. As acetyl cellulose mixtures having different acetyl values, triacetyl cellulose and diacetyl cellulose mixtures, triacetyl cellulose and monoacetyl cellulose mixtures, triacetyl cellulose and diacetyl cellulose and monoacetyl cellulose mixtures, and diacetyl cellulose and monoacetyl cellulose mixtures are preferably used. Can do.

  Particularly preferred is a mixture of triacetyl cellulose and diacetyl cellulose. 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.

Among acetylcelluloses preferably used as organic polymers having a polysaccharide structure,
A surface saponified product of acetyl cellulose described in JP-A No. 2003-128691 is particularly preferable. Examples of the surface saponified product of acetyl cellulose (hereinafter, sometimes simply referred to as “saponified product”) include those obtained by saponifying a mixture of acetyl cellulose having different acetyl values, a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose, A saponified product of a mixture of triacetyl cellulose and monoacetyl cellulose, a saponified product of a mixture of triacetyl cellulose, diacetyl cellulose and monoacetyl cellulose, or a saponified product of a mixture of diacetyl cellulose and monoacetyl cellulose can be preferably used. More preferably, a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose is used, and the mixing ratio (mass ratio) is also in the same range as described above for the mixture of acetyl cellulose having different acetyl values. In this case, the amount (density) of hydroxyl groups on the surface of the solid phase can be controlled by the degree of saponification treatment (saponification rate), which is preferable. From the viewpoint of improving 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. Further, from the viewpoint of increasing the surface area having hydroxyl groups in the solid phase, it is preferable to saponify acetyl cellulose to obtain a solid phase. The solid phase may be a front and back symmetric porous membrane, but a front and back asymmetric porous membrane can be preferably used.

  In order to obtain the above saponified product, a saponification treatment is performed. Here, the saponification treatment refers to bringing an organic polymer having an ester group into contact with a saponification treatment solution (for example, a sodium hydroxide aqueous solution). Thereby, the part which contacted the saponification process liquid, ie, the surface of an organic material, is saponified. In the case of acetylcellulose, the portion in contact with the saponification solution becomes regenerated cellulose and hydroxyl groups are introduced. The regenerated cellulose thus produced 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 solid phase containing regenerated cellulose 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).

The solid phase in the present invention is a solid phase composed of an organic polymer, and the organic polymer itself may be used as a solid phase or may be used by coating the above organic polymer on an arbitrary material ( In this case, an arbitrary material is covered with a solid phase made of an organic polymer.) The material coated with the organic polymer may be either an organic material or an inorganic material as long as it can be coated with the organic polymer. The shape of the solid phase may be any of a shape in which the solution is in contact with the surface, such as a bead shape or a fiber shape, or a shape in which a solution such as a filter shape can pass through.
The solid phase in the present invention is preferably used in the form of a filter or a membrane that allows the solution to pass inside the solid phase. In this case, the thickness of the solid phase is preferably 10 μm to 500 μm, more preferably 50 μm to 250 μm. It is preferable from this viewpoint that it is within this range. In addition, the solid phase through which such a solution can pass (hereinafter also referred to as “solution-flowing solid phase”) is preferably a porous membrane. The average pore diameter of the pores of the porous membrane is preferably 0.1 μm to 10 μm, more preferably 1 μm to 5 μm. Within this range, a surface area sufficient for adsorbing nucleic acids can be obtained, and clogging is also difficult, which is preferable. The average pore diameter of such a solid phase can be measured using a bubble point method (according to ASTM F316-86, JIS K-3832).

The solution-permeable solid phase is preferably an asymmetric porous membrane. Here, the front and back asymmetry indicates a property in which the physical property or chemical property of the membrane changes 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 a porous membrane having an asymmetric average pore size is used in the present invention, the average pore size is preferably changed 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.

Moreover, it is preferable that said solution flow-through solid phase has the following various properties.
Porosity: 50 to 95%, and further 65 to 80%.
Bubble point: 0.1 to 10 kgf / cm ² , and further 0.2 to 4 kgf / cm ² .
Pressure loss: 0.1 to 100 kPa, further 0.5 to 50 kPa. Within this range, a uniform pressure can be obtained during overpressure, which is preferable. Here, the pressure loss is the minimum pressure required to pass water per 100 μm thickness of the membrane.
Water permeability when water is passed at 25 ° C. with a pressure of 1 kg / cm ² (1 minute per 1 cm ^{2 of} membrane): 1 to 5000 mL, and further 5 to 1000 mL.
Nucleic acid adsorption amount per 1 mg of porous membrane: 0.1 μg or more, further 0.9 μg or more.

In order to obtain an appropriate contact time of the liquid with the solid phase, the flow rate when the nucleic acid adsorption solution is passed through the solid phase is ² to 1500 μL / second, further 5 to 700 μL per 1 cm ^{2 of the} solid phase area. / Second is preferred. If the contact time of the liquid to the solid phase is not less than the lower limit value, it is preferable because a sufficient separation and purification effect can be obtained, and if it is not more than the upper limit value, it is preferable from the viewpoint of operability.

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

<Washing process>
Hereinafter, the cleaning process will be described.
After the nucleic acid is adsorbed to the solid phase as described above, the recovery amount and purity of the nucleic acid are improved by bringing the washing solution into contact with the solid phase and washing the solid phase with the nucleic acid adsorbed. The amount of animal tissue containing the necessary nucleic acid can be made minute. In addition, it is preferable to automate the cleaning process and the recovery process described later, because 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.

