JP2006063031A - Method for separating and purifying nucleic acid and nucleic acid-adsorbing porous membrane - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating and purifying nucleic acids, which has a high yield, excellent separation performance, improved cleaning efficiency, simplicity, quickness, excellent automation and miniaturization fitness and uses a porous membrane that has substantially the same separation performance and can be produced in large amounts. <P>SOLUTION: In the method for separating and purifying nucleic acids comprising (1) a process for passing a nucleic acid-containing sample solution through a nucleic acid-adsorbing membrane to adsorb the nucleic acid in the porous membrane, (2) a process for passing a washing solution through the nucleic acid-adsorbing porous membrane to wash the porous membrane in an adsorbed state of nucleic acids and (3) a process for passing a recovering solution through the nucleic acid-adsorbing porous membrane to release nucleic acids from the porous membrane, the nucleic acid-adsorbing porous membrane is a porous membrane that comprises a polysaccharide ester derivative except acetyl cellulose and a saponified polysaccharide ester derivative and adsorbs nucleic acids by interaction in which ionic bond does not substantially participate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

  Conventional nucleic acid extraction methods include those using a centrifugal method, using magnetic beads, and using a filter. For example, a method for separating and purifying nucleic acid by adsorbing the nucleic acid to a solid phase such as silicon dioxide, silica polymer, magnesium silicate, and subsequent operations such as washing and desorption is known (for example, Patent Document 1). reference).

Japanese Patent Publication No. 7-51065

However, the conventional nucleic acid separation and purification method as described above is excellent in separation performance, but is still not sufficient in terms of yield. Moreover, it is difficult to industrially produce a large amount of adsorption media having the same performance, and further improvements have been demanded.
Accordingly, an object of the present invention is to provide a nucleic acid separation and purification method in which a nucleic acid in a specimen is adsorbed on a nucleic acid-adsorbing porous membrane and then desorbed through washing or the like. It is to provide a method capable of separation and purification with high yield.

  As a result of diligent studies to solve the above problems, the present inventors have found that in the method for separating and purifying nucleic acid including the process of adsorbing and desorbing nucleic acid to and from the porous membrane, the porous membrane may be a polysaccharide other than acetylcellulose. Nucleic acid can be separated and purified with high yield and high purity from a sample containing nucleic acid by using a porous membrane made of an ester derivative of saponified product or a saponified product thereof, which adsorbs nucleic acid through an interaction that does not involve ionic bonds. I found that I can do it. The present invention has been completed based on these findings. That is, the present invention is as follows.

1. (1) passing a sample solution containing nucleic acid through a nucleic acid-adsorptive porous membrane and adsorbing the nucleic acid in the nucleic acid-adsorptive porous membrane;
(2) passing the washing solution through the nucleic acid-adsorptive porous membrane and washing the nucleic acid-adsorptive porous membrane with the nucleic acid adsorbed thereon; and
(3) A method for separating and purifying nucleic acid, comprising a step of passing a recovered liquid through the nucleic acid-adsorbing porous membrane to desorb the nucleic acid from the nucleic acid-adsorbing porous membrane,
A method for separating and purifying a nucleic acid, wherein the material forming the nucleic acid-adsorptive porous membrane comprises an ester derivative of a polysaccharide other than acetylcellulose or a saponified product of an ester derivative of a polysaccharide other than acetylcellulose.

2. Polysaccharide ester derivatives other than acetylcellulose are monoester derivatives, diester derivatives, triester derivatives, mixtures of monoester derivatives and diester derivatives, mixtures of monoester derivatives and triester derivatives, diester derivatives and triester derivatives. Or a method for separating and purifying nucleic acid as described in 1 above, which is a mixture of a monoester derivative, a diester derivative and a triester derivative.
3. The nucleic acid according to 1 or 2 above, wherein the ester derivative of a polysaccharide other than acetylcellulose contains at least one of a carboxylic acid ester, a nitrate ester, a sulfate ester, a phosphate ester, and a pyrophosphate ester. Separation and purification method.
4). 4. The method for separating and purifying nucleic acid according to any one of 1 to 3, wherein the material forming the nucleic acid-adsorptive porous membrane is a saponified product of an ester derivative of a polysaccharide other than acetylcellulose.

5. 5. The method for separating and purifying nucleic acid according to any one of 1 to 4, wherein the material forming the nucleic acid-adsorptive porous membrane is a mixture of two or more polymer species.
6). 6. The method for separating and purifying nucleic acid according to any one of 1 to 5, wherein the material forming the nucleic acid-adsorbing porous membrane is a saponified product of a mixture of two or more polymer species.
7). 7. The method for separating and purifying nucleic acid according to any one of 1 to 6, wherein the saponification rate of the ester derivative of polysaccharide other than acetylcellulose is 5% or more.
8). 8. The material according to any one of 1 to 7 above, wherein the material forming the nucleic acid-adsorptive porous membrane is made of regenerated cellulose formed from a copper ammonia solution of cellulose, an alkali solution of cellulose, or a viscose solution. A method for separating and purifying nucleic acids.

9. 9. The method for separating and purifying nucleic acid according to any one of 1 to 8, wherein the nucleic acid-adsorbing porous membrane has a thickness in the range of 10 μm to 500 μm.
10. 10. The method for separating and purifying nucleic acid according to any one of 1 to 9, wherein the nucleic acid-adsorbing porous membrane has a minimum pore diameter of 0.22 μm or more.
11. 11. The method for separating and purifying nucleic acid according to any one of 1 to 10 above, wherein the nucleic acid-adsorptive porous membrane is front-back symmetrical.
12 11. The method for separating and purifying nucleic acid according to any one of 1 to 10 above, wherein the nucleic acid-adsorbing porous membrane is asymmetric on the front and back.

13. The method for separating and purifying nucleic acid according to any one of 1 to 12, wherein the nucleic acid-adsorbing porous membrane has a ratio of the maximum pore diameter to the minimum pore diameter of 1: 2 or more.
14 14. The method for separating and purifying nucleic acid according to any one of 1 to 13, wherein the nucleic acid-adsorbing porous membrane has a porosity of 50 to 95%.
15. The method for separating and purifying nucleic acid according to any one of 1 to 14 above, wherein the bubble point of the nucleic acid-adsorptive porous membrane is 0.1 to 10 kgf / cm ² .
16. The method for separating and purifying nucleic acid according to any one of 1 to 15, wherein the nucleic acid-adsorptive porous membrane has a pressure loss of 0.1 to 100 kPa.
17. The water-permeable amount when the nucleic acid-adsorptive porous membrane allows water to pass through at a pressure of 1 kg / cm ² at 25 ° C. is in the range of 1 to 5000 mL per minute per 1 cm ^{2 of} the membrane. The method for separating and purifying a nucleic acid according to any one of the above.
18. 18. The method for separating and purifying nucleic acid according to any one of 1 to 17, wherein the nucleic acid-adsorbing porous membrane has an adsorption amount of nucleic acid per mg of membrane of 0.1 μg or more.

19. A nucleic acid-adsorbing porous membrane comprising an ester derivative of a polysaccharide other than acetylcellulose or a saponified product of an ester derivative of a polysaccharide other than acetylcellulose.
20. Polysaccharide ester derivatives other than acetylcellulose are monoester derivatives, diester derivatives, triester derivatives, mixtures of monoester derivatives and diester derivatives, mixtures of monoester derivatives and triester derivatives, diester derivatives and triester derivatives. 20. The nucleic acid-adsorptive porous membrane as described in 19 above, which is a mixture of a monoester derivative, a diester derivative and a triester derivative.

  According to the present invention, a nucleic acid in a specimen is adsorbed on a nucleic acid-adsorbing porous membrane, and then desorbed through washing or the like, so that the nucleic acid can be efficiently separated and purified with high purity and high yield. it can. Furthermore, the method for separating and purifying nucleic acid of the present invention is excellent in separation performance, good washing efficiency, simple, rapid, and excellent in automation and downsizing. Moreover, the nucleic acid-adsorbing porous membrane of the present invention can produce a large amount of porous membranes having substantially the same separation performance.

First, the nucleic acid-adsorbing porous membrane used in the method for separating and purifying nucleic acid of the present invention will be described.
The nucleic acid-adsorbing porous membrane used in the present invention is formed from a saponified product of an ester derivative of a polysaccharide other than acetylcellulose or an ester derivative having a polysaccharide other than acetylcellulose.
The nucleic acid-adsorbing porous membrane formed from this ester derivative or a saponified product thereof is characterized in that it is a porous membrane that adsorbs nucleic acids by an interaction that does not substantially involve ionic bonds. This “interaction in which ionic bonds are not substantially involved” means that the ion is not “ionized” under the conditions of use on the porous membrane side. By changing the polarity of the environment, the nucleic acid and the porous membrane Is presumed to be an attractive action. As a result, the nucleic acid can be isolated and purified with excellent separation performance and good washing efficiency. The nucleic acid-adsorptive porous membrane preferably has a hydrophilic group, and it is presumed that the hydrophilic group of the nucleic acid and the porous membrane 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 the hydrophilic group, one having a moderate interaction with water is good (see Chemical Encyclopedia, Kyoritsu Shuppan Co., Ltd., “Hydrophilic group”, “Group that is not very hydrophilic”), Examples thereof include a hydroxyl group, a carboxyl group, a cyano group, an oxyethylene group, an amino group, and an amide group. Preferably it is a hydroxyl group.
Hereinafter, the material for forming the porous film will be described in detail.

[Ester derivatives of polysaccharides other than acetylcellulose or saponified products thereof]
Examples of the polysaccharide constituting the ester derivative or saponified product thereof include cellulose, hemicellulose, dextran, agarose, dextrin, amylose, amylopectin, starch, glycogen, pullulan, mannan, glucomannan, lichenan, isolikenan, laminaran, carrageenan, xylan, Examples include fructan, alginic acid, hyaluronic acid, chondroitin, chitin, and chitosan. The porous membrane in the present invention is preferably composed of any of the above-mentioned polysaccharide ester derivatives or saponified products thereof.

  Examples of the ester in the ester derivative include carboxylic acid ester, nitric acid ester, sulfuric acid ester, sulfonic acid ester, phosphoric acid ester, phosphonic acid ester, and pyrophosphoric acid ester, and the ester derivative is at least one selected from these esters. It is preferable to have. Further, saponified products of carboxylic acid esters, nitrate esters, sulfate esters, sulfonate esters, phosphate esters, phosphonate esters, and pyrophosphate esters can be further preferably used.

  The carboxylic acid ester is preferably selected from one or more of an alkyl carbonyl ester, an alkenyl carbonyl ester, an aromatic carbonyl ester, and an aromatic alkyl carbonyl ester. Further, alkyl carbonyl esters, alkenyl carbonyl esters, aromatic carbonyl esters, and saponified products of aromatic alkyl carbonyl esters can be further preferably used.

