JP2005118007A - Method for separation and purification of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an automatable means for easily and quickly producing a specimen solution containing nucleic acids in high efficiency and reproducibility in a method for the separation and purification of a nucleic acid by adsorbing the nucleic acid in a nucleic acid-containing specimen solution to a nucleic acid-adsorbing porous membrane and desorbing the acid after washing, etc. <P>SOLUTION: The method for producing the specimen solution containing nucleic acids contains a step to add a solution of a nucleic acid solubilizing reagent containing a surfactant to a specimen containing a cell or a virus to mix the specimen with the nucleic acid solubilizing reagent solution and a step to add a water-soluble organic solvent to the mixed liquid. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for preparing a sample solution containing nucleic acid, and a method for separating and purifying nucleic acid using the same. More specifically, the present invention prepares a sample solution containing a nucleic acid using a surfactant, and then a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane in a container having at least two openings, and pressure The present invention relates to a method for separating and purifying nucleic acid from a sample solution containing the prepared nucleic acid using a difference generator.

  Nucleic acids are used in various forms in various fields. For example, in the area of recombinant nucleic acid technology, it is required to use nucleic acids in the form of probes, genomic nucleic acids, and plasmid nucleic acids.

  In the diagnostic field, nucleic acids are used in various ways. For example, nucleic acid probes are routinely used for detection and diagnosis of human pathogens. Similarly, nucleic acids are used to detect genetic disorders. Nucleic acids are also used to detect food contaminants. In addition, nucleic acids are routinely used in the localization, identification and isolation of nucleic acids of interest for a variety of reasons ranging from genetic mapping to cloning and recombinant expression.

  In many cases, nucleic acids are available only in very small amounts, and the isolation and purification operations are cumbersome and time consuming. This often time-consuming and cumbersome operation tends to lead to the loss of nucleic acids. The purification of sample nucleic acids from serum, urine and bacterial cultures also adds the risk of contamination and false positive results.

  As one of 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.

Japanese Patent Publication No. 7-51065

  An object of the present invention is to provide a method for separating and purifying nucleic acid by allowing nucleic acid in a sample solution containing nucleic acid to be adsorbed on a porous membrane capable of adsorbing nucleic acid and then desorbing it through washing or the like. Another object of the present invention is to provide a method for obtaining a sample solution containing nucleic acid having excellent reproducibility and automation. Another object of the present invention is to use a porous membrane that has excellent separation performance, good washing efficiency, is simple, rapid, excellent in automation, and capable of mass production of substantially the same separation performance. It is to provide a method for separating and purifying nucleic acid.

  As a result of diligent studies to solve the above problems, the present inventors have prepared a sample solution containing a nucleic acid from a specimen to be performed prior to the separation and purification of the nucleic acid including the step of adsorbing and desorbing the nucleic acid to the porous membrane. It has been found that a sample solution containing a nucleic acid can be efficiently obtained from a specimen by using a surfactant. The present invention has been completed based on these findings.

  That is, according to the present invention, a step of adding a nucleic acid solubilizing reagent solution containing a surfactant to a sample containing cells or viruses and mixing the sample and the nucleic acid solubilizing reagent solution, and adding a water-soluble organic solvent to the mixed solution A method for preparing a sample solution containing a nucleic acid, comprising the step of adding, is provided. Specific examples of the present invention include (a) a step of injecting a specimen containing cells or viruses into a container, and (b) adding a nucleic acid solubilizing reagent solution containing a chaotropic salt, a surfactant and a proteolytic enzyme to the container. And (c) incubating the mixed solution obtained in step (b), (d) adding a water-soluble organic solvent to the incubated mixed solution. A method for preparing a sample solution containing nucleic acid is provided.

  The surfactant may be any of a nonionic surfactant, a cationic surfactant, an anionic surfactant or an amphoteric surfactant, and one or more surfactants can be used. For example, the surfactant is composed of two types of a nonionic surfactant and an anionic surfactant, two types of a nonionic surfactant and a cationic surfactant, or a nonionic surfactant and an amphoteric surfactant. It consists of two types. Preferably, the nonionic surfactant is a surfactant other than a polyoxyethylene alkylphenyl ether surfactant. Preferably, the nonionic surfactant is a polyoxyethylene alkyl ether surfactant. Preferably, the nonionic surfactant is a fatty acid alkanolamide. Preferably, the cationic surfactant is cetyltrimethylammonium bromide, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, or cetylpyridinium chloride. Preferably, the chaotropic salt is a guanidinium salt. Preferably, the water-soluble organic solvent is selected from methanol, ethanol, isopropanol, n-propanol or mixtures thereof. Preferably, the nucleic acid in the sample solution does not form a precipitate due to the water-soluble organic solvent.