[Cleaning liquid]
The cleaning liquid is preferably a solution containing a water-soluble organic solvent and / or a water-soluble salt. Further, it may contain a buffer and a surfactant as necessary. The washing liquid needs to have a function of washing out impurities in the nucleic acid adsorption solution adsorbed together with the nucleic acid on the solid phase. 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. Further, the addition of a water-soluble salt is preferable because the effect of adsorbing nucleic acids is enhanced, and the selective removal action of impurities and unnecessary components is improved.

[Water-soluble organic solvent]
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.

[Water-soluble salt]
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, potassium salt or lithium salt, most preferably a sodium salt. .
The pH of the cleaning solution may be 3 to 11, preferably 5 to 10, more preferably 6.5 to 8.5.

  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.

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 (polymerase chain reaction) is often inhibited. Therefore, it is ideal that the washing solution does not contain a chaotropic substance in consideration of the subsequent enzyme reaction or the like. In addition, since chaotropic substances are corrosive and harmful, it is extremely advantageous for the experimenter from the viewpoint of safety of the test operation that the chaotropic substances need not be used. Here, the chaotropic substance is urea, guanidine salt, sodium isothiocyanate, sodium iodide, potassium iodide or the like.
By not containing a chaotropic salt, the measured value 260/230 nm of the spectrophotometer is larger than 1.5, and a highly pure nucleic acid can be purified. This makes it possible to conduct highly accurate experiments on purified nucleic acids without causing inconveniences when performing molecular biological experiments such as PCR.

  Conventionally, during the washing step in the nucleic acid separation and purification method, since the washing liquid has high wettability with respect to the container used in the nucleic acid separation and purification method, the washing liquid often remains in the container and is used as a collection liquid during the collection step following the washing step. This is a cause of a decrease in the purity of nucleic acid to be mixed and separated, and a decrease in reactivity when the next step (for example, PCR reaction) is performed after the recovery step. Therefore, when nucleic acid is adsorbed and desorbed using a container, residual washing liquid remains in the container so that the liquid used for adsorption and washing, particularly the washing liquid, does not affect the recovery process or the process after the recovery process. It is desirable not to do so.

Therefore, the surface tension of the cleaning liquid should be less than 0.035 J / m ^{2 in} order to prevent the cleaning liquid in the cleaning process from entering the recovery liquid in the recovery process and to keep the cleaning liquid remaining in the container to a minimum. Is preferred. A low surface tension is preferable because the wettability between the cleaning liquid and the container is improved and the amount of the remaining liquid can be suppressed.

Although it is possible to increase the cleaning efficiency by increasing the proportion of water in the cleaning liquid, in this case, the surface tension of the cleaning liquid increases, thereby increasing the amount of remaining liquid. 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 container. By increasing the water repellency of the container, droplets can be formed, and the amount of liquid remaining by the droplets flowing down can be suppressed. As a method for enhancing water repellency, there are means such as coating the surface of the container with a water repellent such as silicon, or kneading a water repellent such as silicon when molding the container, 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.
The washing solution that can be introduced at a time can be arbitrarily selected within the capacity range of the nucleic acid separation and purification unit. For example, if the cartridge capacity is 800 μl, the range of 50 to 800 μl is good. Preferably, it can also be 400-800 microliters.
The total amount of the cleaning liquid can be from 1/4 to 10 times the amount of the sample solution charged in the cartridge. Preferably, it can be selected from the range of ½ amount to 4 times amount.

In the washing step, the flow rate when the washing solution is brought into contact with the solid phase is preferably 2 to 1500 μL / sec, more preferably 5 to 700 μL / sec per 1 cm ² of the solid phase unit area. 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. In the washing step, mechanical vibration and ultrasonic stirring can be applied to the container used for the nucleic acid separation and purification method simultaneously with the washing step. Alternatively, it can be washed by centrifugation.

  When the target nucleic acid to be collected is DNA before or during the washing step, RNA can be degraded in advance by bringing the RNase solution into contact with the solid phase. Moreover, when the target nucleic acid is RNA, DNA can also be decomposed in advance by bringing a DNA degrading enzyme solution into contact with the solid phase. In any case, it is desirable that the solid phase is subsequently washed with a washing solution to remove RNA-degrading enzyme or DNA-degrading enzyme from the solid phase.

<Recovery process>
Next, in order to desorb the nucleic acid adsorbed on the solid phase, the recovered liquid, which is a solution capable of desorbing the nucleic acid, is brought into contact with the washed solid phase. Since the target nucleic acid is contained in the recovered solution after contact with the solid phase (hereinafter also referred to as “purified solution”), amplification of the nucleic acid by subsequent steps such as PCR (polymerase chain reaction) To provide.

  Nucleic acid can be desorbed by adjusting the volume of the recovered solution with respect to the volume of the sample solution containing nucleic acid prepared from animal tissue. The amount of recovered liquid used depends on the amount of animal tissue that is then subjected to nucleic acid separation and purification. In general, the amount of recovered liquid used is from several tens to several hundreds of microliters. However, when the amount of animal tissue is extremely small, or when it is desired to separate and purify a large amount of nucleic acid, the amount of recovered liquid is from 1 μL to several It can be varied in the range of 10 mL.

