JP2004242622A - Washing solution for separating and refining nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a washing solution used in a separating and refining method of a nucleic acid including a step for absorbing the nucleic acid into a solid phase composed of an organic polymer having a hydroxy group on the surface and desorbing the nucleic acid from the solid phase and capable of washing away only impurity nonspecifically absorbed to the solid phase without detaching the nucleic acid absorbed in the solid phase. <P>SOLUTION: The washing solution is composed of an aqueous solution of a water-soluble alcohol and used in the separating and refining method of the nucleic acid including the step for absorbing the nucleic acid into the solid phase composed of the organic polymer having the hydroxy group on the surface and desorbing the nucleic acid from the solid phase. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００５３】

【００５４】
（２）核酸精製操作
λＤＮＡ（ライフテクノロジー社製）を１００ｎｇ／ｍｌの濃度に調整し、これをサンプルとして２００μｌに、吸着バッファー２００μｌとプロテアーゼＫ２０μｌを添加して、６０℃で１０分間インキュベートした。インキュベート後、エタノール２００μｌを加え、攪拌した。攪拌後、図１〜図６に構造を示す核酸精製用カートリッジにこの液を注入した。注入後、ピストンにて液を押し出した。さらに、洗浄液５００μｌを注入し、ピストンにて液を押し出すことにより、カートリッジおよび吸着固相上の不純物を洗浄した。最後に、蒸留水２００μｌを注入し、ピストンにて液を押し出して、この液を回収した。
【００５５】
（３）核酸の回収量の定量
（２）の操作により精製されたＤＮＡの収量を以下の表１に示す。値は比較例及び本発明とも５回の繰り返しの平均値である。ＯＤは５ｍｍセルにて測定した。 表１の結果より、比較例よりも本発明の場合の方がＤＮＡの収量が高いことが分かる。
【００５６】
【表１】

【００５７】
【発明の効果】
表面に水酸基を有する有機高分子から成る固相に核酸を吸着及び脱着させる工程を含む核酸の分離精製方法において本発明の洗浄液を用いることにより、固相に吸着された核酸を脱着させることなく固相に非特異的に吸着された不純物だけを洗い流すことができる。
【図面の簡単な説明】
【図１】本発明の核酸分離精製装置を示す側断面図である。
【図２】本発明の核酸分離精製装置の分解斜視図である。
【図３】シリンジと固相保持部材の接合方法の他の実施の形態を示す側断面図である。
【図４】固相保持部材の拡大断面図である。
【図５】シリンジの先端部の縦断面図である。
【図６】シリンジの先端部内において、収納部内から外側へ押し出される液体が流れる様子を示す説明図である。
【符号の説明】
１ 核酸の分離精製装置
３ シリンジ
５ ピストン部材
７ 固相保持部材
９ シリンジの先端部
１１ 第１開口部
１３ 指掛け部
１５ シリンジの基端部
１７ 第２開口部
１９ 収納部
２１ テーパ
２３ 雄ねじ
２５ 雌ねじ
２７ 液体案内面
２９ 先端部の先端
３１ プランジャ
３３ ピストン栓
３５ 本体部
３７ 端板
３９ ノズル
４１ 流通孔
４３ 固相支持面
４５ 固相
４６ ポリプロピレン焼結フィルター
４７ テーパ
４９ 縮径部[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a washing solution for use in separation and purification of nucleic acids. More specifically, the present invention relates to a washing solution for use in a method for separating and purifying nucleic acid, which comprises a step of adsorbing and desorbing nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface.
[0002]
[Prior art]
Nucleic acids are used in various forms in various fields. For example, in the area of recombinant nucleic acid technology, it is required to use nucleic acids in the form of probes, genomic nucleic acids, and plasmid nucleic acids.
[0003]
In the diagnostic field, nucleic acids are used in various ways. For example, nucleic acid probes are routinely used for detection and diagnosis of human pathogens. Similarly, nucleic acids are used to detect genetic disorders. Nucleic acids are also used to detect food contaminants. In addition, nucleic acids are routinely used in the localization, identification and isolation of nucleic acids of interest for a variety of reasons ranging from genetic mapping to cloning and recombinant expression.
[0004]
In many cases, nucleic acids are available only in very small amounts, and the isolation and purification operations are cumbersome and time consuming. This often time-consuming and cumbersome operation tends to lead to the loss of nucleic acids. The purification of sample nucleic acids from serum, urine and bacterial cultures also adds the risk of contamination and false positive results.