  According to another aspect of the present invention, a nucleic acid in a sample solution containing a nucleic acid prepared by the above method using a nucleic acid separation / purification unit containing a nucleic acid-adsorbing porous membrane in a container having at least two openings. There is provided a method for separating and purifying nucleic acid, which adsorbs and desorbs.

  Preferably, (a) a nucleic acid adsorbing porous membrane, (b) a container having at least two openings for accommodating the nucleic acid adsorbing porous membrane, and (c) pressure difference generation coupled to one opening of the container Provided is a method for separating and purifying nucleic acid, which uses a nucleic acid separation and purification unit including an apparatus to adsorb and desorb nucleic acid in a sample solution containing the nucleic acid prepared by the above method.

  More preferably, (a) nucleic acid separation containing a nucleic acid-adsorbing porous membrane that allows the sample solution containing the nucleic acid prepared by the above method to pass through a container having at least two openings by a pressure difference. A step of injecting into one opening of the purification cartridge; (b) a nucleic acid separating and purifying cartridge being pressurized using the pressure difference generator coupled to the one opening of the nucleic acid separating and purifying cartridge; A step of allowing the nucleic acid-adsorbing porous membrane to adsorb the nucleic acid-adsorbing porous membrane by passing the sample solution containing the nucleic acid-adsorbing porous membrane through the nucleic acid-adsorbing porous membrane and discharging the sample solution from the other opening of the nucleic acid-separating and purifying cartridge; A step of injecting a washing solution into the one opening; (d) in a nucleic acid separation / purification cartridge using a pressure difference generator coupled to the one opening of the nucleic acid separation / purification cartridge; (E) a step of washing the nucleic acid-adsorbing porous membrane in a state where the nucleic acid is adsorbed by allowing the injected cleaning solution to pass through the nucleic acid-adsorbing porous membrane and discharging it from other openings. Injecting the recovered liquid into the one opening of the separation / purification cartridge; and (f) placing the nucleic acid separation / purification cartridge in a pressurized state using a pressure difference generator connected to the one opening of the nucleic acid separation / purification cartridge. A step of allowing the injected recovery liquid to pass through the nucleic acid-adsorbing porous membrane and discharging it from another opening, thereby desorbing the nucleic acid from the nucleic acid-adsorbing porous membrane and discharging it out of the cartridge for nucleic acid separation and purification. A method for separating and purifying nucleic acids is provided.

  Preferably, the nucleic acid-adsorbing porous membrane is a porous membrane made of an organic polymer that adsorbs nucleic acids with a weak interaction that does not involve ionic bonds. Preferably, the porous film made of an organic polymer is a porous film made of an organic polymer having a hydroxyl group. Preferably, the porous film made of an organic polymer having a hydroxyl group is a porous film obtained by saponifying a porous film of acetylcellulose.

  Preferably, the cleaning liquid is a solution containing 20 to 100% by weight of methanol, ethanol, isopropanol or n-propanol. Preferably, the recovered solution is a solution having a salt concentration of 0.5M or less. Preferably, the pressure difference generator is a pump detachably coupled to one opening of the nucleic acid separation / purification cartridge.

  According to still another aspect of the present invention, an apparatus for performing the above-described nucleic acid separation and purification method is provided. According to still another aspect of the present invention, a reagent kit for performing the nucleic acid separation and purification method is provided.

  The method for preparing a sample solution containing a nucleic acid according to the present invention and the method for separating and purifying a nucleic acid using the same can separate and purify a nucleic acid from a sample containing the nucleic acid with good separation performance, simply and rapidly. Can also be automated.

Embodiments of the present invention will be described below.
The present invention relates to a method for preparing a sample solution containing a nucleic acid. Specifically, a nucleic acid solubilizing reagent solution containing a surfactant is added to a sample containing cells or viruses, and the sample and the nucleic acid solubilizing reagent solution are added. And a step of adding a water-soluble organic solvent to the mixed solution.

  The surfactant used in the present invention may be any of a nonionic surfactant, a cationic surfactant, an anionic surfactant, or an amphoteric surfactant. The nonionic surfactant is preferably a surfactant other than a polyoxyethylene alkylphenyl ether surfactant (for example, Triton-X100, etc.), such as a polyoxyethylene alkyl ether surfactant or a fatty acid alkanolamide. Is preferred. Specific examples of the polyoxyethylene alkyl ether surfactant include cetyltrimethylammonium bromide, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride or cetylpyridinium chloride. Specific examples of the anionic surfactant include POE diethyl ether, POE lauryl ether, POE decyl ether, POE tridecyl ether, POE alkylene decyl ether, POE sorbitan monolaurate, POE sorbitan monooleate, POE sorbitan monostearate, Examples include tetraoleic acid polyoxyethylene sorbitol, POE alkylamine, and POE acetylene glycol.