[Recovered liquid]
As the recovery liquid, preferably, purified distilled water, Tris buffer, Tris / EDTA buffer or the like can be used. The pH of the recovery liquid is preferably pH 2-11. Furthermore, it is preferable that it is pH 5-9. In particular, ionic strength and salt concentration have an effect on the elution of adsorbed nucleic acids. The recovered liquid is preferably a solution having a salt concentration of 0.5 mol / L or less. The recovered liquid preferably has an ionic strength of 500 mol / L or less, more preferably 290 mmol / L or less, and particularly 90 mmol / L or less. More preferably, it is 20 mmol / L. By doing so, the recovery rate of nucleic acids can be improved, and more nucleic acids can be recovered.
The temperature of the recovered liquid can be 5 ° C to 90 ° C. The temperature may be preferably 10 ° C to 50 ° C, more preferably 15 ° C to 35 ° C.

  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. These methods are preferable because they can provide a method for obtaining a nucleic acid solution in which nucleic acid is more concentrated than the sample solution obtained in the step of adding a tissue lysate to animal tissue.

  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.

<Nucleic acid separation and purification unit>
The nucleic acid separation and purification method of the present invention preferably uses a nucleic acid separation and purification unit in which a solid phase is accommodated in a container having at least two openings. Hereinafter, a nucleic acid separation and purification unit in which a solid phase is accommodated in a container having at least two openings is also referred to as a nucleic acid separation and purification cartridge.
As a nucleic acid separation and purification unit used in the nucleic acid separation and purification method of the present invention, further, (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,
It is preferable to contain. Hereinafter, the nucleic acid separation and purification unit will be described.

  There are no particular limitations on the material of the container, as long as it can accommodate the solid phase and can be provided with at least two openings, but plastic is preferred from the standpoint of ease of manufacture. For example, it is preferable to use a transparent or opaque plastic such as polystyrene, polymethacrylate, polyethylene, polypropylene, polyester, nylon, polycarbonate or the like.

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

  The internal volume of the container is preferably determined by the amount of the sample solution to be processed, and 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.

  It is preferable that the end surface of the solid phase in contact with the container is in close contact with the inner wall surface of the container so that the sample solution or the like does not pass through.

  When viewed from the solid phase of the container having at least two openings, the side (open side from the solid phase in the container) used for the inlet of the sample solution or the like is not in close contact with the inner wall of the container. It is preferable to provide a structure in which the sample solution or the like diffuses as evenly as possible over the entire surface of the solid phase.

  When the nucleic acid separation and purification unit couples the pressure difference generator to one opening of the container, the container has a solid phase container, and the container can store the solid phase and adsorb the nucleic acid to the solid phase. The solid phase does not come out of the housing portion when the solution (a solution for adsorbing nucleic acid) or the like is sucked and discharged, and it is only necessary that a pressure difference generating device such as a syringe can be joined to the opening. 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. In order to prevent the solid phase from going out of the storage unit, it is also a preferable aspect to place meshes made of a material that does not contaminate the nucleic acid above and below the solid phase.

  The container is usually produced in a form divided into a main body for storing the solid phase and a lid, and each is provided with at least one opening. Openings are used as inlets and outlets for nucleic acid adsorption solution, washing solution and recovery solution (hereinafter sometimes referred to as “nucleic acid adsorption solution etc.”), and pressure difference generation that can cause the inside of the container to be depressurized or pressurized. Connected to the device. 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 or pressurized by the 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.

  Examples of the pressure difference generator include a pump capable of suction (or pressure reduction) and pressurization such as a syringe, pipetter, or 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.

  In addition, when three or more openings are provided in the container, it is preferable that the extra openings can be temporarily blocked in order to allow suction or discharge of the liquid accompanying the decompression or pressurization operation.

[Purification of nucleic acid by nucleic acid separation and purification unit]
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) 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,
Nucleic acid can be adsorbed and desorbed using a nucleic acid separation and purification unit containing

In this case, the first embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(1a) a step of dissolving a tissue by adding a tissue lysis solution to an animal tissue and treating the animal tissue to prepare a sample solution containing a nucleic acid;
(1b) a step of adding a pretreatment liquid to the sample solution containing the nucleic acid to prepare a solution for adsorbing the nucleic acid on the solid phase (nucleic acid adsorption solution);
(2a ′) inserting one opening of the container constituting the nucleic acid separation and purification unit into the nucleic acid adsorption solution into the solid phase,
(2a ″) a step of reducing the pressure inside the container using a pressure difference generator coupled to the other opening of the container, sucking the nucleic acid adsorption solution into the solid phase, and bringing it into contact with the solid phase;
(2b ′) The inside of the container is pressurized using a pressure difference generator connected to the other opening of the container, and the nucleic acid adsorption solution in the container that has been aspirated is discharged out of the container. Contacting the solution with a solid phase in a container to adsorb nucleic acid to the solid phase;
(3a ′) inserting one opening of the container into the cleaning liquid;
(3a ″) a step of depressurizing the inside of the container using a pressure difference generator coupled to the other opening of the container, and sucking the cleaning liquid into contact with the solid phase;
(3b ') The inside of the container is pressurized using a pressure difference generator connected to the other opening of the container, and the cleaning liquid in the container is sucked out and discharged outside the container, so that the cleaning liquid is again contained in the container. Washing the solid phase by contacting with the solid phase inside,
(4a ′) inserting one opening of the container into the recovered liquid;
(4a ″) a step of depressurizing the inside of the container using a pressure difference generator coupled to the other opening of the container, and sucking the recovered liquid into contact with the solid phase; and (4b ′) other of the container The container is pressurized using a pressure difference generator connected to the opening of the container, and the recovered liquid in the container is aspirated and discharged out of the container to bring the recovered liquid into contact with the solid phase in the container. The step of desorbing the nucleic acid adsorbed on the solid phase and discharging it out of the container.