[0005]
As one of the widely known purification methods, there is a method in which a nucleic acid is adsorbed on the surface of silicon dioxide, silica polymer, magnesium silicate, etc. and purified by subsequent operations such as washing and desorption (for example, Japanese Patent Publication No. 7-51065). Publication). Although this method is excellent in separation performance, there are problems such as difficulty in industrial mass production of adsorption media having the same performance, inconvenience in handling, and difficulty in processing into various shapes. Further, in order to wash away only the impurities nonspecifically adsorbed on the solid phase without desorbing the nucleic acid adsorbed on the solid phase, it is necessary to add a chaotropic substance to the washing solution. However, there is a high possibility that the washing solution is contaminated with the step of desorbing the nucleic acid in the final step, and contamination of the chaotropic substance in the washing solution with the extracted nucleic acid sample causes fatal inhibition in the subsequent analysis and reaction (for example, PCR reaction). cause.
[0006]
[Problems to be solved by the invention]
An object of the present invention is a washing solution for use in a method for separating and purifying nucleic acid comprising a step of adsorbing and desorbing nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface, the nucleic acid adsorbed on the solid phase It is an object of the present invention to provide a washing solution that can wash away only impurities that are non-specifically adsorbed on the solid phase without desorbing the.
[0007]
[Means for Solving the Problems]
As a result of diligent studies to solve the above problems, the present inventors have found that a method for separating and purifying nucleic acid from a water-soluble alcohol aqueous solution includes a process of adsorbing and desorbing nucleic acid on a solid phase composed of an organic polymer having a hydroxyl group on the surface. The present invention is completed by finding that only the impurities non-specifically adsorbed on the solid phase can be washed away without desorbing the nucleic acid adsorbed on the solid phase by washing the nucleic acid using the washing solution comprising It came to.
[0008]
That is, according to the present invention, there is provided a cleaning solution comprising a water-soluble alcohol aqueous solution for use in a method for separating and purifying nucleic acid, which comprises a step of adsorbing and desorbing nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface. Is done.
[0009]
Preferably, the cleaning liquid of the present invention is a cleaning liquid that does not contain a chaotropic substance.
Preferably, the water-soluble alcohol concentration is 20% by weight or more and 100% by weight or less.
Preferably, the water soluble alcohol is ethanol.
Preferably, the organic polymer having a hydroxyl group on the surface is a surface saponified product of acetylcellulose, and more preferably a surface saponified product of triacetylcellulose.
Preferably, acetyl cellulose is a porous membrane.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below.
(1) Cleaning liquid of the present invention The cleaning liquid of the present invention is a cleaning liquid for use in a method for separating and purifying nucleic acid, including a step of adsorbing and desorbing nucleic acid on a solid phase composed of an organic polymer having a hydroxyl group on the surface. It is an aqueous solution containing a basic alcohol. The cleaning liquid of the present invention preferably does not contain a chaotropic substance (for example, guanidinium salt, sodium iodide, sodium perchlorate, etc.). Further, a cleaning solution composed of only a water-soluble alcohol and water and not containing various salts and surfactants is particularly preferable.
[0011]
In the present invention, the water-soluble alcohol concentration is particularly preferably 20% to 100% by weight, more preferably 30% to 80% by weight, and particularly preferably 40% to 80% by weight. .
The water-soluble alcohol used in the present invention is preferably an alcohol having 1 to 4 carbon atoms, and examples thereof include methanol, ethanol, isopropanol, n-isopropanol, and butanol. As the water-soluble alcohol, ethanol is particularly preferable.
[0012]
The cleaning liquid of the present invention can contain a buffer and / or a surfactant in addition to the water-soluble alcohol described above. As the buffer, a known buffer can be used, and examples thereof include Tris. The preferred concentration when using Tris is 10-100 mM. As the surfactant, a known surfactant can be used, for example, Triton-X100 can be used. In addition, SDS, sodium cholate or sarcosine sodium and other anionic surfactants, Tween 20 Alternatively, nonionic surfactants such as megafacs and other various amphoteric surfactants may be used. A preferred concentration when using Triton-X is 0.1 to 10% by weight.
[0013]
(2) Method for separating and purifying nucleic acid using the washing solution of the present invention The method for separating and purifying nucleic acid using the washing solution of the present invention adsorbs and desorbs nucleic acid on a solid phase composed of an organic polymer having a hydroxyl group on the surface. The process to perform is performed.