  Specific examples of the cationic surfactant include monoalkylammonium chloride and dialkylammonium chloride.

  Specific examples of amphoteric surfactants include palmitoyl lysolecithin, N-dodecyl-betaine, stearyl-N-betaine, dodecyl-β-alanine, 3- (dodecyldimethylammonio) -1-propanesulfonic acid, 3- (tetra Decylmethylammonio) -1-propanesulfonic acid and the like.

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

  A sample means any sample containing nucleic acid. The type of nucleic acid in the sample may be one type or two or more types. The length of each nucleic acid is not particularly limited, and for example, a nucleic acid having an arbitrary length of several bp to several Mbp can be used. From the viewpoint of handling, the length of the nucleic acid is generally about several bp to several hundreds kbp.

  The type of the nucleic acid-adsorbing porous membrane is not particularly limited. For example, the nucleic acid-adsorbing porous membrane made of an organic polymer having a hydroxyl group on the surface, the nucleic acid-adsorbing porous membrane made of silicon dioxide, silica polymer or magnesium silicate, or A nucleic acid-adsorbing porous membrane made of silica or a derivative thereof, diatomaceous earth, or alumina can be used. Preferably, a nucleic acid-adsorptive porous membrane made of an organic polymer having a hydroxyl group on the surface can be used.

  As the organic polymer having a hydroxyl group on the surface, a saponified product of acetylcellulose is preferable. The acetyl cellulose may be any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose, but triacetyl cellulose is particularly preferable. In the present invention, it is preferable to use surface-saponified acetylcellulose as the nucleic acid-adsorbing porous membrane. 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 structure of the nucleic acid-adsorbing porous membrane remains acetylcellulose, and only the surface of the nucleic acid-adsorbing porous membrane is saponified. Thereby, the amount (density) of hydroxyl groups on the surface of the nucleic acid-adsorbing porous membrane can be controlled by the degree of saponification treatment (saponification degree).

  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. In the nucleic acid-adsorbing porous membrane, it is preferable that the 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 saponification treatment (saponification degree) × pore diameter. Further, since the membrane structure is composed of acetylcellulose, a solid nucleic acid-adsorbing porous membrane can be obtained.

  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.

  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 saponification rate is preferably about 5% or more, and more preferably 10% or more.

  In order to saponify acetylcellulose, the object to be saponified is immersed in an aqueous sodium hydroxide solution. In order to change the saponification rate, the concentration of sodium hydroxide may be changed. The saponification rate is determined by quantifying residual acetyl groups by NMR.

  In the method for separating and purifying nucleic acid of the present invention, preferably, a nucleic acid-adsorbing porous membrane (for example, a nucleic acid-adsorbing porous membrane made of an organic polymer having a hydroxyl group on the surface) is housed in a container having at least two openings. Nucleic acid can be adsorbed and desorbed using a nucleic acid separation and purification unit.

  More preferably, (a) a nucleic acid-adsorbing porous membrane (for example, a nucleic acid-adsorbing porous membrane made of an organic polymer having a hydroxyl group on the surface), and (b) at least two openings accommodating the nucleic acid-adsorbing porous membrane And (c) a nucleic acid separation / purification unit including a pressure difference generator coupled to one opening of the container.

In this case, the first embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(a) inserting one opening of the nucleic acid separation and purification unit into a sample solution containing nucleic acid;
(b) using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit to draw a sample solution containing nucleic acid under reduced pressure in the container and bring it into contact with the nucleic acid-adsorbing porous membrane;
(c) A step of bringing the container into a pressurized state using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit, and discharging the sample solution containing the aspirated nucleic acid out of the container;
(d) inserting one opening of the nucleic acid separation and purification unit into the nucleic acid washing buffer solution;
(e) a step of aspirating the nucleic acid washing buffer solution by bringing the inside of the container into a reduced pressure state using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit, and contacting the nucleic acid adsorbing porous membrane;
(f) a step of bringing the container into a pressurized state using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit, and discharging the aspirated nucleic acid washing buffer solution out of the container;
(g) inserting one opening of the nucleic acid separation and purification unit into a liquid capable of desorbing the nucleic acid adsorbed on the nucleic acid-adsorbing porous membrane;
(h) The pressure inside the container is reduced using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit, and a solution capable of desorbing the nucleic acid adsorbed on the nucleic acid-adsorbing porous membrane is aspirated, and the nucleic acid Contacting the adsorptive porous membrane; and
(i) Using a pressure difference generator connected to the other opening of the nucleic acid separation and purification unit, the inside of the container is pressurized, and the liquid that can desorb the nucleic acid adsorbed on the nucleic acid-adsorbing porous membrane is discharged out of the container. Process.