  In the case of (2a ″), (3a ″), and (4a ″), it is preferable to suck an amount of the solution that is in contact with almost the entire solid phase, but it is sucked into the pressure difference generator. It is preferable to adjust the amount to be sucked to an appropriate amount, after sucking an appropriate amount of solution, pressurize the inside of the unit container using a pressure difference generator, and discharge the sucked solution. It is not necessary to leave an interval before the operation, and it may be discharged immediately after suction.

In the nucleic acid separation and purification unit for carrying out the first embodiment, it is preferable to provide a member having a hole in the substantially center on the solid phase on the side with respect to the opening coupled to the pressure difference generator. This member has the effect of holding down the solid phase and efficiently discharging the solution for adsorbing nucleic acids, etc., and has a funnel-like or bowl-like slope so that the solution collects in the central hole. It is preferable. The size of the hole, the angle of the slope, and the thickness of the member can be appropriately determined by those skilled in the art in consideration of the amount of the nucleic acid adsorption solution to be processed, the size of the container for storing the solid phase, and the like.
Between this member and the opening, it is preferable to provide a space for storing an overflowing solution for adsorbing nucleic acid and the like and preventing the 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 an amount of the solution for adsorbing nucleic acid that is larger than the entire solid phase.

  In addition, in order to prevent the nucleic acid adsorption solution and the like from concentrating directly below the aspirating opening and to allow the nucleic acid adsorption solution and the like to pass through the solid phase relatively uniformly, It is preferable to provide a space between the members. 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.

The second embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(1a) a step of dissolving a tissue by adding a tissue lysis solution to an animal tissue and treating the animal tissue to prepare a sample solution containing a nucleic acid;
(1b) a step of adding a pretreatment liquid to the sample solution containing the nucleic acid to prepare a solution for adsorbing the nucleic acid on the solid phase (nucleic acid adsorption solution);
(2a) Injecting the nucleic acid adsorption solution into one opening of a container constituting the nucleic acid separation and purification unit,
(2b) A pressure difference generator is connected to one opening of the container to bring the inside of the container into a pressurized state, and the injected nucleic acid adsorption solution is discharged from the other opening of the container, whereby the solution is put into the container Contacting the solid phase with the nucleic acid to adsorb the nucleic acid to the solid phase,
(3a) removing the pressure difference generator from one opening of the container and injecting a cleaning liquid into the one opening;
(3b) A pressure difference generator is connected to one opening of the container to put the inside of the container in a pressurized state, and the injected cleaning liquid is discharged from the other opening of the container, whereby the cleaning liquid is put into a solid phase in the container. Washing the solid phase by contacting,
(4a) removing the pressure difference generator from one opening of the container and injecting the recovered liquid into the one opening;
(4b) A pressure difference generator is connected to one opening of the container to bring the inside of the container into a pressurized state, and the collected recovered liquid is discharged from the other opening of the container, so that the recovered liquid is fixed in the container. The step of desorbing the nucleic acid adsorbed on the solid phase by contacting with the phase and discharging it out of the container.

  In the steps (2a), (3a) and (4a) of the second embodiment of the nucleic acid separation and purification method of the present invention, the method for injecting the nucleic acid adsorption solution into the container is not particularly limited. It is preferable to use a laboratory instrument such as a syringe or a dropper. More preferably, these instruments are nuclease-free and pyrogen-free.

  The method of mixing the solution of each stage prepared based on animal tissue is not particularly limited. For example, when mixing, when using a stirrer, it is preferable to mix at 30 to 3000 rpm for 1 second to 3 minutes. This is preferable because the yield of nucleic acid to be separated and purified can be increased. Alternatively, in the case of inversion mixing, mixing is preferably performed by performing 5 to 30 times. Moreover, in the case of pipetting operation, it is preferable to mix by performing 10 to 50 times.

Before the step (4a) above,
(3c) a step of contacting the DNA-degrading enzyme solution with the solid phase and then washing the solid phase with a washing solution;
By performing the above, it is possible to selectively separate and purify RNA alone from a nucleic acid mixture solution containing DNA and RNA. The DNA degrading enzyme solution is not particularly limited, and any known DNase can be used.

  A reagent for use in the nucleic acid separation and purification method can be used as a reagent kit. The reagent kit includes (i) a nucleic acid separation and purification cartridge, (ii) a tissue lysate or proteolytic enzyme, (iii) a pretreatment solution, (iv) a washing solution, and (v) a recovery solution.

  For nucleic acid adsorption prepared based on animal tissue using a nucleic acid separation / purification unit (nucleic acid separation / purification cartridge) which is a container having at least two openings, containing a solid phase inside, and a pressure difference generator. An example of an automatic apparatus that automatically performs a step of separating and purifying nucleic acid from a solution is shown, but the automatic apparatus is not limited to this.