  As the alkylcarbonyl group of the alkylcarbonyl ester, any one of acetyl group, propionyl group, butyroyl group, barrel group, peptanoyl group, octanoyl group, decanoyl group, dodecanoyl group, tridecanoyl group, hexadecanoyl group, octadecanoyl group It is preferable to select from one or more. In addition, an ester group selected from any one or more of acetyl group, propionyl group, butyroyl group, barrel group, peptanoyl group, octanoyl group, decanoyl group, dodecanoyl group, tridecanoyl group, hexadecanoyl group and octadecanoyl group The saponified product of the alkyl carbonyl ester having it can also be preferably used.

  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, the alkenylcarbonyl group preferably has at least one selected from these alkenylcarbonyl groups. Further, a saponified product of alkenylcarbonyl ester can also be preferably used.

  The ester group of the aromatic carbonyl ester is preferably selected from at least one of a benzoyl group and a naphthaloyl group. Further, a saponified product of the aromatic carbonyl ester having an ester group selected from at least one of benzoyl group and naphthaloyl group can be further preferably used.

Examples of the nitrate ester include nitrocellulose, nitrohemicellulose, nitrodextran, nitroagarose, nitrodextrin, nitroamylose, nitroamylopectin, nitroglycogen, nitropullulan, nitromannan, nitroglucomannan, nitrolikenan, nitroisoliquenan, nitrolaminaran, Nitrocarrageenan, nitroxylan, nitrofructan, nitroalginic acid, nitrohyaluronic acid, nitrochondroitin, nitrochitin, nitrochitosan and the like can be preferably used.
Also, nitrocellulose, nitrohemicellulose, nitrodextran, nitroagarose, nitrodextrin, nitroamylose, nitroamylopectin, nitroglycogen, nitropullulan, nitromannan, nitroglucomannan, nitroligenanan, nitroisoliquenan, nitrolaminaran, nitrocarrageenan, nitro Saponified products such as xylan, nitrofructan, nitroalginic acid, nitrohyaluronic acid, nitrochondroitin, nitrochitin and nitrochitosan can also be used more preferably.

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

  The sulfonic acid ester is preferably selected from any one or more of alkyl sulfonic acid esters, alkenyl sulfonic acid esters, aromatic sulfonic acid esters, and aromatic alkyl sulfonic acid esters. Further, alkyl sulfonate esters, alkenyl sulfonate esters, aromatic sulfonate esters, and saponified products of aromatic alkyl sulfonate esters can be further preferably used.

  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 Rickenan phosphate, isolicenan phosphate, laminaran phosphate, carrageenan phosphate, xylan phosphate, fructan phosphate, alginate phosphate, hyaluronic acid phosphate, chondroitin phosphate, chitin phosphate, chitosan phosphate, and the like can be preferably used. Cellulose phosphate, hemicellulose phosphate, dextran phosphate, agarose phosphate, dextrin phosphate, amylose phosphate, amylopectin phosphate, glycogen phosphate, pullulan phosphate, mannan phosphate, glucomannan phosphate, lichenan phosphate, isolikenanline Saponified products such as 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 and chitosan phosphoric acid can be further preferably used.

  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. Preferred are acids, lichenan phosphonic acid, isollikenan phosphonic acid, laminaran phosphonic acid, carrageenan phosphonic acid, xylan phosphonic acid, fructan phosphonic acid, alginic acid phosphonic acid, hyaluronic acid phosphonic acid, chondroitin phosphonic acid, chitin phosphonic acid, chitosan phosphonic acid, etc. Can be used. In addition, cellulose phosphonic acid, hemicellulose phosphonic acid, dextran phosphonic acid, agarose phosphonic acid, dextrin phosphonic acid, amylose phosphonic acid, amylopectin phosphonic acid, glycogen phosphonic acid, pullulan phosphonic acid, mannan phosphonic acid, glucomannan phosphonic acid, lichenane phosphonic acid, More preferred are saponified products such as isorikenan 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. Can be used.

  Examples of the pyrophosphate esters 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. Can be preferably used. Cellulose pyrophosphate, hemicellulose pyrophosphate, dextran pyrophosphate, agarose pyrophosphate, dextrin pyrophosphate, amylose pyrophosphate, amylopectin pyrophosphate, glycogen pyrophosphate, pullulan pyrophosphate, mannan pyrophosphate, glucomannan pyrophosphate, lichenan pyrolin Saponification products such as acid, isorikenan pyrophosphate, laminaran pyrophosphate, carrageenan pyrophosphate, xylan pyrophosphate, fructan pyrophosphate, alginate pyrophosphate, hyaluronic acid pyrophosphate, chondroitin pyrophosphate, chitin pyrophosphate, chitosan pyrophosphate Furthermore, it can be preferably used.

The ester derivative of the polysaccharide may be a monoester derivative, a diester derivative, or a triester derivative, but is preferably a mixture of ester derivatives having different ester values.
As a mixture of ester derivatives having different ester values, a mixture of triester derivatives and diester derivatives, a mixture of triester derivatives and monoester derivatives, a mixture of triester derivatives and diester derivatives and monoester derivatives, or a diester derivative and A mixture with a monoester derivative is mentioned. Of these, a mixture of a triester derivative and a diester derivative is preferable. The mixing ratio (mass ratio) of the triester derivative and diester derivative is preferably 99: 1 to 1:99, and more preferably 90:10 to 50:50.

Moreover, it can use preferably also about what the hydroxyl group which polysaccharide has etherified with arbitrary substitution degrees. For example, when cellulose is used as the polysaccharide, the ether derivatives include methyl cellulose, ethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxyethyl-carbamoylethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, cyano. Ethyl cellulose, carbamoylethyl cellulose, and the like can be used, and preferably, hydroxymethyl cellulose and hydroxyethyl cellulose can be used, but are not limited to these including the type of polysaccharide and the type of ether.
Moreover, it can use preferably also about what the hydroxyl group of polysaccharide was halogenated by arbitrary substitution degrees.

  The case where a cellulose ester derivative is used as an ester derivative of a polysaccharide other than acetylcellulose will be described below as an example. Cellulose, the cellulose ester derivative raw material, includes cotton linter, wood pulp (hardwood pulp, conifer pulp), hemp, natural cellulose such as cellulose produced during the cultivation process of acetic acid bacteria, acid hydrolysis, mechanical crushing, and explosion For example, cellulose ester derivatives obtained from any raw material cellulose can be used, or they may be mixed and used in some cases. Details of these raw material celluloses are described, for example, in Plastic Materials Course (17) Fibrous resin (Maruzawa, Uda, Nikkan Kogyo Shimbun, 1970).

  According to it, the molecular weight of cellulose is wide, for example, natural cellulose is 600,000 to 1,500,000 (degree of polymerization approx. 3500 to 10,000), and purified linter is 80,000 to 500,000 (degree of polymerization approx. 500 to 3000). The wood pulp is 80,000 to 1,340,000 (the degree of polymerization is approximately 500 to 2100). Here, the molecular weight greatly affects the strength properties of cellulose or its derivatives. When the molecular weight decreases, the mechanical strength suddenly decreases from a certain degree of polymerization. However, as a raw material for the nucleic acid-adsorbing porous membrane of the present invention, Any molecular weight can be used without problems.

  It is desirable to use the above-mentioned raw material cellulose as it is as a raw material for cellulose ester derivatives, but to use purified linter and refined high-quality wood pulp by refining linter or pulp. Linter is a short fiber having a short fiber length among cotton fibers, has a high α-cellulose content (for example, 88 to 92% by mass), has a high purity, and has few impurities. This crude linter can be obtained by purifying litter, alkali cooking, bleaching, acid treatment, dehydration and drying. Details of these are described on pages 25 to 28 of the plastic material course (17) Fibrous resin (Maruzawa, Uda, Nikkan Kogyo Shimbun, published in 1970), and the characteristics are described in Tables 2.3. Yes. In the present invention, it is preferable to use linter purified by this method.

  The refined pulp is also described on pages 28 to 32 of the same book, and the characteristics are also described in Tables 2 and 4. In the present invention, pulp refined by such a method is also preferable as the cellulose ester derivative raw material. Here, refined cotton linter and pulp may be mixed and used. In that case, the mixing ratio is not particularly limited, but is preferably 5/95 to 95/5, and more preferably 10/90 to 90/10. By mixing, the solubility of the cellulose ester derivative can be improved during the production of the porous membrane, and the surface state and mechanical properties of the porous membrane can be improved.

  The α-cellulose content, which is an indicator of pulp purity, can be selected from a range of about 80 to 100% by mass. In wood pulp, it is usually about 85 to 98% by mass. In the present invention, low-purity pulp, for example, pulp having an α-cellulose content of about 80 to 96% by mass (particularly 92 to 96% by mass) can also be used. Of these pulps, usually wood pulp can be used.

The neutral constituent sugar component in pulp or cotton is mainly composed of glucose, but the nucleic acid-adsorbing porous membrane of the present invention may further contain mannose and xylose. The neutral constituent sugar component in cotton is described in JP-A-11-130301.
The ratio in the case of containing mannose and xylose is not particularly limited, but mannose / xylose (molar ratio) = 0.35 / 1 to 3.0 / 1, preferably 0.35 / 1 to 2.5 / 1, Preferably it is 0.35 / 1-2/1. Furthermore, in the cellulose triester derivative produced in that case, the total content of mannose and xylose is preferably 0.01 to 5 mol%, more preferably 0.1 to 4 mol%. “Mannose” and “xylose” are the main constituent sugars of hemicellulose (xylan, glucomannan, etc.) contained in pulp. Specifically, the constituent sugar components of these raw material pulp and the obtained cellulose triester derivative can be analyzed by the method described in JP-A-11-130301.

  In the nucleic acid-adsorptive porous membrane of the present invention, the viscosity average polymerization degree of the cellulose ester derivative is preferably 200 to 3000. Moreover, it is preferable that the ratio of the weight average molecular weight and the number average molecular weight of the cellulose ester derivative is 0.8-2. Furthermore, the cellulose ester derivative preferably contains an acid having an acid dissociation index of 1.93 to 4.5 or a salt thereof.

  The amount of residual acetic acid or the fatty acid having 3 to 22 carbon atoms in the cellulose ester derivative is preferably 0.5% by mass or less. Moreover, it is preferable that the cellulose ester derivative contains 1 ppb to 10000 ppm of at least one kind of alkali metal and / or alkaline earth metal. Further, the cellulose ester derivative is made of aluminum, bismuth, silicon, heavy metals (chromium, manganese, iron, cobalt, nickel, copper, zinc, arsenic, silver, cadmium, tin, antimony, gold, platinum, mercury, lead, etc.) It is preferable to contain at least one kind from 1 ppb to 1000 ppm.