The second embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(a) preparing a sample solution containing nucleic acid using a specimen and injecting the sample solution containing the nucleic acid into one opening of the nucleic acid separation and purification unit;
(b) The container is pressurized by using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the sample solution containing the injected nucleic acid is discharged from the other opening to thereby remove the nucleic acid. Contacting the adsorptive porous membrane;
(c) injecting a nucleic acid washing buffer into the one opening of the nucleic acid separation and purification unit;
(d) The container is pressurized using a pressure difference generator connected to the one opening of the nucleic acid separation and purification unit, and the injected nucleic acid washing buffer is discharged from the other opening, whereby the nucleic acid adsorptivity Contacting the porous membrane;
(e) injecting a liquid capable of desorbing the nucleic acid adsorbed on the nucleic acid-adsorbing porous membrane into the one opening of the nucleic acid separation and purification unit;
(f) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and a liquid capable of desorbing the injected nucleic acid is discharged from the other opening, A step of desorbing the nucleic acid adsorbed on the nucleic acid adsorbing porous membrane and discharging it out of the container.

  The method for separating and purifying nucleic acid according to the present invention will be described more specifically. In the present invention, the nucleic acid in the sample solution is preferably adsorbed by bringing the sample solution containing nucleic acid into contact with a nucleic acid-adsorbing porous membrane (for example, a nucleic acid-adsorbing porous membrane made of an organic polymer having a hydroxyl group on the surface). The nucleic acid adsorbed on the porous porous membrane is then desorbed from the nucleic acid adsorbable porous membrane using a suitable solution described below. More preferably, the sample solution containing nucleic acid is obtained by adding a water-soluble organic solvent to a solution in which nucleic acid is dispersed in a solution by treating a sample containing cells or viruses with a solution that dissolves cell membranes and nuclear membranes. It is a solution to which a surfactant is added.

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

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

  For cell membrane lysis and nucleic acid solubilization, for example, when the target sample is whole blood, 1) removal of erythrocytes, 2) removal of various proteins, and 3) lysis of leukocytes and lysis of the nuclear membrane. Necessary. 1) Removal of red blood cells and 2) removal of various proteins should be performed in order to prevent non-specific adsorption to the nucleic acid-adsorbing porous membrane and clogging of the porous membrane. Each is required to solubilize certain 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. For example, the above 1), 2) and 3) can be achieved simultaneously by incubating at 60 ° C. for 10 minutes with guanidine hydrochloride, surfactant and protease K (manufactured by SIGMA) added.

  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 to 6M, preferably 1M to 5M.

  As the surfactant, those described above in this specification can be used. The concentration of the surfactant in the solution is usually 0.05% to 10% by weight, particularly preferably 0.1% to 5% by weight.

  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.

  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 the nucleic acid-adsorbing porous membrane. By this operation, the nucleic acid in the sample solution is adsorbed to the nucleic acid-adsorbing porous membrane (for example, an 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 the nucleic acid-adsorbing porous membrane, the water-soluble organic solvent is mixed with the solubilized nucleic acid mixture, and the obtained nucleic acid mixture It is necessary that salt be present therein.

  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 the nucleic acid in this state is brought into contact with the nucleic acid-adsorbing porous membrane, it interacts between the polar group on the nucleic acid surface and the polar group on the nucleic acid-adsorbing porous membrane, and the nucleic acid is adsorbed on the surface of the nucleic acid-adsorbing porous membrane. It is thought to do. In the method of the present invention, the nucleic acid can be brought into an unstable state by mixing a water-soluble organic solvent with the solubilized nucleic acid mixture and the presence of a salt in the obtained nucleic acid mixture.

  Examples of the water-soluble organic solvent 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.

  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.

  Next, the nucleic acid-adsorbing porous membrane to which the nucleic acid has been adsorbed is brought into contact with the nucleic acid washing buffer solution. This solution has a function of washing out impurities in the sample solution adsorbed on the nucleic acid adsorbing porous membrane together with the nucleic acid. Therefore, it is necessary to have a composition that does not desorb nucleic acids from the porous nucleic acid-adsorbing membrane but desorbs impurities. The nucleic acid washing buffer solution is composed of an aqueous solution containing a main agent, a buffering agent, and, if necessary, a surfactant. As the main agent, an aqueous solution of about 10 to 100% by weight (preferably about 20 to 100% by weight, more preferably about 40 to 80% by weight) such as methanol, ethanol, isopropanol, n-isopropanol, butanol, and acetone is used as a buffering agent. Examples of the surfactant include the above-described buffer and surfactant. Among these, a solution containing ethanol, Tris and a surfactant is preferable. Preferred concentrations of Tris and surfactant are 10 to 100 mM and 0.1 to 10% by weight, respectively.