The automatic apparatus described above uses a nucleic acid separation / purification cartridge containing a solid phase that adsorbs nucleic acid through which the solution can pass, and the nucleic acid separation / purification cartridge uses a solution (nucleic acid adsorption) to adsorb nucleic acid to the solid phase. Solution) is injected and pressurized to adsorb the nucleic acid in the nucleic acid adsorption solution to the solid phase, and then a washing solution is dispensed into the nucleic acid separation and purification cartridge and pressurized to remove impurities, and then the nucleic acid A nucleic acid separation and purification apparatus for automatically performing a separation and purification operation, in which a separation liquid is dispensed into a separation and purification cartridge, and the nucleic acid adsorbed on the solid phase is desorbed and collected together with the collection liquid. Pressurized air is introduced into the raw material liquid container that contains the nucleic acid adsorption solution, the waste liquid container that contains the cleaning liquid discharge liquid, the mounting mechanism that holds the collection container that contains the nucleic acid-containing recovery liquid, and the nucleic acid separation and purification cartridge. Pressurized air supply mechanism for, as well as to characterized in that it comprises a dispensing mechanism for dispensing the washing solution and a recovering solution to the nucleic acid separation-purification cartridge.

  The mounting mechanism includes a stand mounted on the apparatus main body, 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 a position relative to the nucleic acid separation and purification cartridge below the cartridge holder. What comprises the said waste-liquid container and the container holder holding the said collection | recovery container in the exchangeable state 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 that supports the air nozzle and is held by 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.

  Further, the dispensing mechanism holds a cleaning liquid dispensing nozzle for dispensing the cleaning liquid, a recovered liquid dispensing nozzle for dispensing the recovered liquid, the cleaning liquid dispensing nozzle and the recovered liquid dispensing nozzle, A nozzle moving table that can be moved in order on the cartridge for nucleic acid separation and purification held by the cartridge holder in the mounting mechanism, a cleaning liquid supply pump that sucks the cleaning liquid from the cleaning liquid bottle containing 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 containing the liquid 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 after injecting and pressurizing a solution for adsorbing nucleic acid to the solid phase into the nucleic acid separation and purification cartridge equipped with the solid phase member to adsorb the nucleic acid to the solid phase member After removing the impurities by washing and discharging the cleaning solution, the nucleic acid separation and purification process is automatically performed to separate and recover the nucleic acid adsorbed on the solid-phase membrane member by dispensing the recovery solution and efficiently in a short time. A mechanism that can automatically separate and purify nucleic acid in a sample solution can be configured compactly.

  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.

  Furthermore, when the pressurized air supply mechanism is configured to include 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, a simple mechanism ensures reliable pressurized air flow. Supply is possible.

  Furthermore, the dispensing mechanism includes a washing liquid dispensing nozzle, a collected liquid dispensing nozzle, a nozzle moving table that can be moved 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 Separation / Purification Unit (Nucleic Acid Separation Cartridge) A container (nucleic acid separation / purification cartridge) having an inner diameter of 7 mm and having a portion containing a solid phase of a nucleic acid-adsorbing porous membrane is resistant. Made of impact polystyrene.

(3) A porous membrane obtained by saponifying a porous membrane of triacetyl cellulose (pore size: 2.5 μm, diameter: 7 mm, thickness: 100 μm, saponification rate: 95%) is used as the nucleic acid-adsorbing porous membrane. The nucleic acid separation and purification cartridge prepared in 1) was housed in the porous membrane solid phase container.

(4) Preparation of 3% calcium hydroxide solution The following 3% calcium hydroxide solution was prepared.

[3% calcium hydroxide solution]
Calcium hydroxide {Wako Pure Chemical Industries, Ltd.} 30g
970 g of distilled water

(5) Preparation of tissue lysis solution, pretreatment solution and washing solution A tissue lysis solution, pretreatment solution, washing solution and recovery solution for DNA separation and purification from animal tissues of the following formulation were prepared.

[Tissue lysate (for separation and purification of DNA from tissue)]
1 mol / L Tris-HCL {Wako Pure Chemical Industries, Ltd.} 26g
Sodium chloride 3g
0.5 mol / L EDTA {Wako Pure Chemical Industries, Ltd.} 140 g
10% by mass SDS {manufactured by Wako Pure Chemical Industries, Ltd.} 160 g
350g of distilled water
pH 8.2

[Pretreatment solution (for separation and purification of DNA from tissues)]
Guanidine hydrochloride {manufactured by Wako Pure Chemical Industries, Ltd.} 380 g
Tween-20 (ICN) 120g
3 g of acetylene glycol (manufactured by Air Products)
Silicone oil {GE Toshiba Silicone Co., Ltd.} 0.6g
BiS-Tris 15.5g
275 g of distilled water
pH 6.0

[Washing solution (for separating and purifying DNA from tissue)]
1 mol / L Tris hydrochloric acid {Wako Pure Chemical Industries, Ltd.} 8g
Sodium chloride {Wako Pure Chemical Industries, Ltd.} 4.5g
Ethanol {Wako Pure Chemical Industries, Ltd.} 360g
350g of distilled water
pH 7.6

[Recovered solution (for separation and purification of DNA from tissues)]
1 mol / L Tris hydrochloric acid {manufactured by Wako Pure Chemical Industries, Ltd.} 2.5 g
250g distilled water
pH 9.0

(4) Alkaline treatment of animal tissue Bovine decalcified bone (Ocein) was placed in a nuclease-free and pyrogen-free 1.5 mL microtube {platinum tube; manufactured by BM Instruments Co., Ltd.}. The prepared 3% calcium hydroxide solution was added and stirred at 20 ° C. or lower for 20 days. Thereafter, it was washed with a large amount of water while stirring. The ossein that has been subjected to the alkali treatment is referred to as L-ossein.