X-ray analysis can be used as a means for evaluating the structure of cellulose. In the X-ray analysis image, the cellulose molecules are preferably arranged in parallel to the fiber axis direction, attracted by hydrogen bonds, and one unit cell is formed by five cellulose molecules. Further, according to the X-ray method, the crystallinity of natural cellulose is about 70%, and the crystallinity of cellulose used for the cellulose ester derivative raw material used in the present invention is:
It is preferable that it is 30 to 70%.

  Here, polysaccharides such as cellulose used also in the present invention have been variously analyzed. For example, for cellulose, ASTM standard Part 15, TAPPI Standard (Technical Association of the Pulp and Paper Industry) and JIS are used. P 8101 and the like. Measurement items include ash, calcium oxide and magnesium oxide content, α-cellulose, β-cellulose, copper value, and the like.

  The porous membrane is preferably prepared by dissolving a cellulose ester derivative in a solvent to prepare a dope solution and casting the dope solution. Such a production method is described in US Pat. No. 3,129,159.

As the porous membrane of cellulose, a porous membrane of regenerated cellulose can be preferably used. Regenerated cellulose is a cellulose ester derivative solid surface or whole celluloseated by saponification treatment, cellulose made from copper ammonia solution, cellulose viscose solution, cellulose alkaline solution Can be mentioned. Regenerated cellulose differs from the original cellulose obtained from the above-mentioned cotton linter, wood pulp, etc. in terms of crystal state and the like. Although cellulose has crystal forms of I, II, III, and IV (original cellulose is a crystal form of I), any crystal form can be preferably used in the present invention, and I, II, Each of the crystal forms III and IV may be contained in any ratio.
The regenerated cellulose porous membrane and the production method thereof will be described below, but the regenerated cellulose porous membrane that can be used in the present invention is not limited to the following.
As the regenerated cellulose porous membrane prepared from the acetylcellulose porous membrane, those obtained by the raw materials and methods described in JP-B No. 45-4633, JP-A No. 56-1000060, and the like can be used. .
Regarding the regenerated cellulose porous membrane prepared from a copper ammonia solution of cellulose, JP-A-58-89625, JP-A-58-89626, JP-A-58-89627, JP-A-58- No. 89628, JP-A-59-45333, JP-A-59-45334, JP-A-59-199728, JP-A-61-274707, JP-A-62-1403, Those obtained by the raw materials and methods described in JP-A-63-161972 and JP-A-7-330945 can be used.
Further, a regenerated cellulose porous membrane can be obtained from a viscose solution obtained by reacting an alkali and carbon disulfide with cellulose to devise the stock solution composition and coagulation method, and can be used in the present invention.
Further, a regenerated cellulose porous membrane prepared from an alkaline solution of cellulose can be produced by the raw materials and methods described in JP-A-62-240328, JP-A-62-240329, JP-A-1-188539, and the like. What is obtained can be used.

  Regarding the cellulose ester derivatives described above, JP-A-10-45803, JP-A-11-269304, JP-A-8-231761, JP-A-8-231761, JP-A-10-60170, Japanese Laid-Open Patent Publication Nos. 9-40792, 11-5851, 11-269304, 9-90101, 57-182737, 4-277530, and It is also preferable to use the cellulose ester derivatives described in JP-A-11-292989, JP-A-12-131524, JP-A-12-137115, and the like. These materials are not particularly limited to the nucleic acid-adsorbing porous membrane of the present invention.

  Saponification is performed to obtain a saponified product of an ester derivative of a polysaccharide other than acetylcellulose. Saponification refers to bringing an ester derivative into contact with a saponification solution (for example, an aqueous sodium hydroxide solution). Thereby, the ester site | part of the ester derivative which contacted the saponification process liquid is hydrolyzed, and a hydroxyl group is introduce | transduced. In order to change the saponification rate, the saponification treatment may be performed by changing the concentration of sodium hydroxide, the treatment temperature, and the treatment time. The saponification rate can be easily measured by NMR, IR, or XPS (for example, it can be determined by the degree of peak reduction of the carbonyl group).

It is preferable to saponify the surface of the porous membrane prepared by including the ester derivative. In this case, the amount (density) of hydroxyl groups on the solid surface can be controlled by the degree of saponification treatment (saponification rate). In order to increase the nucleic acid separation efficiency, it is preferable that the amount (density) of hydroxyl groups is large. For example, the saponification rate (surface saponification rate) of a porous membrane prepared containing an ester derivative is preferably 5% or more and 100% or less, and more preferably 10% or more and 100% or less. In order to increase the surface area of the organic polymer having a hydroxyl group, it is preferable to saponify the porous membrane of the cellulose triester derivative.
A porous membrane is preferable because it can be manufactured without distinguishing the front and back of the membrane when a porous membrane with front and back symmetry is used, and the risk of clogging can be reduced by using a porous membrane with asymmetrical front and back. It can be preferably used.

  In the method for separating and purifying nucleic acid of the present invention, a porous membrane made of the ester derivative or a saponified product thereof and a porous membrane made of an organic material or an inorganic material can be used in combination. The porous film that can be used in combination preferably has a hydrophilic group. For example, a porous film made of an organic material having a hydrophilic group, or a porous film in which a hydrophilic group is introduced by treating an organic material having no hydrophilic group An organic material having no hydrophilic group is coated with a material having a hydrophilic group to introduce a hydrophilic group, a porous film made of an inorganic material having a hydrophilic group, or an inorganic material having no hydrophilic group is treated. For example, a porous film in which a hydrophilic group is introduced, a porous film in which a hydrophilic group is introduced by coating an inorganic material having no hydrophilic group with a material having a hydrophilic group, and the like can be used. Among them, it is preferable to use an organic material such as an organic polymer because of the ease of processing of the material forming the porous film that can be used in combination.

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

  First, in a method of attaching a porous film made of an organic material and a graft polymer chain by chemical bonding, a polymer having a functional group that reacts with the porous film at the terminal or side chain of the polymer is used. And the functional group of the porous membrane can be chemically reacted with each other. The functional group that reacts with the porous membrane is not particularly limited as long as it can react with the functional group of the porous membrane. For example, a silane coupling group such as alkoxysilane, an isocyanate group, an amino group, and a hydroxyl group. Carboxyl group, carbonyl group, sulfonic acid group, phosphoric acid group, epoxy group, allyl group, methacryloyl group, acryloyl group, amide group, hydrazide group, aldehyde group, thiol group, succinimide group and the like.

  Particularly useful compounds as a polymer having a reactive functional group at the end of the polymer or in the side chain are a polymer having a trialkoxysilyl group at the polymer end, a polymer having an amino group at the polymer end, and a polymer having a carboxyl group at the polymer end. , A polymer having an epoxy group at the polymer end, and a polymer having an isocyanate group at the polymer end. The polymer used at this time is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption. Specifically, polyhydroxyethylacrylic acid, polyhydroxyethylmethacrylic acid and salts thereof , Polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, polylactic acid, polyketone, polyetherimide, polyamideimide, polyphenylenesulfone, polyphenylenesulfidesulfone, nylon, N-methyl modified nylon, N-alkoxymethyl modified nylon, N-alkylthiomethyl modified nylon, polysulfone, polyethersulfone, polyarylsulfone, polyallylamine, polyurethane, fibroin, polyamino acid, polypeptide, polyactyl Luamide, poly-N-isopropylacrylamide, nylon 46, nylon 66, nylon 610, nylon 612, nylon 6, nylon 7, nylon 11, nylon 12, N-methyl modified nylon, N-alkoxymethyl modified nylon, N-alkylthiomethyl Examples thereof include modified nylons such as modified nylon, polysaccharide ester derivatives and saponified products thereof, mixtures of polysaccharide ester derivatives having different ester values, and saponified products thereof.

A method of polymerizing a compound having a polymerizable double bond based on a porous membrane to form a graft polymer chain is generally called surface graft polymerization. The surface graft polymerization method is a method of polymerizing a compound having a polymerizable double bond disposed so as to be in contact with the porous film by giving active species on the surface of the substrate by a method such as plasma irradiation, light irradiation, and heating. It refers to the method of bonding with a porous membrane.
A compound useful for forming a graft polymer chain bound to a substrate has a double bond that can be polymerized and has a hydrophilic group that participates in nucleic acid adsorption. It is necessary to be. As these compounds, any polymer, oligomer, or monomer compound having a hydrophilic group can be used as long as it has a double bond in the molecule. Particularly useful compounds are monomers having hydrophilic groups.

  Specific examples of the particularly useful monomer having a hydrophilic group include the following monomers. For example, a hydroxyl group-containing monomer such as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and glycerol monomethacrylate can be particularly preferably used. In addition, carboxyl group-containing monomers such as acrylic acid and methacrylic acid, or alkali metal salts and amine salts thereof, acrylamide, and the like can also be preferably used.

  As another method for introducing a hydrophilic group into an organic material, a material having a hydrophilic group can be coated. The material used for the coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of easy work. Examples of the polymer include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, polylactic acid, polyketone and polyether. Imide, polyamideimide, polyphenylenesulfone, polyphenylenesulfidesulfone, nylon, N-methyl modified nylon, N-alkoxymethyl modified nylon, N-alkylthiomethyl modified nylon, polysulfone, polyethersulfone, polyarylsulfone, polyallylamine, polyurethane, fibroin , Polyamino acid, polypeptide, polyacrylamide, poly-N-isopropylacrylamide, nylon 46, nylon 66, nylon 610, Modified nylons such as Iron 612, nylon 6, nylon 7, nylon 11, nylon 12, N-methyl modified nylon, N-alkoxymethyl modified nylon, N-alkylthiomethyl modified nylon, ester derivatives of polysaccharides and saponified products thereof, Mention may be made, for example, of ester derivative mixtures of polysaccharides having different ester values and saponified products thereof.

  In addition, after coating a porous film of an organic material having no hydrophilic group with acetyl cellulose or a mixture of acetyl celluloses having different acetyl values, the coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values is saponified. You can also In this case, the saponification rate is preferably about 5% or more and 100% or less. Furthermore, the saponification rate is preferably about 10% or more and 100% or less.