  Next, the washed nucleic acid adsorbing porous membrane is brought into contact with a solution capable of desorbing the nucleic acid adsorbed on the nucleic acid adsorbing porous membrane. 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.

The nucleic acid separation and purification unit used in the present invention contains a nucleic acid-adsorbing porous membrane (for example, a nucleic acid-adsorbing porous membrane made of an organic polymer having a hydroxyl group on the surface) in a container having at least two openings. Separation and purification unit.
There is no particular limitation on the material of the container, and it is sufficient that the nucleic acid-adsorbing porous membrane can be accommodated and at least two openings can be provided. However, plastic is preferable from the viewpoint of ease of manufacture. For example, it is preferable to use a transparent or opaque resin such as polystyrene, polymethacrylic ester, polyethylene, polypropylene, polyester, nylon, and polycarbonate.

  The container has a housing portion for the nucleic acid-adsorbing porous membrane, and the housing portion can contain the nucleic acid-adsorbing porous membrane so that the nucleic acid-adsorbing porous membrane does not come out of the housing portion when the sample liquid or the like is sucked and discharged. It is sufficient that a pressure difference generating device such as a syringe can be joined to the opening. For this purpose, it is preferable that the container is initially divided into two parts and can be integrated after accommodating the nucleic acid-adsorbing porous membrane. Further, in order to prevent the nucleic acid-adsorbing porous membrane from going out of the accommodating portion, meshes made of a material that does not contaminate DNA can be placed on the upper and lower sides of the nucleic acid-adsorbing porous membrane.

  There is no particular limitation on the shape of the nucleic acid-adsorbing porous membrane accommodated in the container, and in the case of a circle, a square, a rectangle, an ellipse, a membrane, a cylindrical shape, a scroll shape, or an organic polymer having a hydroxyl group on the surface. Any shape such as coated beads may be used, but from the viewpoint of production suitability, highly symmetrical shapes such as circles, squares, cylinders, and scrolls, and beads are preferred.

  One opening of the container is inserted into a sample solution containing nucleic acid, and the sample solution is brought into contact with the nucleic acid-adsorbing porous membrane by sucking from the other opening, and then discharged, and then the nucleic acid washing buffer solution is sucked. The target nucleic acid can be obtained by discharging and then sucking and discharging a solution capable of desorbing the nucleic acid adsorbed on the nucleic acid-adsorbing porous membrane, and collecting the discharged liquid.

  The nucleic acid separation and purification unit used in the present invention contains (a) a nucleic acid adsorbing porous membrane (for example, a nucleic acid adsorbing porous membrane made of an organic polymer having a hydroxyl group on the surface) and (b) the nucleic acid adsorbing porous membrane. And a container having at least two openings, and (c) a pressure difference generator coupled to one opening of the container. Hereinafter, the nucleic acid separation and purification unit will be described.

  The container is usually produced in a manner divided into a main body for accommodating the nucleic acid-adsorbing porous membrane and a lid, and each is provided with at least one opening. One is used as an inlet and an outlet for a sample solution containing nucleic acid, a nucleic acid washing buffer solution, and a liquid (hereinafter referred to as “sample solution etc.”) that can desorb nucleic acid adsorbed on the nucleic acid-adsorbing porous membrane. Is connected to a pressure difference generator capable of reducing the pressure in the container. The shape of the main body is not particularly limited, but it is preferable to make the cross section circular in order to facilitate manufacture and to facilitate diffusion of the sample solution and the like over the entire surface of the nucleic acid-adsorbing porous membrane. It is also preferable to make the cross section quadrangular in order not to generate cutting scraps of the nucleic acid adsorbing porous membrane.

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

  The internal volume of the container is determined only by the amount of the sample solution to be processed, but is usually represented by the volume of the nucleic acid-adsorbing porous membrane to be accommodated. That is, it is preferable to have a size that accommodates about 1 to 6 nucleic acid-adsorbing porous membranes having a thickness of about 1 mm or less (for example, about 50 to 500 μm) and a diameter of about 2 mm to 20 mm.

  The end surface of the nucleic acid-adsorptive porous membrane is preferably in close contact with the inner wall surface of the container so that the sample solution or the like does not pass through.

  Under the nucleic acid-adsorbing porous membrane facing the opening used for the sample solution entrance, a space is provided without being in close contact with the inner wall of the container, so that the sample solution etc. diffuses as evenly as possible across the entire surface of the nucleic acid-adsorbing porous membrane. To make the structure.