(5) Dissolution treatment of animal tissue 250 mg of L-Ocein was placed in a nuclease-free and pyrogen-free 1.5 mL microtube {platinum tube; manufactured by BM Instruments Co., Ltd.}, and the tissue prepared in (3) above. 180 μL of the lysis solution and 20 μL of protease K 20 mg / mL (manufactured by SIGMA) solution of protease were stirred and incubated at 55 ° C. for 2 hours to dissolve the animal tissue. Undissolved residual tissue was precipitated by centrifugation, and the supernatant was placed in another nuclease-free and pyrogen-free 1.5 mL microtube {platinum tube; manufactured by BM Instruments Co., Ltd.}.

(6) Preparation of Nucleic Acid Adsorption Solution To each sample solution obtained in (5) above, 180 μL of the pretreatment solution prepared in (3) above was added, stirred, and incubated at 70 ° C. for 10 minutes. Subsequently, 240 μL of ethanol was added and stirred to obtain a solution (nucleic acid adsorption solution) for adsorbing nucleic acids to the solid phase.

(7) -1: Separation and purification operation of nucleic acid (Example)
Next, the nucleic acid adsorption solution obtained in the above (6) is injected into one opening of the nucleic acid separation and purification cartridge having the porous membrane solid phase prepared in the above (2), and then into the one opening. A pressure difference generator (tubing pump) is connected, the inside of the nucleic acid separation and purification cartridge is pressurized (80 kpa), and the injected solution for adsorbing nucleic acid is passed through the solid phase of the nucleic acid-adsorbing porous membrane. Thus, the nucleic acid in the solution for adsorbing nucleic acid was adsorbed to the solid phase of the porous membrane by bringing it into contact with the solid phase of the porous membrane and discharging from the other opening of the nucleic acid separation and purification cartridge. Subsequently, 750 μl of the cleaning solution prepared in (3) above is injected into one opening of the nucleic acid separation and purification cartridge, and a tubing pump is connected to the one opening, and the inside of the nucleic acid separation and purification cartridge is pressurized ( 80 kpa), the injected washing solution was passed through the solid phase of the porous membrane and discharged from the other openings, thereby washing the nucleic acid adsorbed on the porous membrane. This operation was performed three times. Subsequently, 200 μl of the recovery liquid prepared in (3) above is injected into one opening of the nucleic acid separation / purification cartridge, and a tubing pump is coupled to one opening of the nucleic acid separation / purification cartridge. The inside is pressurized (80 kpa), the injected recovery liquid is passed through the solid phase of the porous membrane, and discharged from other openings, so that the nucleic acid adsorbed on the porous membrane is desorbed and contains the nucleic acid. It recovered as a liquid (solution after purification). This nucleic acid separation and purification operation (from the time when the sample solution containing the nucleic acid was injected into the cartridge until it was collected) was performed at room temperature, and the time required for this operation was 12 minutes.

(7) -2: Separation and purification operation of nucleic acid (comparative example)
A solution containing nucleic acid (purified solution) was collected from 250 mg of ossein by the same method as in (5) to (7) -1.

(8) Quantification and electrophoresis of nucleic acid FIG. 1 shows the results of DNA agarose gel electrophoresis of the purified solution collected in the above Examples. Furthermore, the yield of DNA converted from the UV measurement of the solution after purification is shown in Table 1.

  As is apparent from the results of the electrophoresis in FIG. 1, the example using the method of the present invention can purify a large amount of DNA from L-ossein obtained by alkaline treatment of ossein. it can. Further, as is apparent from the results in Table 1, it can be seen that nucleic acids can be separated and purified from animal tissues very efficiently by using the method of the present invention.

  As described above, the method of the present invention makes it possible to efficiently recover DNA from tissues that were difficult to extract before.

<Example 2>
(1) Preparation of cartridge for separation and purification of nucleic acid and (2) Housing of porous membrane The cartridge was prepared in the same manner as in Example 1.

(3) Dissolution treatment of animal tissue-derived components The L-ossein obtained in Example 1 (4) was placed in a nuclease-free and pyrogen-free 1.5 mL microtube {platinum tube; manufactured by BM Instruments Co., Ltd.}. Then, distilled water was added and hydrothermal treatment was performed at 45 ° C. for 5 hours. After the treatment, the supernatant was collected and dried. To 250 mg of this dried extract, 180 μL of the tissue lysate prepared in Example 1 (3) and 20 μL of protease K20 mg / mL (manufactured by SIGMA) solution of proteolytic enzyme are added and stirred, and incubated at 55 ° C. for 2 hours. This dissolved the animal tissue. Undissolved residual tissue was precipitated by centrifugation, and the supernatant was placed in another nuclease-free and pyrogen-free 1.5 mL microtube {platinum tube; manufactured by BM Instruments Co., Ltd.}.

(4) Preparation of Nucleic Acid Adsorption Solution To each sample solution obtained in (2) above, 180 μL of the pretreatment solution prepared in Example 1 (3) was added and stirred, and incubated at 70 ° C. for 10 minutes. . Subsequently, 240 μL of ethanol was added and stirred to obtain a solution (nucleic acid adsorption solution) for adsorbing nucleic acids to the solid phase.