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

  As another method for introducing a hydrophilic group into a porous film made of an inorganic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for the coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of easy work. Examples of the polymer include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, polylactic acid, polyketone and polyether. Imide, polyamideimide, polyphenylenesulfone, polyphenylenesulfidesulfone, nylon, N-methyl modified nylon, N-alkoxymethyl modified nylon, N-alkylthiomethyl modified nylon, polysulfone, polyethersulfone, polyarylsulfone, polyallylamine, polyurethane, fibroin , Polyamino acid, polypeptide, polyacrylamide, poly-N-isopropylacrylamide, nylon 46, nylon 66, nylon 610, Niro 612, nylon 6, nylon 7, nylon 11, nylon 12, N-methyl modified nylon, N-alkoxymethyl modified nylon, N-alkylthiomethyl modified nylon and other modified nylons, polysaccharide ester derivatives and their saponified products, ester values And a mixture of polysaccharide ester derivatives different from each other and saponified products thereof.

  As a porous film made of an inorganic material having no hydrophilic group, a porous film produced by processing a metal such as aluminum, ceramics such as glass, cement, ceramics, or new ceramics, silicon, activated carbon, aluminosilicate, etc. Can be mentioned.

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

[Additive]
The nucleic acid-adsorbing porous membrane used in the method for separating and purifying nucleic acid of the present invention includes various additives (for example, plasticizers, antistatic agents, anti-degradation agents) according to applications in order to improve various characteristics of the membrane. Agents, UV inhibitors, surfactants, release agents, colorants, reinforcing agents, cross-linking agents, and the like).
In addition, when the additive is added, for example, when a nucleic acid-adsorptive porous membrane is prepared by a solution casting method, it can be added at any time during the preparation step of the dope solution. Alternatively, a so-called last addition method of adding and mixing at the end of the preparation step, that is, immediately before casting the dope solution, may be employed. Moreover, it can also add to a film | membrane by impregnating after film forming.

(Plasticizer)
Examples of the plasticizer include phosphoric acid esters and carboxylic acid esters described in JP-A-2002-265636, polyhydric alcohols described in JP-A-2-6826, JP-A-5-194788, and JP-A-60-250053. JP-A-4-2277941, JP-A-6-16869, JP-A-5-271471, JP-A-7-286068, JP-A-5-5047, JP-A-11-80381, JP-A-7-20317. (Di) pentaerythritol esters described in JP-A-8-57879, JP-A-10-152568, JP-A-10-120824, JP-A-11-124445, glycerol esters described in JP-A-11-246704, Diglycerol esters described in JP-A No. 2000-63560 and quees described in JP-A No. 11-92574 Esters, substituted phenyl phosphates of JP-A 11-90946 Patent forth, plasticizer agents listed like in the JP-A No. 56-100604. The utilization method and characteristics of the plasticizer are described in each of these publications and can be applied to the present invention.

(Antistatic agent)
The antistatic agent can be added for the purpose of preventing the film from being charged when the film is handled. Examples of the antistatic agent include ionic conductive substances, conductive fine particles, and organic electronic conductive organic compounds.
The ion conductive substance is a substance containing ions serving as a carrier that shows electric conductivity and carries electricity. Specific examples include ionic polymer compounds. Examples of the ionic polymer compound include anionic polymer compounds such as those described in JP-B-49-23828, JP-B-49-23827, and JP-B-47-28937; A dissociation group in the main chain as shown in Japanese Patent Publication Nos. 50-54772, 59-14735, 57-18175, 57-18176, 57-56059, etc. Ionene type polymers having: JP-B 53-13223, JP-B 57-15376, JP-B 53-45231, JP-B 55-145783, JP-B 55-65950, JP-B 55-67746, JP-B Japanese Patent Nos. 57-11342, 57-19735, 58-56858, JP 61-27853, 62-9346 As seen in, cationic pendant polymer having a cationic dissociative group in the side chain; and the like can be given.

The conductive fine particle is a particulate conductive substance, and it is preferable that the particulate conductive substance is dispersed in the nucleic acid-adsorbing porous film by being finely dispersed and added. The conductive fine particles include conductive fine particles made of a metal oxide or a composite oxide thereof and a dispersible granular polymer (for example, ionene conductive polymer described in JP-A-9-203810, or fourth having intermolecular crosslinking. It is desirable to select from secondary ammonium cation conductive polymer particles. The preferred particle size is in the range of 5 nm to 10 μm, and the more preferred range depends on the type of conductive material used.
As an example of the metal oxide, ZnO, TiO ₂ , SnO ₂ , Al ₂ O ₃ , In ₂ O ₃ , SiO ₂ , MgO, BaO, MoO ₂ , V ₂ O ₅ , or a composite oxide thereof is preferable. In particular, ZnO, TiO ₂ and SnO ₂ are preferred. Examples of containing different atoms include, for example, addition of Al, In, etc. to ZnO, addition of Nb, Ta, etc. to TiO ₂ , and addition of Sb, Nb, halogen elements, etc. to SnO ₂ . Addition is effective. The addition amount of these different atoms is preferably in the range of 0.01 to 25 mol% in the conductive fine particles, but is particularly preferably in the range of 0.1 to 15 mol%.
Further, the volume resistivity of the conductive fine particles made of these metal oxides or complex oxides thereof is preferably 10 ⁷ Ωcm or less, and particularly preferably 10 ⁵ Ωcm or less.
The primary particle diameter of the conductive fine particles made of a metal oxide or a composite oxide thereof is preferably from 100 to 0.2 μm, and the long diameter of the higher order structure is preferably from 30 nm to 6 μm. The conductive fine particles having such a particle size are preferably contained in the nucleic acid-adsorptive porous membrane in a volume fraction of 0.01% to 20%.

A “dispersible granular polymer” is a polymer that appears as a clear or slightly turbid solution by visual observation but appears as a granular dispersion under an electron microscope. The volume resistivity of the dispersible granular polymer is preferably 10 ⁷ Ωcm or less, particularly preferably 10 ⁵ Ωcm or less. The dispersed granular polymer is preferably contained in the nucleic acid adsorbing porous membrane in a volume fraction of 0.01% to 20%.
As the dispersible granular polymer, cationic conductive polymer particles having intermolecular crosslinking are preferable. The feature of this cationic conductive polymer particle having intermolecular crosslinking is that it has high concentration and high density of the cationic component in the particle, so it not only has excellent conductivity, but also has low relative humidity. In the case of the film shape, the adhesion of the particles is good in the film-forming process after casting. It is also strong and has excellent chemical resistance. The cationic conductive polymer particles having intermolecular crosslinks are generally in the range of about 10 nm to 1000 nm, and preferably in the range of 20 nm to 300 nm.

Examples of the organic electronic conductive organic compound include polythiophene, polypyrrole, polyaniline, polyacetylene, polyphosphazene and the like. These are preferably used in a complex with polystyrene sulfonic acid, perchloric acid or the like as an acid donor.
The volume resistivity of the organic electronic conductive organic compound is preferably 10 ⁷ Ωcm or less, and particularly preferably 10 ⁵ Ωcm or less. The organic electronic conductive organic compound is preferably contained in the nucleic acid adsorbing porous membrane in a volume fraction of 0.01% to 20%.

(Deterioration inhibitor, UV protection agent)
Examples of the deterioration inhibitor include antioxidants, peroxide decomposers, radical inhibitors, metal deactivators, acid scavengers, and amines. As for the deterioration preventing agent and the ultraviolet ray preventing agent, JP-A-60-235852, JP-A-3-199201, JP-A-5-190707, JP-A-5-194789, JP-A-5-271471, JP-A-6-271471. 107854, JP-A-6-118233, JP-A-6-148430, JP-A-7-11056, JP-A-7-11055, JP-A-7-11056, JP-A-8-29619, JP-A-8-239509. JP-A-7-11056, JP-A-2000-204173, JP-A-5-97073, JP-A-5-194789, JP-A-6-107854, JP-A-60-235852, JP-A-12-193821 JP-A-8-29619, JP-A-6-118233, JP-A-6-148430, JP-A-2002-265636, JP-A-2002-265636 Like each publication of JP 5-197073 may be preferably used those described in. As a particularly preferable example of the deterioration preventing agent, dibutylhydroxytoluene (BHT) can be mentioned.

The addition amount of the deterioration inhibitor and the ultraviolet light inhibitor is preferably 0.01 to 1% by mass in the dope solution, and more preferably 0.01 to 0.08% by mass. If the addition amount is within the above range, the deterioration preventing agent or the ultraviolet ray preventing agent does not bleed out on the porous membrane surface, and the effect of the deterioration preventing agent or the ultraviolet ray preventing agent is recognized, which is preferable. In addition, it is also possible to use a deterioration inhibitor and an ultraviolet light inhibitor in combination.
It is preferable that the deterioration preventing agent or the ultraviolet ray preventing agent has a boiling point of 200 ° C. or higher and a liquid at 25 ° C., or a melting point of 25 to 250 ° C. and a solid at a standard temperature of 20 ° C. More preferably, it has a boiling point of 250 ° C. or higher and is liquid at 25 ° C., or a melting point of 25 to 200 ° C. and is solid at a standard temperature of 20 ° C. When the deterioration inhibitor or the ultraviolet light inhibitor is a liquid, purification is usually carried out by distillation under reduced pressure, but a higher vacuum is preferable, for example, 100 Pa or less is preferable. Moreover, it is also preferable to refine | purify using a molecular distillation apparatus etc. In the case where the deterioration preventing agent or the ultraviolet ray preventing agent is a solid, it is generally carried out by recrystallization using a solvent, filtering, washing and drying.

(Surfactant)
As for surfactants, JP-A No. 2002-265636, Japanese Patent Publication No. 55-31418, List of surfactants, etc. 2001 edition (Japan Surfactant Industry Association), Application of surfactants (Shoshobo, Kori Those described in Takao Yone, published on September 1, 1980) are preferably used, but are not limited thereto. In the present invention, the preferred surfactant is not particularly limited in its kind and amount of use, and may be any amount that provides the desired surface active characteristics.

(paint remover)
The release agent can be used for the purpose of reducing the load when peeling the porous membrane from the support during the production of the nucleic acid-adsorbing porous membrane. Surfactants are effective as the release agent, and the surfactants that can be used as the release agent are not particularly limited, such as phosphoric acid type, sulfonic acid type, carboxylic acid type, nonionic type, and cationic type. These are described in, for example, JP-A-61-243837 and JP-A-2000-99847.
Moreover, as a release agent, the acid dissociation index pKa 1.93 to 4.50 described in JP-A-10-316701 [preferably 2.0 to 4.4, more preferably 2.2 to 4.3 (for example, , 2.5 to 4.0), particularly 2.6 to 4.3 (for example, about 2.6 to 4.0)] or an acid thereof is preferable. These may be either inorganic acids or organic acids. For the pKa of acids, you can refer to “Revised 3rd edition, Chemical Handbook, Fundamentals II” (edited by Chemical Society of Japan, published by Maruzen Co., Ltd.).
Moreover, it can use preferably also about the peeling agent as described in Unexamined-Japanese-Patent No. 2002-265636.
The utilization method or the characteristics of the release agent described in each of the above documents can be similarly applied to the nucleic acid-adsorbing porous membrane according to the present invention.