  On the nucleic acid-adsorbing porous membrane facing the other opening, that is, the opening coupled to the pressure difference generator, a member having a hole in the center is preferably provided. This member has the effect of holding down the nucleic acid-adsorptive porous membrane and efficiently discharging the sample solution, etc., and has a sloped shape such as a funnel or bowl so that the liquid collects in the central hole. It is preferable to do. The size of the hole, the angle of the slope, and the thickness of the member can be appropriately determined by those skilled in the art in consideration of the amount of the sample solution to be processed, the size of the container that contains the nucleic acid-adsorbing porous membrane, and the like. . It is preferable to provide a space between the member and the opening for storing the overflowed sample solution or the like and preventing the sample solution from being sucked into the pressure difference generator. The size of this space can also be appropriately selected by those skilled in the art. In order to efficiently collect nucleic acids, it is preferable to aspirate a sample solution containing nucleic acid in an amount more than the entire nucleic acid-adsorbing porous membrane is immersed.

  In addition, the sample solution is prevented from concentrating only in the part directly under the aspirating opening, so that the sample solution can pass through the nucleic acid adsorbing porous membrane relatively uniformly. It is preferable to provide a space between the membrane and this member. For this purpose, it is preferable to provide a plurality of protrusions from the member toward the nucleic acid-adsorptive porous membrane. The size and number of the protrusions can be appropriately selected by those skilled in the art, but it is preferable to keep the opening area of the nucleic acid-adsorbing porous membrane as large as possible while maintaining the space.

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

  The pressure difference generator first sucks a sample solution containing nucleic acid by reducing the pressure in the container containing the nucleic acid-adsorbing porous membrane. Examples of the pressure difference generating device include a pump capable of suction and pressurization such as a syringe, a pipetter, or a peristaltic pump. Of these, a syringe is suitable for manual operation, and a pump is suitable for automatic operation. Also, the pipetter has the advantage that it can be easily operated by one hand. Preferably, the pressure difference generator is detachably coupled to one opening of the container.

  Next, a nucleic acid purification method using the above-described nucleic acid separation and purification unit will be described. First, one opening of the nucleic acid separation and purification unit is inserted into a sample solution containing nucleic acid. Next, the inside of the purification unit is depressurized using a pressure difference generator connected to the other opening, and the sample solution is sucked into the container. By this operation, the sample solution comes into contact with the nucleic acid adsorbing porous membrane, and the nucleic acid in the sample solution is adsorbed on the nucleic acid adsorbing porous membrane. At this time, it is preferable to suck an amount of the sample solution in contact with almost the entire nucleic acid-adsorptive porous membrane. However, if the sample solution is sucked into the pressure difference generating device, the device is contaminated, so the amount is adjusted to an appropriate amount.

  After sucking an appropriate amount of the sample solution, the inside of the unit container is pressurized using a pressure difference generator, and the sucked liquid is discharged. It is not necessary to leave an interval before this operation, and it may be discharged immediately after suction.

  Next, the nucleic acid washing buffer solution is sucked into the container by the same decompression-pressurization operation as described above, and discharged from the container to wash the inside of the container. This solution has a function of washing away the sample solution remaining in the container and washing out impurities in the sample solution adsorbed on the nucleic acid-adsorbing porous membrane together with the nucleic acid. Therefore, it is necessary to have a composition that does not desorb nucleic acids from the porous nucleic acid-adsorbing membrane but desorbs impurities. The nucleic acid washing buffer solution is composed of an aqueous solution containing a main agent, a buffering agent, and, if necessary, a surfactant. The main agent is about 10 to 90% (preferably about 50 to 90%) such as methyl alcohol, ethyl alcohol, butyl alcohol, acetone and the like. 90%) of the aqueous solution, the buffering agent and the surfactant include the above-described buffering agent and surfactant.

Next, a solution capable of desorbing the nucleic acid adsorbed on the nucleic acid-adsorptive porous membrane is introduced into the container by the same vacuum-pressurization operation as described above, and discharged from the container. 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).
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1
(1) Preparation of Nucleic Acid Separation / Purification Container A nucleic acid separation / purification container having an inner diameter of 7 mm and a portion accommodating the nucleic acid-adsorbing porous membrane was prepared from high impact polystyrene.

(2) Preparation of nucleic acid separation and purification cartridge A porous membrane obtained by saponifying a porous membrane of triacetyl cellulose is used as the nucleic acid adsorptive porous membrane, and is accommodated in the nucleic acid adsorptive porous membrane storage section of the nucleic acid separation and purification container prepared above. did.