(5) -1: Separation and purification operation of nucleic acid (Example)
Next, the nucleic acid adsorption solution obtained in the above (4) is injected into one opening of the nucleic acid separation and purification cartridge having the porous membrane solid phase prepared in the above (2), and then into the one opening. A pressure difference generator (tubing pump) is connected, the inside of the nucleic acid separation and purification cartridge is pressurized (80 kpa), and the injected solution for adsorbing nucleic acid is passed through the solid phase of the nucleic acid-adsorbing porous membrane. Thus, the nucleic acid in the solution for adsorbing nucleic acid was adsorbed to the solid phase of the porous membrane by bringing it into contact with the solid phase of the porous membrane and discharging from the other opening of the nucleic acid separation and purification cartridge. Subsequently, 750 μl of the cleaning solution prepared in Example 1 (3) is injected into one opening of the nucleic acid separation and purification cartridge, and a tubing pump is connected to the one opening to pressurize the inside of the nucleic acid separation and purification cartridge. The nucleic acid adsorbed on the porous membrane was washed by passing the washing solution injected into the state (80 kpa) through the solid phase of the porous membrane and discharging it from other openings. This operation was performed three times. Subsequently, 200 μl of the recovery liquid prepared in (3) above is injected into one opening of the nucleic acid separation / purification cartridge, and a tubing pump is coupled to one opening of the nucleic acid separation / purification cartridge. The inside is pressurized (80 kpa), the injected recovery liquid is passed through the solid phase of the porous membrane, and discharged from other openings, so that the nucleic acid adsorbed on the porous membrane is desorbed and contains the nucleic acid. It recovered as a liquid (solution after purification). This nucleic acid separation and purification operation (from the time when the sample solution containing the nucleic acid was injected into the cartridge until it was collected) was performed at room temperature, and the time required for this operation was 12 minutes.

(5) -2: Separation and purification operation of nucleic acid (comparative example)
A solution containing nucleic acid (purified solution) was collected from 250 mg of ossein by the same method as in (1) to (5) -1.

(6) Nucleic Acid Electrophoresis FIG. 2 shows the results of DNA agarose gel electrophoresis of the purified solution collected in Example 2 above.

  As is clear from the results of the electrophoresis of FIG. 2 as a result of the above Example 2, the example using the method of the present invention purifies DNA from an extract obtained by treating ossein with an alkali treatment and a hot water treatment. be able to.

2 is a photograph obtained by subjecting a nucleic acid separated and purified according to the method of the present invention, a nucleic acid separated and purified as a comparative example, and a molecular weight marker to agarose gel electrophoresis. 2 is a photograph obtained by subjecting a nucleic acid separated and purified according to the method of the present invention, a nucleic acid separated and purified as a comparative example, and a molecular weight marker to agarose gel electrophoresis.

Explanation of symbols

1: Examples, L-Ocein 2: Comparative Example, Ossein 3: Examples, Gelatin M: 100 bp Ladder

Claims (33)