In the present invention, the “nucleic acid” may be either single-stranded or double-stranded, and there is no molecular weight limitation.
[0014]
The organic polymer having a hydroxyl group on the surface is preferably a surface saponified product of acetylcellulose. The acetyl cellulose may be any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose, but triacetyl cellulose is particularly preferable. In the present invention, surface saponified acetylcellulose is preferably used as the solid phase. Here, the surface saponification means that only the surface in contact with the saponification solution (for example, NaOH) is saponified. In the present invention, it is preferable that the solid phase structure remains acetylcellulose and only the surface of the solid phase is saponified. Thereby, the amount (density) of hydroxyl groups on the surface of the solid phase can be controlled by the degree of surface saponification treatment (surface saponification degree).
[0015]
In order to increase the surface area of the organic polymer having a hydroxyl group on the surface, it is preferable to form a film of the organic polymer having a hydroxyl group on the surface. Acetylcellulose may be a porous membrane or a non-porous membrane, but it is more preferable to make the membrane porous. When the solid phase is a porous membrane, it is preferable that the membrane structure remains acetylcellulose and only the surface of the structure is saponified. Thereby, the amount (density) of the spatial hydroxyl group can be controlled by the degree of surface saponification treatment (surface saponification degree) × pore diameter. Further, since the membrane structure is composed of acetylcellulose, a solid solid phase can be obtained. Here, saponifying acetylcellulose and introducing a hydroxyl group only on the surface means that the structure remains as acetylcellulose and the surface is celluloseized. When cellulose is used as a raw material, it cannot be made into a liquid, so that a porous membrane or a flat membrane cannot be produced industrially.
[0016]
For example, a film of triacetylcellulose is commercially available from Fuji Photo Film under the trade name TAC base, and a microfilter FM500 (manufactured by Fuji Photo Film Co., Ltd.) is available as a porous film of triacetylcellulose.
Further, for example, it is also preferable to form a triacetyl cellulose film on the surface of polyethylene beads and saponify the surface to give a hydroxyl group on the surface. In this case, triacetyl cellulose is coated on the beads. The material of the beads is not limited to polyethylene as long as it does not contaminate the nucleic acid.
[0017]
In order to increase the nucleic acid separation efficiency, a larger number of hydroxyl groups is preferred. For example, in the case of acetylcellulose such as triacetylcellulose, the surface saponification rate is preferably about 5% or more, more preferably 10% or more.
In order to saponify the surface of acetylcellulose, an object to be saponified is immersed in an aqueous sodium hydroxide solution. In order to change the surface saponification rate, the concentration of sodium hydroxide may be changed. The surface saponification rate is determined by quantifying residual acetyl groups by NMR.
[0018]
In the method for separating and purifying nucleic acid described above, preferably, nucleic acid is adsorbed and desorbed using a nucleic acid separation and purification unit containing a solid phase composed of an organic polymer having a hydroxyl group on the surface in a container having at least two openings. It can be carried out.
[0019]
More preferably, (a) a solid phase made of an organic polymer having a hydroxyl group on the surface, (b) a container having at least two openings containing the solid phase, and (c) one opening of the container. Nucleic acid can be adsorbed and desorbed using a nucleic acid separation and purification unit including a combined pressure difference generator.
[0020]
The first embodiment of the method for separating and purifying nucleic acid can include the following steps.
(A) inserting one opening of the nucleic acid separation and purification unit into a sample solution containing nucleic acid;
(B) A solid phase composed of an organic polymer having a hydroxyl group on the surface by sucking a sample solution containing nucleic acid by reducing the pressure in the container using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit. Contacting with,
(C) a step of pressurizing the inside of the container using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit, and discharging the sample solution containing the aspirated nucleic acid out of the container;
(D) inserting one opening of the nucleic acid separation and purification unit into the nucleic acid washing solution;
(E) Using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit, the container is depressurized and the nucleic acid washing solution is sucked and brought into contact with a solid phase composed of an organic polymer having a hydroxyl group on the surface. Process,
(F) a step of bringing the inside of the container into a pressurized state using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit, and discharging the aspirated nucleic acid washing solution out of the container;
(G) inserting one opening of the nucleic acid separation and purification unit into a liquid capable of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface;
(H) Depressurize the inside of the container using a pressure difference generator connected to the other opening of the nucleic acid separation and purification unit to desorb the nucleic acid adsorbed on the solid phase composed of an organic polymer having a hydroxyl group on the surface. A step of aspirating the liquid to be brought into contact with the solid phase, and (i) an organic compound having a hydroxyl group on the surface thereof, wherein the inside of the container is pressurized using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit. A step of discharging a liquid capable of desorbing a nucleic acid adsorbed on a solid phase composed of a polymer to the outside of the container.