(Coloring agent)
Colorants include known dyes, dyes, pigments, oxidative coloring dyes, reducing coloring dyes, pH indicators, fluorescent dyes, coupling dyes, ultraviolet absorbing dyes, infrared absorbing dyes, near infrared absorbing dyes, pressure sensitive dyes, and photochromic dyes. , Thermochromic dyes, electrochromic dyes, organic luminescent dyes, photoelectric conversion dyes, sensitizing dyes, dichroic dyes, electroluminescent dyes, food dyes, organic nonlinear optical dyes, chemiluminescent dyes, pharmaceutical dyes, medical diagnostics Dyes, cosmetic dyes, semiconductor laser dyes, dyes for sublimation transfer, dyes for melt transfer, heat sensitive dyes, leuco dyes, electromagnetic wave absorbing dyes, photoconductive dyes, chargeable dyes, etc. A single or a plurality of complex colorants can be used at a desired concentration, but is not limited thereto. Further, the colorant can be used in combination with a dispersant such as a surfactant or a protective polymer, and in that case, it can be used in a desired ratio.

(Reinforcing agent)
The reinforcing agent can be added for the purpose of improving the film strength. Reinforcing agents include glass fiber, carbon fiber, silicon fiber, cellulose fiber, pulp fiber, potassium titanate fiber, silicon carbide whisker, silicon nitride whisker, zinc oxide whisker, aluminum borate whisker, basic magnesium sulfate and fibrous zonotlite , Potassium titanate whisker, silicon carbide (SiC) whisker, whisker-like calcium carbonate and the like are preferably used, but are not limited to these, and any fiber or needle-like crystal may be used. Can be used. In addition, a synthetic polymer can be added to improve folding resistance, and polyurethanes described in JP-A No. 54-11081 can be preferably used, but are not limited thereto. Absent.

(Crosslinking agent)
As the crosslinking agent, known ones can be used, but it is preferable to select an appropriate type depending on the functional group of the nucleic acid-adsorbing porous membrane. When the functional group is a hydroxyl group, the crosslinking agents described in JP-A-7-256066, JP-A-3-68431 and the like can be preferably used, but are not limited thereto.

(Wetting agent)
As the wetting agent, those described in JP-A-63-262550, JP-A-63-262549, and JP-B-55-31418 can be preferably used, but are not limited thereto.

[Properties of nucleic acid-adsorbing porous membrane]
In the nucleic acid-adsorbing porous membrane according to the present invention, it is more preferable that the solution can pass through the inside, and the thickness is in the range of 10 μm to 500 μm. More preferably, the thickness is in the range of 50 μm to 250 μm. Since it is excellent in washing | cleaning efficiency by being in this range, it is preferable.

  The nucleic acid-adsorbing porous membrane preferably has a minimum pore diameter of 0.22 μm or more, and more preferably a minimum pore diameter of 0.5 μm or more. The nucleic acid-adsorbing porous membrane preferably has a ratio of the maximum pore diameter to the minimum pore diameter of 1: 2 or more. If it exists in this range, since sufficient surface area for nucleic acid adsorption | suction is obtained and it is hard to clog, it is preferable. More preferably, the ratio of the maximum pore diameter to the minimum pore diameter is 1: 5 or more.

The porosity of the nucleic acid-adsorptive porous membrane is preferably 50 to 95%, and more preferably 65 to 80%.
Moreover, it is preferable that the bubble point of a nucleic acid adsorptive porous membrane is 0.1-10 kgf / cm < ² >, More preferably, it is 0.2-4 kgf / cm < ² >.

  The pressure loss of the nucleic acid-adsorptive porous membrane is preferably a porous membrane having a pressure loss of 0.1 to 100 kPa. Thereby, a uniform pressure is obtained at the time of overpressure. More preferably, a porous film having a pressure loss of 0.5 to 50 kPa can be used. Here, the pressure loss is the minimum pressure required to pass water per 100 μm thickness of the membrane.

The nucleic acid-adsorptive porous membrane preferably has a water permeation amount of 1 to 5000 mL per minute per 1 cm ^{2 of} membrane when water is passed at 25 ° C. at a pressure of 1 kg / cm ² . More preferably, the water permeation amount when water is passed at 25 ° C. and a pressure of 1 kg / cm ² is 5 to 1000 mL per minute per 1 cm ^{2 of} membrane.

  As the nucleic acid-adsorbing porous membrane, it is preferable to use a porous membrane in which the amount of nucleic acid adsorbed per 1 mg of the porous membrane is 0.1 μg or more. More preferably, a porous membrane having a nucleic acid adsorption amount of 0.9 μg or more per 1 mg of the porous membrane can be used.

  The nucleic acid-adsorbing porous membrane is preferably a porous membrane made of a cellulose derivative that does not dissolve within 1 hour but dissolves within 48 hours when a square porous membrane having a side of 5 mm is immersed in 5 mL of trifluoroacetic acid. Can be used. Further, a porous membrane made of a cellulose derivative which dissolves within 1 hour when immersed in 5 mL of trifluoroacetic acid but is not dissolved within 24 hours when immersed in 5 mL of dichloromethane is preferable. Can be used.

[Nucleic acid separation and purification cartridge]
The nucleic acid-adsorbing porous membrane can be used by being housed in a container having at least two openings. Hereinafter, this container is referred to as a “nucleic acid separation and purification cartridge”.
The nucleic acid separation / purification cartridge may contain one or more nucleic acid-adsorbing porous membranes. In this case, the plurality of nucleic acid-adsorptive porous membranes may be the same or different.

The nucleic acid separation and purification cartridge can contain a combination of the nucleic acid-adsorbing porous membrane described above and a porous membrane made of another organic material or a porous membrane made of an inorganic material. As such a combination, for example, a combination of a regenerated cellulose and a porous membrane of a glass filter can be exemplified. The porous film made of an organic material or the porous film made of an inorganic material may be nucleic acid adsorbent or non-nucleic acid non-adsorbable. Examples of the nucleic acid non-adsorbing organic material include nylon and polysulfone.
The nucleic acid-adsorptive porous membrane described above can be formed in a form other than the membrane depending on the shape of the cartridge to be accommodated. For example, a chip shape or a block shape can be used.

  It is preferable that the nucleic acid separation and purification cartridge does not contain any other member other than containing the nucleic acid-adsorbing porous membrane in a container having at least two openings. As the material of the container, plastics such as polypropylene, polystyrene, polycarbonate, polyvinyl chloride and the like can be used. Biodegradable materials can also be preferably used. The container may be transparent or colored.

As the nucleic acid separation / purification cartridge, a nucleic acid separation / purification cartridge provided with means for identifying individual nucleic acid separation / purification cartridges can be used. Examples of means for identifying individual nucleic acid separation and purification cartridges include bar codes and magnetic tapes.
In addition, a nucleic acid separation and purification cartridge having a structure capable of easily taking out the nucleic acid-adsorbing porous membrane from a container having at least two openings can be used.

Next, each step of the nucleic acid separation and purification method will be described.
The nucleic acid separation and purification method of the present invention comprises:
(1) a step of passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid in the nucleic acid-adsorbing porous membrane (hereinafter also referred to as “adsorption step”),
(2) a step of passing a washing solution through the nucleic acid-adsorptive porous membrane and washing the nucleic acid-adsorptive porous membrane with the nucleic acid adsorbed thereon; Process "),
(3) A step of passing the recovery liquid through the nucleic acid-adsorptive porous membrane to desorb nucleic acids from the nucleic acid-adsorptive porous membrane (hereinafter also referred to as “recovery step”)
Is included at least.

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

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

When the nucleic acid separation / purification cartridge containing the nucleic acid-adsorbing porous membrane is used, the nucleic acid can be separated and purified by the following steps. That is,
(a) a step of injecting a sample solution containing a nucleic acid into one opening of a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane in a container having at least two openings;
(b) The nucleic acid separation and purification cartridge is pressurized using the pressure difference generator connected to the one opening of the nucleic acid separation and purification cartridge, and the sample solution containing the injected nucleic acid is converted into a nucleic acid-adsorbing porous membrane. The nucleic acid is adsorbed in the nucleic acid-adsorptive porous membrane by passing through and discharging from the other opening of the nucleic acid separation and purification cartridge,
(c) a step of injecting a washing liquid into the one opening of the cartridge for separating and purifying nucleic acid,
(d) Using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification cartridge, pressurize the inside of the nucleic acid separation and purification cartridge, pass the injected washing solution through the nucleic acid-adsorbing porous membrane, A step of washing the nucleic acid-adsorbing porous membrane with the nucleic acid adsorbed by discharging it from the opening of
(e) a step of injecting a recovery solution into the one opening of the cartridge for separating and purifying nucleic acid,
(f) The inside of the nucleic acid separation and purification cartridge is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification cartridge, and the injected recovery liquid is passed through the nucleic acid-adsorbing porous membrane. By discharging from other openings, a step of desorbing the nucleic acid from the inside of the nucleic acid-adsorbing porous membrane and discharging it out of the cartridge for nucleic acid separation and purification can be mentioned.

  In the method for separating and purifying nucleic acid of the present invention, the steps from injecting the sample solution containing the first nucleic acid to obtaining the nucleic acid outside the cartridge for separating and purifying nucleic acid are rapidly completed within 10 minutes, and within 2 minutes in a suitable situation. It is possible. In the nucleic acid fraction purification step, the nucleic acid can be obtained in a yield of 50% by mass or more with respect to the total amount contained in the sample, and in a suitable situation, the yield is 90% by mass or more.

  The nucleic acid separation and purification method of the present invention can recover nucleic acids having a molecular weight ranging from 1 kbp to 300 kbp, particularly from 20 kbp to 300 kbp. That is, long-chain nucleic acids can be recovered as compared with the conventional spin column method using a glass filter.

  In the method for separating and purifying nucleic acid of the present invention, the measurement value (260 nm / 280 nm) with an ultraviolet-visible spectrophotometer is 1.6 to 2.0 in the case of DNA, and 1.8 to 2.2 in the case of RNA. A nucleic acid having a high purity can be recovered, and a high purity nucleic acid with a small amount of impurities can be steadily obtained. Furthermore, a nucleic acid having a purity of about 1.8 when the measurement value (260 nm / 280 nm) with an ultraviolet-visible spectrophotometer is DNA and about 2.0 when RNA is RNA can be recovered.