(3) Preparation of Nucleic Acid Solubilizing Reagent Solution and Washing Solution Nucleic acid solubilizing reagent solution and washing solution having the formulation shown in Table 1 were prepared.

(Table 1)
Nucleic acid solubilizing reagent solution (Example of the present invention)
382 g guanidine hydrochloride (Life Technology)
Tris (Life Technology) 12.1g
POE lauryl ether (Kao) 10g
1000ml distilled water

Cleaning liquid
10 mM Tris-HCl 65% ethanol

(4) Nucleic acid purification operation A cancerous human bone marrow cell (HL60) culture solution was prepared. 200 μl of the nucleic acid solubilizing reagent solution described in Table 1 and 20 μl of protease K (manufactured by SIGMA) solution are added to 200 μl of this culture solution and incubated at 60 ° C. for 10 minutes. After incubation, add 200 μl of ethanol and stir. After stirring, the mixture is injected into one opening of the nucleic acid separation / purification cartridge having the nucleic acid adsorbing porous membrane prepared in (1) and (2), and then a pressure difference generator is connected to the one opening to separate the nucleic acids. The inside of the purification cartridge is pressurized, and the sample solution containing the injected nucleic acid is passed through the nucleic acid-adsorbing porous membrane to be brought into contact with the nucleic acid-adsorbing porous membrane and discharged from the other opening of the nucleic acid separation and purification cartridge. Subsequently, a washing liquid is injected into the one opening of the nucleic acid separation and purification cartridge, a pressure difference generator is connected to the one opening, the inside of the nucleic acid separation and purification cartridge is brought into a pressurized state, and the injected washing liquid is adsorbed to the nucleic acid. Through the porous porous membrane and discharged from the other openings. Subsequently, the recovered liquid is injected into the one opening of the nucleic acid separation / purification cartridge, and a pressure difference generator is connected to the one opening of the nucleic acid separation / purification cartridge so that the inside of the nucleic acid separation / purification cartridge is pressurized and injected. The recovered liquid was passed through the nucleic acid-adsorptive porous membrane, discharged from other openings, and this liquid was recovered.

  Further, as a comparative example, a solution containing a nucleic acid is obtained by operating under the same conditions as above except that the nucleic acid solubilizing reagent solution shown in Table 2 is used instead of the nucleic acid solubilizing reagent solution shown in Table 1. Was recovered.

(Table 2)
Nucleic acid solubilizing reagent solution (comparative example)
382 g guanidine hydrochloride (Life Technology)
Tris (Life Technology) 12.1g
1000ml distilled water

(5) Quantification of recovery amount of nucleic acid The experiment of the above-mentioned Examples and Comparative Examples was repeated twice. The amount of DNA recovered was quantified by measuring the light absorption of the recovered liquid at 260 nm. Table 3 shows the measurement results.

(Table 3)
229.8 259.6 280.3 320 399.8 260/280
Comparative example (first time) 0.260 0.140 0.110 0.01 0.00 1.857
(Second time) 0.330 0.183 0.131 0.01 0.04 1.803
Example (first time) 0.460 0.390 0.230 0.010 0.020 1.727
(Second time) 0.320 0.390 0.220 0.010 0.010 1.810

  As is apparent from the results in Table 3, it can be seen that by using the method of the present invention, nucleic acids can be efficiently recovered and purified with excellent reproducibility.

Claims (30)