[0021]
The second embodiment of the method for separating and purifying nucleic acid can include the following steps.
(A) preparing a sample solution containing a nucleic acid using a specimen and injecting the sample solution containing the nucleic acid into one opening of the nucleic acid separation and purification unit;
(B) Using a pressure difference generator connected to the one opening of the nucleic acid separation and purification unit, pressurize the inside of the container, and discharge the sample solution containing the injected nucleic acid from the other opening. Contacting with a solid phase comprising an organic polymer having a hydroxyl group on
(C) a step of injecting a nucleic acid washing solution into the one opening of the nucleic acid separation and purification unit;
(D) The container is pressurized using the pressure difference generator connected to the one opening of the nucleic acid separation and purification unit, and the injected nucleic acid washing solution is discharged from the other opening, whereby hydroxyl groups are formed on the surface. Contacting with a solid phase comprising an organic polymer having,
(E) a step of injecting a liquid capable of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface into the one opening of the nucleic acid separation and purification unit;
(F) By using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, pressurizing the inside of the container, and discharging a liquid capable of desorbing the injected nucleic acid from the other opening, A step of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface and discharging it out of the container.
[0022]
The method for separating and purifying nucleic acid using an organic polymer having a hydroxyl group on the surface will be described more specifically. In the present invention, preferably, the nucleic acid in the sample solution is adsorbed on the solid phase by bringing the sample solution containing the nucleic acid into contact with the solid phase composed of an organic polymer having a hydroxyl group on the surface, and then adsorbed on the solid phase. Nucleic acids are desorbed from the solid phase using a suitable solution as described below. More preferably, the sample solution containing nucleic acid is a solution obtained by adding a water-soluble organic solvent to a solution obtained by dispersing a nucleic acid in a solution by treating a specimen containing cells or viruses with a solution that dissolves cell membranes and nuclear membranes. is there.
[0023]
The sample solution containing the nucleic acid that can be used in the present invention is not limited, but in the diagnostic field, for example, whole blood collected as a specimen, plasma, serum, urine, stool, semen, saliva and other body fluids, or plants (or their) Particular), animals (or parts thereof), etc., or solutions prepared from biological materials such as lysates and homogenates thereof.
[0024]
First, these specimens are treated with an aqueous solution containing a reagent that dissolves the cell membrane and solubilizes the nucleic acid. As a result, the cell membrane and the nuclear membrane are dissolved, and the nucleic acid is dispersed in the aqueous solution.
For cell membrane lysis and nucleic acid solubilization, for example, when the target sample is whole blood, (1) removal of red blood cells, (2) removal of various proteins, and (3) lysis of white blood cells and nuclear membrane Must be dissolved. (1) Removal of red blood cells and (2) removal of various proteins are performed in order to prevent nonspecific adsorption to the solid phase and clogging of the porous membrane. (3) Lysis of leukocytes and nuclear membranes are subject to extraction. Are required to solubilize the nucleic acids. In particular, (3) lysis of leukocytes and lysis of the nuclear membrane are important steps. In the method of the present invention, it is necessary to solubilize nucleic acids in this step. In the examples described below in the present specification, the above (1), (2) and 3 ▼ is achieved at the same time.
[0025]
Examples of the nucleic acid solubilizing reagent used in the present invention include a solution containing a guanidine salt, a surfactant and a proteolytic enzyme.
As the guanidine salt, guanidine hydrochloride is preferable, but other guanidine salts (guanidine isothiocyanate, guanidine thiocyanate) can also be used. The concentration of the guanidine salt in the solution is 0.5M or more and 6M or less, preferably 1M or more and 5M or less.
[0026]
As the surfactant, Triton-X100 can be used, but in addition to this, an anionic surfactant such as SDS, sodium cholate or sarcosine sodium, a nonionic surfactant such as Tween 20 or MegaFac, Various other amphoteric surfactants can also be used. In the present invention, it is preferable to use a nonionic surfactant such as polyoxyethylene octylphenyl ether (Triton-X100). The concentration of the surfactant in the solution is usually 0.05% to 10% by weight, particularly preferably 0.1% to 5% by weight.