Hereinafter, the (1) adsorption step, (2) washing step, and (3) recovery step in the method for separating and purifying nucleic acid of the present invention will be described in detail.
[Adsorption process]
In the method for separating and purifying nucleic acid of the present invention, as described above, in the adsorption step (1), a sample containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane.
When passing a sample solution containing nucleic acid through a nucleic acid-adsorptive porous membrane, passing the sample solution from one surface to the other surface allows the solution to contact the porous membrane uniformly. It is preferable. When a sample solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane, it is preferable that the sample solution is passed from the side having the larger pore size of the nucleic acid-adsorbing porous membrane to the smaller side because clogging is difficult. .

The flow rate when the sample solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane is ² to 1500 μL / sec per 1 cm ^{2 of} membrane area in order to obtain an appropriate contact time of the liquid with the porous membrane. It is preferable. If the contact time of the liquid with the porous membrane is too short, a sufficient separation and purification effect cannot be obtained, and if it is too long, it is not preferable from the viewpoint of operability. Furthermore, the flow rate is preferably 5 to 700 μL / sec per 1 cm ^{2 of} membrane area.
One or more nucleic acid-adsorptive porous membranes can be used. The plurality of nucleic acid-adsorbing porous membranes may be the same or different.

(Sample solution containing nucleic acid)
The specimen that can be used in the present invention is not particularly limited as long as it contains a nucleic acid. For example, in the diagnostic field, whole blood collected as a specimen, plasma, serum, urine, stool, semen, saliva and other body fluids, or This includes biological materials such as plants (or parts thereof), animals (or parts thereof), bacteria, viruses, etc., or lysates and homogenates thereof.

  The specimen containing the nucleic acid may be a specimen containing a single nucleic acid or a specimen containing a plurality of different types of nucleic acids. Further, the number of specimens may be one or plural. That is, a plurality of samples may be processed in parallel using a plurality of containers. The length of the nucleic acid to be recovered used in the nucleic acid separation and purification method is not particularly limited, and may be a nucleic acid having an arbitrary length of several bp to several Mbp, for example. From the viewpoint of handling, it is generally preferable that the length of the nucleic acid to be recovered is about several bp to several hundreds kbp. The nucleic acid separation and purification method can take out a relatively long nucleic acid quickly compared to a conventional nucleic acid separation and purification method, that is, a simple nucleic acid separation and purification method. Preferably, it can be used for recovering a nucleic acid of 20 to 300 kbp, more preferably 50 to 200 kbp, still more preferably 70 to 140 kbp. In the step of obtaining a sample solution containing a nucleic acid from a specimen, which will be described later, it is preferable to gently agitate and pipette from the viewpoint of recovering longer DNA or RNA. The type of nucleic acid to be collected is not particularly limited, such as DNA or RNA.

  The specimen is preferably dissolved in a cell membrane, a nuclear membrane, etc., and nucleic acid is dispersed in an aqueous solution to obtain a sample solution containing the nucleic acid.

  For example, when the target sample is whole blood, E. Removal of red blood cells Removal of various proteins, and C.I. It is preferable to lyse leukocytes and lysate the nuclear envelope. A. E. Removal of red blood cells and B. The removal of various proteins is performed in order to prevent non-specific adsorption to the membrane and clogging of the porous membrane. Leukocyte lysis and nuclear membrane lysis are preferably performed to solubilize the nucleic acid to be extracted. In particular, in the method for separating and purifying nucleic acid of the present invention, C.I. It is preferable to lyse leukocytes and lysate the nuclear envelope. In the method for separating and purifying nucleic acid of the present invention, when the sample is whole blood, it is preferable to solubilize the nucleic acid by performing these steps.

In the present invention, it is preferable to use a nucleic acid solubilizing reagent in order to solubilize the nucleic acid by dissolving the cell membrane and the nuclear membrane. More preferably, as a method of dissolving a cell membrane and a nuclear membrane, solubilizing a nucleic acid, and obtaining a sample solution containing the nucleic acid from a specimen,
(I) a step of injecting the specimen into the container;
(II) adding a nucleic acid solubilizing reagent solution to the container, mixing the specimen and the nucleic acid solubilizing reagent solution, and obtaining a mixed solution;
(III) incubating the mixed solution obtained above,
(IV) The method including the process of adding a water-soluble organic solvent to the liquid mixture after incubation can be mentioned.

  The sample is preferably homogenized before the step (I) or after the step (I) and before the step (II). Thereby, automation processing appropriateness can be improved. As the homogenization treatment, for example, ultrasonic treatment, use of sharp protrusions, use of high-speed stirring treatment, treatment of extruding from fine voids, treatment using glass beads, and the like can be performed.

  In the step (II), examples of the nucleic acid solubilizing reagent include a solution containing a compound selected from a proteolytic enzyme, a chaotropic salt, a surfactant, an antifoaming agent, and a nucleic acid stabilizer.

  In particular, the use of a nucleic acid solubilizing reagent containing a proteolytic enzyme is preferable because the amount and efficiency of recovery of nucleic acid can be improved, and the amount of specimen containing the necessary nucleic acid can be reduced and accelerated.

Examples of the proteolytic enzyme 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 nucleic acid solubilizing reagent solution is preferably 0.001 IU to 10 IU, more preferably 0.01 IU to 1 IU per 1 ml of the total volume at the time of addition.

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

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

As the chaotropic salt, a guanidine salt (for example, guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride), sodium isothiocyanate, sodium iodide, potassium iodide and the like can be used. Of these, guanidine salts are preferable, and guanidine hydrochloride is more preferable. These salts may be used alone or in combination. In addition, urea or the like can also be used as the chaotropic substance.
The concentration of the chaotropic salt in the nucleic acid solubilizing reagent solution is preferably 0.5M or more, more preferably 0.5M to 4M, and further preferably 1M to 3M.

Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. In the present invention, nonionic surfactants and cationic surfactants can be preferably used.
As the nonionic surfactant, a polyoxyethylene alkylphenyl ether surfactant, a polyoxyethylene alkyl ether surfactant, or a fatty acid alkanolamide can be used. Preferably, a polyoxyethylene alkyl ether surfactant is used. Can be used. Polyoxyethylene alkyl ether surfactants include POE decyl ether, POE lauryl ether, POE tridecyl ether, POE alkylene decyl ether, POE sorbitan monolaurate, POE sorbitan monooleate, POE sorbitan monostearate, tetraolein It is preferably selected from acid polyoxyethylene sorbit, POE alkylamine, and POE acetylene glycol. These surfactants may be used alone or in combination.

  As the cationic surfactant, cetyltrimethylammonium promide, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, cetylpyridinium chloride and the like are preferable. These surfactants may be used alone or in combination. The concentration of these surfactants in the nucleic acid solubilizing reagent solution is preferably 0.1 to 20% by mass.

Examples of antifoaming agents include silicone-based antifoaming agents (eg, silicone oil, dimethylpolysiloxane, silicone emulsion, modified polysiloxane, silicone compound, etc.), alcohol-based antifoaming agents (eg, acetylene glycol, heptanol, ethylexanol, Higher alcohols, polyoxyalkylene glycols, etc.), ether type antifoaming agents (for example, heptyl cellosolve, nonyl cellosolv-3-heptylcorbitol, etc.), fats and oils type antifoaming agents (for example, animal and vegetable oils), fatty acid type antifoaming agents (Eg, stearic acid, oleic acid, palmitic acid, etc.), metal soap antifoaming agents (eg, aluminum stearate, calcium stearate, etc.), fatty acid ester antifoaming agents (eg, natural wax, tributyl phosphate, etc.), phosphorus Phosphoric acid Tell-based antifoaming agent (for example, sodium octyl phosphate), amine-based antifoaming agent (for example, diamylamine), amide-based antifoaming agent (for example, stearamide), and other antifoaming agents (for example, sulfuric acid) Ferric iron, bauxite, etc.).
Especially, as an antifoamer, a silicon type antifoamer and an alcohol type antifoamer are preferable. Particularly preferably, the antifoaming agent is a combination of two components, a silicon antifoaming agent and an alcohol antifoaming agent. As the alcohol-based antifoaming agent, it is preferable to use an acetylene glycol-based surfactant.

  Examples of the nucleic acid stabilizer include those having an action of inactivating nuclease activity. Depending on the sample, a nuclease or the like that degrades the nucleic acid may be contained. When the nucleic acid is homogenized, the nuclease acts on the nucleic acid, and the yield may be drastically reduced. The nucleic acid stabilizer is preferable because the nucleic acid in the sample can be stably present, thereby improving the recovery amount and recovery efficiency of the nucleic acid, enabling the sample to be reduced in volume and speeding up.

As the nucleic acid stabilizer having an action of inactivating the nuclease activity, compounds generally used as a reducing agent can be used. Examples of the reducing agent include hydrogen, hydrogen iodide, hydrogen sulfide, lithium aluminum hydride, hydrogenated compounds such as sodium borohydride, alkali metals, metals with high electrical positiveity such as magnesium, calcium, aluminum, and zinc, or Amalgams, aldehydes, saccharides, organic acids such as formic acid and oxalic acid, mercapto compounds and the like. Of these, mercapto compounds are preferred. Examples of the mercapto compound include cysteine, 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 nucleic acid solubilizing reagent solution is preferably 0.1 to 20% by mass, and more preferably 0.3 to 15% by mass. When a mercapto compound is used as the nucleic acid stabilizer, the concentration in the pretreatment liquid is preferably 0.1 to 10% by mass, and more preferably 0.5 to 5% by mass.

When recovering nucleic acids other than RNA, such as DNA, it is preferable to add RNase to the nucleic acid solubilizing reagent solution. When an RNase is added to the nucleic acid solubilizing reagent solution, interference caused by RNA coexisting with the recovered nucleic acid can be reduced. It is also preferable to add a DNA degrading enzyme inhibitor to the nucleic acid solubilizing reagent solution.
On the other hand, when recovering nucleic acids other than DNA such as RNA, it is preferable to add a DNA degrading enzyme to the nucleic acid solubilizing reagent solution. In this case, interference by DNA coexisting with the recovered nucleic acid can be reduced. It is also preferable to add an RNase inhibitor. As the RNase inhibitor, those that specifically inhibit RNase are preferable.
The RNA degrading enzyme is not particularly limited, and for example, an RNA-specific degrading enzyme such as ribonuclease H (RNase H) can be preferably used.
The DNA degrading enzyme is not particularly limited, and for example, a DNA-specific degrading enzyme such as DNase I can be preferably used.
The nucleolytic enzyme and the nucleolytic enzyme inhibitor can be used at concentrations usually used. Moreover, it can be heated as usual. The heating treatment is preferably performed simultaneously with the treatment with the proteolytic enzyme. This heating process will also be described in the section “Incubation” described later.