A nucleic acid comprising a step of adding a nucleic acid solubilizing reagent solution containing a surfactant to a sample containing cells or viruses, mixing the sample and the nucleic acid solubilizing reagent solution, and adding a water-soluble organic solvent to the mixed solution. A method for preparing a sample solution. (A) A step of injecting a specimen containing cells or viruses into a container, (b) a nucleic acid solubilizing reagent solution containing a chaotropic salt, a surfactant and a proteolytic enzyme is added to the container, and the specimen and the nucleic acid solubilizing reagent are added. The method according to claim 1, comprising a step of mixing the solution, (c) incubating the mixed solution obtained in step (b), and (d) adding a water-soluble organic solvent to the incubated mixed solution. . The method according to claim 1 or 2, wherein the surfactant is one type of nonionic surfactant. The method according to claim 1 or 2, wherein the surfactant is one type of cationic surfactant. The method according to claim 1 or 2, wherein the surfactant is one type of anionic surfactant. The method according to claim 1 or 2, wherein the surfactant is one type of amphoteric surfactant. The method according to claim 1 or 2, wherein the surfactant comprises two types, a nonionic surfactant and an anionic surfactant. The method according to claim 1 or 2, wherein the surfactant comprises two types, a nonionic surfactant and a cationic surfactant. The method according to claim 1 or 2, wherein the surfactant comprises two types, a nonionic surfactant and an amphoteric surfactant. The method in any one of Claims 1-3 or 7-9 whose nonionic surfactant is surfactants other than a polyoxyethylene alkylphenyl ether type surfactant. The method in any one of Claims 1-3 or 7-9 whose nonionic surfactant is a polyoxyethylene alkyl ether type surfactant. The method according to any one of claims 1 to 3 or 7 to 9, wherein the nonionic surfactant is a fatty acid alkanolamide. The method according to claim 1, wherein the cationic surfactant is cetyltrimethylammonium bromide. The method according to any one of claims 1, 2, 4 and 8, wherein the cationic surfactant is dodecyltrimethylammonium chloride. The method according to any one of claims 1, 2, 4 and 8, wherein the cationic surfactant is tetradecyltrimethylammonium chloride. The method according to claim 1, wherein the cationic surfactant is cetylpyridinium chloride. The method of claim 2, wherein the chaotropic salt is a guanidinium salt. 18. A process according to any of claims 1 to 17, wherein the water-soluble organic solvent is selected from methanol, ethanol, isopropanol, n-propanol or mixtures thereof. The method according to claim 1, wherein the nucleic acid in the sample solution does not form a precipitate due to the water-soluble organic solvent. The nucleic acid in the sample solution containing the nucleic acid prepared by the method according to any one of claims 1 to 19, using a nucleic acid separation and purification unit containing a nucleic acid-adsorbing porous membrane in a container having at least two openings. A method for separating and purifying nucleic acid, wherein adsorption and desorption are performed. (a) a nucleic acid-adsorbing porous membrane, (b) a container having at least two openings for accommodating the nucleic acid-adsorbing porous film, and (c) a pressure difference generator coupled to one opening of the container. A method for separating and purifying nucleic acid, wherein a nucleic acid separation and purification unit is used to adsorb and desorb nucleic acid in a sample solution containing the nucleic acid prepared by the method according to any one of claims 1 to 19. (A) A nucleic acid-adsorbing porous membrane capable of allowing a sample solution containing a nucleic acid prepared by the method according to any one of claims 1 to 19 to pass through a container having at least two openings by a pressure difference (B) injecting into the nucleic acid separation / purification cartridge using the pressure difference generator connected to the one opening of the nucleic acid separation / purification cartridge. And allowing the sample solution containing the injected nucleic acid to pass through the nucleic acid-adsorbing porous membrane and discharging it from the other opening of the nucleic acid separation and purification cartridge, thereby adsorbing the nucleic acid in the nucleic acid-adsorbing porous membrane, (c) A step of injecting a washing solution into the one opening of the cartridge for nucleic acid separation and purification; (d) a nucleic acid separation and purification cart using a pressure difference generator connected to the one opening of the nucleic acid separation and purification cartridge; A step of washing the nucleic acid-adsorbing porous membrane in a state where nucleic acids are adsorbed by making the inside of the wedge pressurized and passing the injected cleaning solution through the nucleic acid-adsorbing porous membrane and discharging from the other openings, (E) a step of injecting the recovery liquid into the one opening of the nucleic acid separation and purification cartridge; and (f) the inside of the nucleic acid separation and purification cartridge using the pressure difference generator coupled to the one opening of the nucleic acid separation and purification cartridge. The pressurized recovery solution is passed through the nucleic acid-adsorbing porous membrane and discharged from the other openings, thereby desorbing the nucleic acid from the nucleic acid-adsorbing porous membrane and discharging it out of the cartridge for nucleic acid separation and purification. A method for separating and purifying nucleic acid, comprising a step. The method according to any one of claims 20 to 22, wherein the nucleic acid-adsorptive porous membrane is a porous membrane made of an organic polymer that adsorbs nucleic acids with a weak interaction not involving ionic bonds. The method according to claim 23, wherein the porous film made of an organic polymer is a porous film made of an organic polymer having a hydroxyl group. The method according to claim 24, wherein the porous membrane made of an organic polymer having a hydroxyl group is a porous membrane obtained by saponifying a porous membrane of acetylcellulose. The method according to claim 22, wherein the cleaning liquid is a solution containing 20 to 100% by weight of methanol, ethanol, isopropanol or n-propanol. The method according to claim 22, wherein the recovered liquid is a solution having a salt concentration of 0.5M or less. The method according to claim 22, wherein the pressure difference generating device is a pump removably coupled to one opening of the nucleic acid separation and purification cartridge. An apparatus for performing the method for separating and purifying nucleic acid according to any one of claims 20 to 28. A reagent kit for performing the method for separating and purifying nucleic acid according to any one of claims 20 to 28.