[0027]
Protease K can be used as the proteolytic enzyme, but the same effect can be obtained with other proteases. Since protease is an enzyme, it is preferable to warm it, it is preferable to use it at 37 ° C to 70 ° C, particularly 50 ° C to 65 ° C.
[0028]
A water-soluble organic solvent is added to the aqueous solution in which nucleic acids are dispersed in this manner, and brought into contact with an organic polymer having a hydroxyl group on the surface. By this operation, the nucleic acid in the sample solution is adsorbed to the organic polymer having a hydroxyl group on the surface. In order to adsorb the nucleic acid solubilized by the operation described above in this specification to a solid phase composed of an organic polymer having a hydroxyl group on the surface, a water-soluble organic solvent is mixed with the solubilized nucleic acid mixture. It is necessary that a salt be present in the obtained nucleic acid mixture.
[0029]
That is, by destroying the hydration structure of water molecules present around the nucleic acid, the nucleic acid is solubilized in an unstable state. When a nucleic acid in this state is brought into contact with a solid phase composed of an organic polymer having a hydroxyl group on the surface, the polar group on the nucleic acid surface interacts with the polar group on the solid phase surface, and the nucleic acid is adsorbed on the solid surface. It is thought to do. In the method of the present invention, the nucleic acid can be brought into an unstable state by mixing a water-soluble organic solvent with the solubilized nucleic acid mixture and the presence of a salt in the obtained nucleic acid mixture.
[0030]
Examples of the water-soluble organic solvent used here include ethanol, isopropanol, and propanol. Among them, ethanol is preferable. The concentration of the water-soluble organic solvent is preferably 5% by weight to 90% by weight, and more preferably 20% by weight to 60% by weight. The concentration of ethanol added is particularly preferably as high as possible without causing pseudo-collects.
[0031]
Salts present in the resulting nucleic acid mixture include various chaotropic substances (guanidinium salts, sodium iodide, sodium perchlorate), sodium chloride, potassium chloride, ammonium chloride, sodium bromide, potassium bromide, bromide. Calcium, ammonium bromide and the like are preferred. In particular, a guanidinium salt is particularly preferable because it has the effects of cell membrane lysis and nucleic acid solubilization.
[0032]
Next, an organic polymer having a hydroxyl group on the surface to which the nucleic acid has been adsorbed is brought into contact with the cleaning liquid of the present invention. This washing solution has a function of washing out impurities in the sample solution adsorbed on the organic polymer having a hydroxyl group on the surface together with the nucleic acid. Therefore, it is necessary to have a composition in which nucleic acids are not desorbed from an organic polymer having a hydroxyl group on the surface, but impurities are desorbed. As described above, the cleaning liquid is composed of an aqueous solution containing a water-soluble alcohol and, if necessary, a surfactant.
[0033]
Next, the organic polymer having a hydroxyl group on the surface after the washing is brought into contact with a solution capable of desorbing the nucleic acid adsorbed on the organic polymer having a hydroxyl group on the surface. Since the target nucleic acid is contained in this solution, it is recovered and provided for subsequent operation, for example, amplification of the nucleic acid by PCR (polymerase chain reaction). The solution capable of desorbing nucleic acids preferably has a low salt concentration, and particularly preferably a solution having a salt concentration of 0.5 M or less. As this solution, purified distilled water, TE buffer or the like can be used.
[0034]
(3) Nucleic acid separation / purification apparatus The nucleic acid separation / purification apparatus used in the present invention is a nucleic acid separation / purification apparatus in which a solid phase composed of an organic polymer having a hydroxyl group is contained in a container having at least two openings. It is. Specific examples of the nucleic acid separation and purification apparatus are shown in FIGS.
[0035]
As shown in FIG. 1, the nucleic acid separation and purification apparatus 1 of the present invention includes a cylindrical syringe 3, a piston member 5 inserted into the syringe 3, and a solid phase holding member connected to the distal end portion of the syringe 3. 7. In the present invention, the side on which the liquid in the syringe 3 is pushed out is the “front end” side, and the opposite direction is the “base end” side.
[0036]
The syringe 3 has a distal end portion 9 to which the solid phase holding member 7 is connected, and the first opening portion 11 is formed in the distal end portion 9. Further, the proximal end side of the syringe 3 has a proximal end portion 15 in which a finger hook portion 13 is formed, and a second opening portion 17 is formed in the proximal end portion 15. A storage portion 19 is formed inside the syringe 3, and the storage portion 19 can hold the liquid that has entered from the first opening portion 11 or the second opening portion 17 by a sealing action of a piston stopper described later. .