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

(Mixed with sample)
In the step (II), the method for mixing the specimen and the nucleic acid solubilizing reagent solution is not particularly limited. When mixing, it is preferable to mix at 30 to 3000 rpm for 1 second to 3 minutes with a stirrer. Thereby, the yield of nucleic acid to be separated and purified can be increased. Or it is also preferable to mix by inversion mixing 5 to 30 times. In addition, mixing can also be performed by performing pipetting operations 10 to 50 times. In this case, the yield of nucleic acid separated and purified by a simple operation can be increased.

(Incubation)
In the case of using the nucleic acid solubilizing reagent solution containing the proteolytic enzyme in the step (III) above, by incubating the mixed solution of the sample and the nucleic acid solubilizing reagent solution at the optimal temperature and reaction time of the proteolytic enzyme. The yield of the nucleic acid to be separated and purified can be increased. The incubation temperature is usually 20 ° C. to 70 ° C., preferably the optimum temperature for the proteolytic enzyme, and the incubation time is usually 1 minute to 18 hours, preferably the optimum reaction time for the proteolytic enzyme. The incubation method is not particularly limited, and can be performed by putting it in a hot water bath or a warmer.

(Water-soluble organic solvent)
In the step (IV), examples of the water-soluble organic solvent added to the mixed solution after incubation include alcohols, acetone, acetonitrile, dimethylformamide and the like. In particular, alcohol can be preferably used.
The alcohol may be any of primary alcohol, secondary alcohol, and tertiary alcohol. Among these, methanol, ethanol, propanol and isomers thereof, or butanol and isomers thereof can be preferably used. More preferably, ethanol can be used. These water-soluble organic solvents may be used alone or in combination.
The water-soluble organic solvent may be added to the nucleic acid solubilizing reagent solution, and the concentration in the nucleic acid solubilizing reagent solution is preferably 1 to 20% by mass. Moreover, it is preferable that the final concentration in the sample solution containing the nucleic acid of the water-soluble organic solvent is 5 to 90% by mass.

The pH of the obtained sample solution is preferably pH 5 to 10, more preferably pH 6 to 9, and further preferably pH 7 to 8.
Further, the surface tension of the obtained sample solution is preferably 0.05 J / m ² or less, the viscosity is preferably 1 to 10,000 mPa, and the specific gravity is 0.8 to 1.2. preferable.

[Washing process]
Hereinafter, the cleaning step (2) will be described. By performing the washing, the amount and purity of the nucleic acid recovered can be improved, and the amount of the specimen containing the necessary nucleic acid can be made very small. The cleaning step may be completed by one cleaning for speeding up, and when the purity is more important, the cleaning is preferably repeated a plurality of times.

In the washing step, the washing solution is supplied to the nucleic acid separation / purification cartridge containing the nucleic acid-adsorbing porous membrane using an automatic injection device or a supply means having the same function as these. The supplied cleaning solution is supplied from one opening of the nucleic acid separation and purification cartridge (opening into which the sample solution containing nucleic acid is injected), and the inside of the nucleic acid separation and purification cartridge is pressurized using a pressure difference generator coupled to the opening. It can be passed through the nucleic acid-adsorbing porous membrane in a state and discharged from an opening different from the one opening.
Also, the cleaning liquid can be supplied from one opening and discharged from the same opening. Furthermore, it is also possible to supply and discharge the cleaning liquid from an opening different from the one opening to which the sample solution containing the nucleic acid in the cartridge for nucleic acid separation and purification is supplied. However, a method of supplying from one opening of the nucleic acid separation / purification cartridge, passing through the nucleic acid-adsorptive porous membrane, and discharging from a different opening from the one opening is more preferable because of excellent cleaning efficiency.
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. The upper limit of the amount of the cleaning liquid is preferably 200 μl / mm ² or less, and within this range, operability can be maintained and sample outflow can be suppressed.

In the washing step, the flow rate when the washing solution is passed through the nucleic acid-adsorbing porous membrane is preferably 2 to 1500 μL / sec, and preferably 5 to 700 μL / sec, per unit area (cm ² ) of the membrane. More preferred. If the passage speed is lowered and time is taken, the washing is sufficiently performed. However, the nucleic acid separation and purification operation can be speeded up by setting the flow rate within the above range.

  In the washing step, the temperature of the washing solution is preferably 4 to 70 ° C. Furthermore, it is more preferable that the temperature of the cleaning liquid is room temperature. In the washing step, the nucleic acid separation and purification cartridge can be washed while applying mechanical vibration or ultrasonic stirring. Moreover, you may wash | clean by centrifuging.

  In the washing step, the washing solution generally does not contain an enzyme such as a nucleolytic enzyme, but can contain an enzyme that degrades contaminants such as proteins. In some cases, a DNA degrading enzyme, an RNA degrading enzyme, or the like can be included. By using a washing solution containing a DNA degrading enzyme, only RNA in the sample can be selectively recovered. Conversely, by using a washing solution containing an RNase, only DNA in the sample can be selectively recovered.

  In the washing step, the washing liquid is preferably a solution containing a water-soluble organic solvent and / or a water-soluble salt. The washing liquid needs to have a function of washing out impurities in the sample solution adsorbed together with the nucleic acid on the nucleic acid adsorbing porous membrane. For that purpose, it is necessary to have a composition that does not desorb nucleic acid from the nucleic acid-adsorbing porous membrane but desorbs impurities. For this purpose, for example, a water-soluble organic solvent such as alcohol is suitable for desorbing components other than the nucleic acid while retaining the nucleic acid because the nucleic acid is hardly soluble. In addition, by adding a water-soluble salt, the effect of adsorbing nucleic acids is enhanced, so that the effect of selectively removing impurities and unnecessary components is improved.

  As the water-soluble organic solvent that can be used for the cleaning liquid, alcohol, acetone, or the like can be used, and alcohol is preferable. Examples of the alcohol include methanol, ethanol, isopropanol, n-isopropanol, butanol and the like, and it is preferable to use ethanol among them. The amount of the water-soluble organic solvent contained in the cleaning liquid is preferably 20 to 100% by mass, and more preferably 40 to 80% by mass.

  On the other hand, the water-soluble salt contained in the cleaning liquid is preferably a halide salt, and chloride is particularly preferable. The water-soluble salt is preferably a monovalent or divalent cation, particularly preferably an alkali metal salt or an alkaline earth metal salt. Among them, a sodium salt and a potassium salt are preferable, and a sodium salt is most preferable. When the water-soluble salt is contained in the cleaning liquid, the concentration is preferably 10 mM / L or more, and the upper limit is not particularly limited as long as the solubility of impurities is not impaired, but it is 1 M / L or less. Is preferable, and it is more preferable that it is 0.1 M / L or less. In particular, the water-soluble salt is preferably sodium chloride, and more preferably contains 20 mM / L or more of sodium chloride.

  It is preferable that the cleaning liquid does not contain a chaotropic substance. Thereby, the possibility that the chaotropic substance is mixed in the recovery process subsequent to the cleaning process can be reduced. When a chaotropic substance is mixed during the recovery step, an enzyme reaction such as a PCR reaction is often inhibited. Therefore, it is ideal that the washing liquid does not contain a chaotropic substance in consideration of the subsequent enzyme reaction or the like. In addition, since chaotropic substances are corrosive and harmful, it is extremely advantageous for the experimenter from the viewpoint of safety of the test operation that the chaotropic substances need not be used. Here, the chaotropic substance is the above-mentioned urea, guanidine salt, sodium isothiocyanate, sodium iodide, potassium iodide or the like.

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

Therefore, the surface tension of the cleaning liquid should be less than 0.035 J / m ^{2 in} order to prevent the cleaning liquid in the cleaning process from being mixed into the recovered liquid in the next process and to keep the cleaning liquid remaining in the cartridge to a minimum. Is preferred. If the surface tension is lowered, the wettability between the cleaning liquid and the cartridge is improved, and the remaining liquid volume can be suppressed.

Conversely, for the purpose of reducing the remaining of the cleaning liquid in the cartridge in the cleaning process, the surface tension of the cleaning liquid is set to 0.035 J / m ² or more to increase the water repellency of the cartridge to form droplets. The remaining liquid amount can be suppressed by flowing down. Either surface tension is selected depending on the combination of the porous membrane adsorbing nucleic acid, the recovery liquid, the cleaning liquid, and the like.

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

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

[Recovery process]
The process of desorbing and recovering nucleic acid from the nucleic acid adsorbing polyporous membrane is shown below. In the recovery step, the recovery solution is supplied to the nucleic acid separation / purification cartridge equipped with the nucleic acid-adsorbing porous membrane using an automatic injection device or a supply means having the same function. The recovery liquid is supplied from one opening of the nucleic acid separation and purification cartridge (opening into which the sample solution containing nucleic acid is injected), and the inside of the nucleic acid separation and purification cartridge is pressurized using a pressure difference generator coupled to the opening. Thus, the nucleic acid-adsorbing porous membrane can be passed through and discharged from an opening different from the one opening. Further, the recovery liquid can be supplied from one opening and discharged from the same opening. Furthermore, it is also possible to supply and discharge the recovery liquid from an opening different from the one opening to which the sample solution containing the nucleic acid in the nucleic acid separation and purification cartridge is supplied. However, a method of supplying from one opening of the nucleic acid separation and purification cartridge, passing through the nucleic acid-adsorptive porous membrane, and discharging from an opening different from the one opening is excellent in recovery efficiency and more preferable.

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

  As the recovered liquid, preferably, purified distilled water, Tris / EDTA buffer or the like can be used.

In the recovery step, the nucleic acid recovery solution can be made into a composition that can be used in the subsequent subsequent steps. The separated and purified nucleic acid is often amplified by a PCR (polymerase chain reaction) method. In this case, the separated and purified nucleic acid solution needs to be diluted with a buffer solution suitable for the PCR method. In the recovery step according to this method, a buffer solution suitable for the PCR method is used as the recovery solution, so that the subsequent PCR step can be easily and rapidly transferred. In this case, a buffer solution used in the PCR reaction (for example, an aqueous solution having a final concentration of KCl 50 mmol / L, Tris-Cl 10 mmol / L, MgCl ₂ 1.5 mmol / L) can also be used as the recovery solution.

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

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

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

  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 nucleolytic enzyme inhibitor include EDTA. As another embodiment, a stabilizer may be added to the collection container in advance.

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

  The step of separating and purifying nucleic acid from a sample containing nucleic acid using the nucleic acid separation and purification cartridge containing the nucleic acid-adsorbing porous membrane and the pressure generator can be performed using an automatic device that automatically performs the step. . The use of an automatic apparatus not only simplifies and speeds up the operation, but also makes it possible to obtain a certain level of nucleic acid regardless of the skill of the operator.