(1) a step of preparing a solution for adsorbing nucleic acid on a solid phase;
(2) A step of bringing the solution for adsorbing nucleic acid onto the solid phase into contact with the solid phase to adsorb the nucleic acid onto the solid phase;
(3) bringing the washing solution into contact with the solid phase and washing the solid phase with the nucleic acid adsorbed; and (4) bringing the recovery solution into contact with the solid phase to desorb the nucleic acid from the solid phase. Process,
In the method for separating and purifying nucleic acid having
The solution for adsorbing nucleic acid to the solid phase contains nucleic acid obtained by adding a tissue lysate after acid or alkali treatment of animal tissue, animal tissue containing hard protein or animal tissue-derived components A method for separating and purifying nucleic acid, which is a solution obtained by further adding a pretreatment liquid to a sample solution, wherein the solid phase is a solid phase composed of an organic polymer having a polysaccharide structure. (1) a step of preparing a solution for adsorbing nucleic acid on a solid phase;
(2) A step of bringing the solution for adsorbing nucleic acid onto the solid phase into contact with the solid phase to adsorb the nucleic acid onto the solid phase;
(3) bringing the washing solution into contact with the solid phase and washing the solid phase with the nucleic acid adsorbed; and (4) bringing the recovery solution into contact with the solid phase to desorb the nucleic acid from the solid phase. Process,
In the method for separating and purifying nucleic acid having
A solution for adsorbing nucleic acid to the solid phase is obtained by adding a pretreatment solution and a proteolytic enzyme to an animal tissue, an animal tissue containing hard protein, or an ingredient derived from animal tissue after acid or alkali treatment. A method for separating and purifying nucleic acid, wherein the solid phase is a solid phase comprising an organic polymer having a polysaccharide structure.   The method for separating and purifying nucleic acid according to claim 1 or 2, comprising an extraction step by hydrothermal treatment after the acid or alkali treatment.   The method for separating and purifying nucleic acid according to any one of claims 1 to 3, wherein the animal tissue containing the hard protein is selected from bone, cartilage, tooth, tendon, skin, nail and hair.   The tissue lysate is a solution containing at least one selected from a surfactant, a buffer, a nucleic acid stabilizer, an alkali metal halide, a chaotropic salt, a proteolytic enzyme, and an antifoaming agent. 3. The method for separating and purifying nucleic acid according to 3 or 4.   The nucleic acid separation according to any one of claims 1 to 5, wherein the pretreatment liquid is a solution containing at least one selected from an antifoaming agent, a nucleic acid stabilizer, a chaotropic salt, a surfactant, and a buffer. Purification method.   The method for separating and purifying nucleic acid according to claim 5 or 6, wherein the surfactant is a nonionic surfactant.   The method for separating and purifying nucleic acid according to claim 7, wherein the nonionic surfactant is a polyoxyethylene-based surfactant.   The method for separating and purifying nucleic acid according to claim 8, wherein the polyoxyethylene-based surfactant is a polyoxyethylene sorbitan-based surfactant.   The method for separating and purifying nucleic acid according to claim 5, wherein the buffer is BisTris (Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane).   The method for separating and purifying nucleic acid according to any one of claims 1 to 10, wherein the organic polymer having a polysaccharide structure is acetylcellulose.   The method for separating and purifying nucleic acid according to any one of claims 1 to 10, wherein the organic polymer having a polysaccharide structure is an organic polymer obtained by saponifying acetyl cellulose or a mixture of acetyl celluloses having different acetyl values.   The method for separating and purifying nucleic acid according to claim 12, wherein the saponification rate of the mixture of acetylcelluloses having different acetyl values after saponification is 5% or more.   The method for separating and purifying nucleic acid according to claim 12, wherein the saponification rate of the mixture of acetylcelluloses having different acetyl values after saponification is 10% or more.   The method for separating and purifying nucleic acid according to any one of claims 1 to 10, wherein the organic polymer having a polysaccharide structure is regenerated cellulose.   The method for separating and purifying nucleic acid according to claim 1, wherein the solid phase is a porous membrane.   The method for separating and purifying nucleic acid according to claim 16, wherein the porous membrane is a front and back asymmetric porous membrane.   The method according to claim 16 or 17, wherein the porous membrane is a porous membrane having an average pore diameter of 0.1 to 10.0 µm.   The method according to claim 16, 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 1 to 19, wherein a solution for adsorbing nucleic acid to a solid phase is obtained by further adding a water-soluble organic solvent.   The method for separating and purifying nucleic acid according to claim 20, wherein the water-soluble organic solvent contains at least one selected from methanol, ethanol, propanol, and butanol.   The method for separating and purifying nucleic acid according to any one of claims 1 to 21, further comprising a step of removing undissolved residual tissue components from a sample solution obtained by adding a tissue lysis solution to animal tissue.   The method for separating and purifying nucleic acid according to any one of claims 1 to 22, 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) 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 nucleic acid separation and purification method according to any one of claims 1 to 22, wherein the nucleic acid is adsorbed and desorbed using a nucleic acid separation and purification unit comprising:   The method for separating and purifying nucleic acid according to claim 24, wherein the pressure difference generator is a pressurizing device.   The method for separating and purifying nucleic acid according to claim 24, wherein the pressure difference generator is an apparatus for reducing pressure. 27. The method for separating and purifying nucleic acid according to claim 24, 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 24 or 25, comprising the following steps.
(1a) a step of dissolving a tissue by adding a tissue lysis solution to an animal tissue and treating the animal tissue to prepare a sample solution containing a nucleic acid;
(1b) adding a pretreatment solution to the sample solution containing the nucleic acid to prepare a solution for adsorbing the nucleic acid on the solid phase;
(2a) injecting a solution for adsorbing nucleic acid on the solid phase into one opening of a container having at least two openings for containing the solid phase;
(2b) A pressure difference generator is connected to the one opening to bring the inside of the container into a pressurized state, and a solution for adsorbing nucleic acid to the injected solid phase is discharged from the other opening, thereby Contacting the solid phase with the nucleic acid to adsorb the nucleic acid to the solid phase,
(3a) removing the pressure difference generator from the one opening and injecting a cleaning liquid into the one opening; (3b) connecting the pressure difference generator to the one opening to bring the inside of the container into a pressurized state; A step of cleaning the solid phase by contacting the cleaning liquid with the solid phase in the container by discharging the injected cleaning liquid from the other opening,
(4a) removing the pressure difference generator from the one opening and injecting the recovered liquid into the one opening; (4b) connecting the pressure difference generator to the one opening to bring the inside of the container into a pressurized state. A step of discharging the injected recovery liquid from the other opening, bringing the recovery liquid into contact with the solid phase in the container, desorbing the nucleic acid adsorbed on the solid phase, and discharging it out of the container. Before the step (4a) above,
(3c) a step of contacting the DNA-degrading enzyme solution with the solid phase and then washing the solid phase with a washing solution;
The method for separating and purifying nucleic acid according to claim 28, comprising:   The method for separating and purifying nucleic acid according to any one of claims 1 to 29, wherein the washing solution is a solution containing 20 to 100% by mass of at least one selected from methanol, ethanol, isopropanol, and n-propanol.   The method for separating and purifying nucleic acid according to any one of claims 1 to 30, wherein the recovered solution is a solution having a salt concentration of 0.5 mol / L or less.   32. Any one of claims 1-31, comprising: (i) a nucleic acid separation and purification unit; (ii) a tissue lysate or proteolytic enzyme; (iii) a pretreatment liquid; (iv) a washing liquid; A reagent kit for performing the method described in 1.   An apparatus for performing the method according to any of claims 1-32.