[0037]
As shown in FIG. 2, the distal end portion 9 of the syringe 3 is formed with a taper 21 whose outer peripheral surface is reduced in diameter toward the distal end so that the solid-phase holding member 7 can be fitted. Instead of such a taper 21, a male screw 23 is formed on the outer peripheral surface of the distal end portion 9 of the syringe 3 as shown in FIG. 3, and a female screw corresponding to the male screw 23 is formed on the inner surface of the solid end holding member 7. 25 can be formed so that the solid-phase holding member 7 can be detachably attached to the distal end portion 9 of the syringe 3 in a screwed manner.
[0038]
Further, as best shown in FIG. 5, an arbitrary longitudinal section of the syringe 3 has an arbitrary vertical cross section from the most contracted diameter portion 49 in the distal end portion 9 toward the distal end side. Thus, a liquid guide surface 27 having a shape curved in a quadratic curve is formed. The region surrounded by the circular cross section formed by the reduced diameter portion 49 and the liquid guide surface has a substantially mortar shape as a whole. The operation of the liquid guide surface 27 having such a shape will be described later. Further, in an arbitrary longitudinal section of the distal end portion 9 of the syringe 3, the distal end 29 of the distal end portion 9 has an acute angle, and is fixed by being pressed against the surface of a solid phase made of an organic polymer having a hydroxyl group on the surface described later. Demonstrate the effect of retaining the phase. This point will be described in detail later.
[0039]
The piston member 5 includes a plunger 31 that extends from the second opening 17 side into the storage portion 19, and a piston plug 33 that is connected to the tip of the plunger 31 and can be in close contact with the inner surface of the storage portion 19. The piston plug 33 is fixed to the tip of the plunger 31, so that the piston plug 33 can reciprocate in the storage portion 19 by pushing or pulling the plunger 31.
[0040]
Next, as shown in FIG. 4, the solid-phase holding member 7 mainly has a main body portion 35 having a substantially cylindrical shape or a slight taper shape, and its proximal end side is in an open state, and the distal end side is formed by an end plate 37. It is blocked. A nozzle 39 protrudes from the center of the end plate 37 toward the front end side, and a flow hole 41 communicating with the inside of the main body 35 is formed inside the nozzle 39. A circular solid-phase support surface 43 substantially perpendicular to the longitudinal axis of the syringe 3 is formed on the inner surface of the end plate 37 on the proximal end side. The solid-phase support surface 43 has an organic group having a hydroxyl group on the surface. A circular flat solid phase 45 made of a polymer is supported. As will be described later in detail, the solid phase 45 is made of a material capable of adsorbing and desorbing nucleic acids in the sample solution.
[0041]
In addition, a polypropylene sintered filter 46 that functions as a cushion is provided on the tip side of the solid phase 45. The polypropylene sintered filter 46 has a circular flat plate shape like the solid phase 45, and is provided between the solid phase 45 and the solid phase support surface 43, so that the solid phase 45 is attached to the distal end portion 9 of the syringe 3. When pressed, the solid phase 45 is supported from its proximal end side to provide a cushioning action. Accordingly, it is possible to prevent the solid phase 45 from being excessively crushed and deformed, or the gap in which the liquid formed in the solid phase 45 circulates from being crushed and the liquid flowability from being deteriorated.
[0042]
A taper 47 is formed on the inner side of the main body 35 of the solid-phase holding member 7 so as to reduce the diameter in the distal direction corresponding to the taper 21 formed at the distal end 9 of the syringe 3. By simply fitting the taper 47 of the solid phase holding member 7 to the outside of the taper 21 formed at the distal end portion 9 of the syringe 3, the syringe 3 and the solid phase holding member 7 are integrated in a watertight state. be able to.
[0043]
When the solid-phase holding member 7 is attached to the distal end portion 9 of the syringe 3, the distal end 29 in which the cross section of the syringe distal end portion 9 is formed at an acute angle as described above is just inside the circular outer peripheral edge of the solid phase 45. Press contact. As a result, the sintered polypropylene filter 46 is somewhat crushed, so that the solid phase 45 is placed between the distal end portion 9 of the syringe 3 and the sintered polypropylene filter 46 by the reaction of the sintered polypropylene filter 46 having a cushioning action. It will be pinched by. As a result, the solid phase 45 is firmly supported by the solid phase support surface 43 via the polypropylene sintered filter 46. As described above, by adopting a configuration in which the tip 29 formed with an acute angle in the cross section of the syringe tip 9 is in pressure contact with the inner side of the circular outer peripheral edge of the solid phase 45, the piston plug 33 can be operated by operating the plunger 31. When reciprocating, the liquid pushed out from the storage unit 19 or the liquid flowing into the storage unit 19 through the circulation hole 41 always passes through the solid phase support surface 43, and the solid phase 45. It is possible to prevent the liquid from circulating around the outer periphery of the circular outer periphery.