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

The automatic apparatus includes a loading mechanism that holds the nucleic acid separation and purification cartridge, a waste liquid container that contains the sample liquid and the discharge liquid of the cleaning liquid, and a collection container that contains the collection liquid containing the nucleic acid, and pressurizes the nucleic acid separation and purification cartridge. A pressurized air supply mechanism that introduces air and a dispensing mechanism that dispenses a cleaning solution and a recovery solution into the nucleic acid separation and purification cartridge are provided.
This automatic apparatus uses a nucleic acid separation / purification cartridge containing a nucleic acid-adsorbing porous membrane through which a solution can pass, and injects and pressurizes a sample liquid containing nucleic acid into the nucleic acid separation / purification cartridge. After adsorbing the nucleic acid in the nucleic acid-adsorbing porous membrane, dispensing the washing liquid to the nucleic acid separation and purification cartridge and pressurizing to remove impurities, and then dispensing the recovered liquid to the nucleic acid separation and purification cartridge This is a nucleic acid separation and purification apparatus that automatically performs a separation and purification operation in which nucleic acid adsorbed on a nucleic acid-adsorbing porous membrane is desorbed and collected together with a collected liquid.

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

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

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

  According to the above automatic apparatus, the nucleic acid separation and purification cartridge, the mounting mechanism that holds the waste liquid container and the recovery container, the pressurized air supply mechanism that introduces pressurized air into the nucleic acid separation and purification cartridge, and the nucleic acid separation and purification cartridge It is equipped with a dispensing mechanism that dispenses the cleaning solution and the recovery solution, and injects and pressurizes the sample solution containing the nucleic acid into the nucleic acid separation and purification cartridge equipped with the nucleic acid-adsorbing porous membrane member to adsorb the nucleic acid to the nucleic acid-adsorbing porous membrane member After that, after washing and dispensing impurities, the recovery solution is dispensed to separate and recover the nucleic acid adsorbed on the nucleic acid-adsorptive porous membrane member automatically. Thus, a mechanism capable of automatically separating and purifying nucleic acid in a sample solution efficiently in a short time can be configured in a compact manner.

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

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

  In addition, the dispensing mechanism includes a washing liquid dispensing nozzle, a recovery liquid dispensing nozzle, a nozzle moving table that can move in sequence on the nucleic acid separation and purification cartridge, and a washing liquid drawn from the washing liquid bottle and supplied to the washing liquid dispensing nozzle. When 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 configured, the cleaning liquid and the recovery liquid can be sequentially dispensed with a simple mechanism. .

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
[Example 1]
(1) Production of nucleic acid purification cartridge A container for a nucleic acid separation and purification cartridge having an inner diameter of 7 mm and having a portion for accommodating a nucleic acid adsorbing porous membrane is produced from high impact polystyrene. A nucleic acid-adsorbing porous membrane obtained by saponifying a porous membrane (film thickness = 70 μm, average pore size = 1.2 μm) of cellulose nitrate (cellulose nitrate ester) with 1.0 N NaOH for 2 hours, The cartridge for nucleic acid separation and purification is housed in a portion for housing the nucleic acid-adsorbing porous membrane of the container for nucleic acid separation and purification cartridge to produce a nucleic acid separation and purification cartridge.

(2) Preparation of Nucleic Acid Solubilizing Reagent and Washing Solution A nucleic acid solubilizing reagent solution and a washing solution having the formulation shown in Table 1 are prepared.

(3) DNA separation and purification operation To 200 μl of human blood sample, 200 μl of the nucleic acid solubilizing reagent prepared in (2) above and 20 μl of protease (“Protease” Type XXIV Bacterial, manufactured by SIGMA) are added, and the temperature is 60 ° C. Incubate for 10 minutes. After incubation, 200 μl of ethanol is added and stirred to prepare a sample solution containing nucleic acid. The sample solution containing the nucleic acid is injected into one opening of the nucleic acid separation and purification cartridge provided with the nucleic acid-adsorbing porous membrane prepared in (1) above. Subsequently, a pressure generator is coupled to the opening, the inside of the cartridge for separating and purifying nucleic acid is pressurized, and the sample solution containing the injected nucleic acid is passed through the porous nucleic acid adsorbing porous membrane, thereby It is brought into contact with the porous membrane and discharged from the other opening of the nucleic acid separation and purification cartridge. Subsequently, the cleaning liquid prepared in (2) is injected into the opening into which the sample solution containing nucleic acid is injected, and the pressure generator is connected to this opening to bring the inside of the cartridge for nucleic acid separation and purification into a pressurized state. Then, it is passed through the nucleic acid-adsorbing porous membrane and discharged from the other opening. Repeat this operation three times. Subsequently, the recovery liquid is injected into the opening into which the sample solution containing the nucleic acid is injected, and a pressure generator is connected to this opening, the inside of the nucleic acid separation / purification cartridge is pressurized, and the injected recovery liquid is added to the nucleic acid adsorption. It passes through a porous porous membrane, is discharged from the other opening, and this liquid is recovered.

(4) Confirmation of separation and purification of nucleic acid Agarose gel electrophoresis was performed using the collected liquid.

[Comparative Example 1]
The operation is performed under the same conditions as in Example 1 except that a glass filter GA100 manufactured by ADVANTEC is used as the nucleic acid-adsorbing porous membrane.

  The results of the agarose gel electrophoresis of Example 1 and Comparative Example 1 are shown in FIG. As can be seen from the results of FIG. 1, the nucleic acid-adsorbing porous membrane made of a saponified cellulose nitrate ester of the present invention can efficiently and efficiently produce nucleic acid in comparison with a nucleic acid-adsorbing porous membrane made of a conventional glass filter. It was confirmed that it could be separated and purified.

2 shows the results of electrophoresis of nucleic acids separated and purified from a sample solution containing nucleic acids according to the practice of the present invention.

Claims (20)

(1) passing a sample solution containing nucleic acid through a nucleic acid-adsorptive porous membrane and adsorbing the nucleic acid in the nucleic acid-adsorptive porous membrane;
(2) passing the washing solution through the nucleic acid-adsorptive porous membrane and washing the nucleic acid-adsorptive porous membrane with the nucleic acid adsorbed thereon; and
(3) A method for separating and purifying nucleic acid, comprising a step of passing a recovered liquid through the nucleic acid-adsorbing porous membrane to desorb the nucleic acid from the nucleic acid-adsorbing porous membrane,
A method for separating and purifying a nucleic acid, wherein the material forming the nucleic acid-adsorptive porous membrane comprises an ester derivative of a polysaccharide other than acetylcellulose or a saponified product of an ester derivative of a polysaccharide other than acetylcellulose.   Polysaccharide ester derivatives other than acetylcellulose are monoester derivatives, diester derivatives, triester derivatives, mixtures of monoester derivatives and diester derivatives, mixtures of monoester derivatives and triester derivatives, diester derivatives and triester derivatives. Or a method for separating and purifying nucleic acid according to claim 1, wherein the mixture is a mixture of a monoester derivative, a diester derivative and a triester derivative.   3. The nucleic acid according to claim 1 or 2, wherein the ester derivative of a polysaccharide other than acetylcellulose contains at least one of carboxylic acid ester, nitrate ester, sulfate ester, phosphate ester, and pyrophosphate ester. Separation and purification method.   The method for separating and purifying nucleic acid according to any one of claims 1 to 3, wherein the material forming the nucleic acid-adsorptive porous membrane is a saponified product of an ester derivative of a polysaccharide other than the acetylcellulose.   The method for separating and purifying nucleic acid according to any one of claims 1 to 4, wherein the material forming the nucleic acid-adsorbing porous membrane is a mixture of two or more polymer species.   The method for separating and purifying nucleic acid according to any one of claims 1 to 5, wherein the material forming the nucleic acid-adsorbing porous membrane is a saponified product of a mixture of two or more polymer species.   The method for separating and purifying nucleic acid according to any one of claims 1 to 6, wherein a saponification rate of an ester derivative of a polysaccharide other than acetylcellulose is 5% or more.   The material for forming the nucleic acid-adsorptive porous membrane is made of regenerated cellulose formed from a copper ammonia solution of cellulose, an alkali solution of cellulose, or a viscose solution. A method for separating and purifying nucleic acids of   The method for separating and purifying nucleic acid according to any one of claims 1 to 8, wherein the nucleic acid-adsorbing porous membrane has a thickness in the range of 10 µm to 500 µm.   The method for separating and purifying nucleic acid according to any one of claims 1 to 9, wherein the nucleic acid-adsorbing porous membrane has a minimum pore diameter of 0.22 µm or more.   The method for separating and purifying nucleic acid according to any one of claims 1 to 10, wherein the nucleic acid-adsorbing porous membrane is front-back symmetrical.   The method for separating and purifying nucleic acid according to any one of claims 1 to 10, wherein the nucleic acid-adsorbing porous membrane is asymmetric on the front and back sides.   The method for separating and purifying nucleic acid according to any one of claims 1 to 12, wherein the nucleic acid-adsorbing porous membrane has a ratio of the maximum pore diameter to the minimum pore diameter of 1: 2 or more.   The method for separating and purifying nucleic acid according to any one of claims 1 to 13, wherein a porosity of the nucleic acid-adsorptive porous membrane is 50 to 95%. Bubble point of the nucleic acid-adsorbing porous membrane is a 0.1 to 10 / cm ^2, The method for separating and purifying nucleic acid according to any one of claims 1 to 14.   The method for separating and purifying nucleic acid according to any one of claims 1 to 15, wherein the nucleic acid-adsorptive porous membrane has a pressure loss of 0.1 to 100 kPa. The amount of water permeation when the nucleic acid-adsorptive porous membrane allows water to pass through at a pressure of 1 kg / cm ² at 25 ° C is in the range of 1 to 5000 mL per minute per 1 cm ^{2 of} the membrane. The method for separating and purifying a nucleic acid according to any one of the above.   The method for separating and purifying nucleic acid according to any one of claims 1 to 17, wherein the nucleic acid-adsorbing porous membrane has an adsorption amount of nucleic acid per mg of membrane of 0.1 µg or more.   A nucleic acid-adsorbing porous membrane comprising an ester derivative of a polysaccharide other than acetylcellulose or a saponified product of an ester derivative of a polysaccharide other than acetylcellulose.   Polysaccharide ester derivatives other than acetylcellulose are monoester derivatives, diester derivatives, triester derivatives, mixtures of monoester derivatives and diester derivatives, mixtures of monoester derivatives and triester derivatives, diester derivatives and triester derivatives. The nucleic acid-adsorptive porous membrane according to claim 19, which is a mixture of a monoester derivative, a diester derivative, and a triester derivative.