[0044]
FIG. 6 is a diagram illustrating a state in which the liquid pushed out from the storage portion 19 flows out in the distal end portion 9 of the syringe 3 in which the liquid guide surface 27 having a shape that curves in a quadratic curve is formed. FIG. As shown in FIG. 6, the liquid guide surface 27 having a shape that curves like a quadratic curve from the diameter-reduced diameter-reduced portion 49 in the distal end portion 9 moves to the distal end side of the longitudinal axis of the syringe 3. Accordingly, the expansion rate of the diameter of the cross section of the liquid guide surface 27 per unit length, that is, the expansion rate is reduced. Therefore, the diameter is suddenly increased near the reduced diameter portion 49, but the degree of diameter expansion gradually becomes gradually as it goes to the tip side. With such a configuration, the flow velocity distribution in the cross section of the liquid flowing out from the inside of the storage portion 19 is substantially constant from the reduced diameter portion 49 to the solid phase 45, and thus over the entire surface of the solid phase 45. An almost constant fluid pressure is applied. As a result, when the liquid flows between the storage unit 19 of the syringe 3 to be described later and the outside, the nucleic acid separation function, the washing function, and the like are effectively exhibited over the entire surface of the solid phase 45, which is efficient. Nucleic acids can be separated and purified.
[0045]
In the nucleic acid separation and purification apparatus described above, the solid phase holding member 7 is a separate member from the syringe 3, and the solid phase holding member 7 is detachable from the syringe 3. The solid phase holding member 7 and the syringe 3 may be formed as an integral member from the beginning.
[0046]
(4) Method for separating and purifying nucleic acid by using nucleic acid separation and purification apparatus A method for separating and purifying nucleic acid using the above-described nucleic acid separation and purification apparatus will be described. First, with the plunger 31 pushed into the distal end side, the flow hole 41 of the nucleic acid separation and purification apparatus 1 is inserted into a sample solution containing nucleic acid. Next, the plunger 31 is pulled to the base end side, the inside of the storage unit 19 is decompressed, the sample solution containing nucleic acid is sucked, and brought into contact with the solid phase 45 made of an organic polymer having a hydroxyl group on the surface. By this operation, the sample solution comes into contact with the solid phase 45 and the nucleic acid in the sample solution is adsorbed on the solid phase 45.
[0047]
After sucking an appropriate amount of the sample solution, the plunger 31 is pushed again to the tip side to pressurize the inside of the storage unit 19 and discharge the sucked liquid. It is not necessary to leave an interval before this operation, and it may be discharged immediately after suction.
[0048]
Next, the cleaning liquid of the present invention is sucked into the storage portion by the pressure-reduction operation in the storage portion 19 similar to the above by the operation of the plunger 31, and is discharged from the storage portion to clean the inside of the storage portion. This solution has a function of washing away the sample solution remaining in the storage section and washing out impurities in the sample solution adsorbed on the solid phase together with the nucleic acid.
[0049]
Next, a solution capable of desorbing the nucleic acid adsorbed on the solid phase 45 is introduced into the storage unit 19 by the pressure reduction-pressurization operation in the storage unit 19 similar to the above by the operation of the plunger 31 and discharged from the storage unit 19. To do. Since this effluent contains the target nucleic acid, it can be recovered and provided for subsequent operation, for example, amplification of the nucleic acid by PCR (polymerase chain reaction).
[0050]
The above method is a method in which a sample solution containing nucleic acid and a washing solution are sucked into the storage unit 19 through the flow hole 41 and discharged, but the plunger 31 is removed from the second opening 17 of the nucleic acid separation and purification apparatus. A sample solution containing a nucleic acid may be injected into the storage unit 19, and the sample solution may be discharged from the circulation hole 41 using the plunger 31. Similarly, a nucleic acid cleaning solution and a solution capable of desorbing the nucleic acid adsorbed on the solid phase 45 are injected into the storage unit 19 from the second opening 17 and the cleaning solution is discharged from the circulation hole 41 using the plunger 31. It may be.
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
[0051]
【Example】
(1) Materials and Reagents Using a cartridge for nucleic acid purification whose structure is shown in FIGS. 1 to 6, a sample, a washing solution, and distilled water are sequentially injected from the second opening side, and a piston member (blanger) is inserted each time. And pressed. Further, Fuji Microfilter FR250 (manufactured by Fuji Photo Film) was used as the nucleic acid adsorption solid phase. As a nucleic acid adsorption solid phase for comparison, a glass filter GF / D (manufactured by Whatman) (film thickness 2 mm) was used. The adsorption buffer solution for nucleic acid purification and the washing solution were prepared as follows.
[0052]

[0053]

[0054]
(2) Nucleic acid purification operation λDNA (manufactured by Life Technology) was adjusted to a concentration of 100 ng / ml, 200 μl of adsorption buffer and 20 μl of protease K were added to this sample as a sample, and incubated at 60 ° C. for 10 minutes. After incubation, 200 μl of ethanol was added and stirred. After stirring, this solution was poured into a cartridge for nucleic acid purification whose structure is shown in FIGS. After the injection, the liquid was pushed out by the piston. Further, 500 μl of the cleaning liquid was injected, and the liquid on the cartridge and the adsorption solid phase was cleaned by pushing out the liquid with a piston. Finally, 200 μl of distilled water was injected, the liquid was pushed out with a piston, and this liquid was collected.
[0055]
(3) Quantification of recovery amount of nucleic acid The yield of DNA purified by the operation of (2) is shown in Table 1 below. The value is an average value of five repetitions in both the comparative example and the present invention. OD was measured with a 5 mm cell. From the results in Table 1, it can be seen that the yield of DNA is higher in the case of the present invention than in the comparative example.
[0056]
[Table 1]

[0057]
【The invention's effect】
In the method for separating and purifying nucleic acid including the step of adsorbing and desorbing nucleic acid to the solid phase comprising an organic polymer having a hydroxyl group on the surface, the nucleic acid adsorbed on the solid phase can be immobilized without desorption by using the washing solution of the present invention. Only impurities that are non-specifically adsorbed to the phase can be washed away.
[Brief description of the drawings]
FIG. 1 is a side sectional view showing an apparatus for separating and purifying nucleic acid of the present invention.
FIG. 2 is an exploded perspective view of the nucleic acid separation and purification apparatus of the present invention.
FIG. 3 is a side sectional view showing another embodiment of a method for joining a syringe and a solid phase holding member.
FIG. 4 is an enlarged cross-sectional view of a solid phase holding member.
FIG. 5 is a longitudinal sectional view of a distal end portion of a syringe.
FIG. 6 is an explanatory diagram showing a state in which liquid pushed out from the storage portion flows in the distal end portion of the syringe.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Nucleic acid separation and purification apparatus 3 Syringe 5 Piston member 7 Solid phase holding member 9 Syringe tip part 11 First opening part 13 Finger hook part 15 Syringe base end part 17 Second opening part 19 Storage part 21 Taper 23 Male screw 25 Female screw 27 Liquid guide surface 29 Tip 31 of the tip portion Plunger 33 Piston plug 35 Main body portion 37 End plate 39 Nozzle 41 Flow hole 43 Solid phase support surface 45 Solid phase 46 Polypropylene sintered filter 47 Taper 49 Reduced diameter portion

Claims (8)

A cleaning solution comprising a water-soluble alcohol aqueous solution for use in a method for separating and purifying nucleic acid, comprising a step of adsorbing and desorbing nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface. The washing | cleaning liquid of Claim 1 which does not contain a chaotropic substance. The washing | cleaning liquid of Claim 1 or 2 which consists only of water-soluble alcohol and water, and does not contain various salts and surfactant. The cleaning liquid according to any one of claims 1 to 3, wherein the water-soluble alcohol concentration is 20 wt% or more and 100 wt% or less. The cleaning liquid according to claim 1, wherein the water-soluble alcohol is ethanol. The cleaning liquid according to any one of claims 1 to 5, wherein the organic polymer having a hydroxyl group on the surface is a surface saponified product of acetylcellulose. The cleaning liquid according to any one of claims 1 to 6, wherein the organic polymer having a hydroxyl group on the surface is a surface saponified product of triacetylcellulose. The cleaning liquid according to any one of claims 1 to 7, wherein acetylcellulose is a porous film.

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