JP2006230237A - Method and apparatus for isolating and purifying nucleic acids - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method that can obtain sample solutions including nucleic acids having reproducibility efficiently, simply, rapidly with excellent automation adequacy in the isolation and purification method for nucleic acids in which the nucleic acids in a sample solution including nucleic acids is adsorbed to a nucleic acid-adsorbing porous membrane, then desorbed via washing and the like and the nuleic acids are isolated and purified. <P>SOLUTION: In the nucleic acid isolation and purification method comprising step (1) where sample solution including nucleic acids is prepared; step (2) where the sample solution is allowed to contact with a solid phase so that the nucleic acids are adsorbed to the solid phase; step (3) where the washing solution is allowed to contact with the solid phase and the solid phase is washed as the nucleic acids are adsorbed; step (4) where the recovering solution is allowed to contact with the solid phase to adsorb the nucleic acids from the solid phase, the particle size of the nucleic acid particles in the sample solution is adjusted to less than 1μm in the step (1) where the sample solution including nucleic acids is prepared. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for obtaining a sample solution containing a nucleic acid from a specimen in order to separate and purify the nucleic acid. More specifically, the present invention relates to a method and apparatus for separating and purifying nucleic acid from a sample containing nucleic acid, wherein the radius of nucleic acid particles in a sample solution containing nucleic acid is adjusted to 1 μm or less.

  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 an excellent method for separating and purifying a nucleic acid, a method is widely known in which a nucleic acid is adsorbed on a solid phase such as silicon dioxide, silica polymer, magnesium silicate, etc., and purified by subsequent operations such as washing and desorption. (Patent Document 1)

Further, in a method for separating and purifying nucleic acid including a process of adsorbing and desorbing nucleic acid on a solid phase, an organic polymer having a hydroxyl group on the surface is used as the solid phase, and the solid phase is placed in a container having two openings. A method of using a housed nucleic acid separation and purification unit has been developed. (Patent Document 2)
According to this method, not only high-purity nucleic acid can be separated from a sample solution containing nucleic acid, but also it is excellent in simplicity, rapidity, and automation, and industrial mass production of adsorption media with the same performance is possible. And is easy to handle and can be processed into various shapes.

Japanese Patent Publication No. 7-51065 JP 2003-128691 A

However, depending on the sample solution containing the nucleic acid, not only does it take a long time to separate and purify the nucleic acid, but also the yield of the nucleic acid is greatly reduced, and there is a problem in its reproducibility.
Therefore, the present invention is an efficient, simple and rapid method for separating and purifying nucleic acid by adsorbing nucleic acid in a sample solution containing nucleic acid to a nucleic acid-adsorbing porous membrane 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.

In the method for obtaining a sample solution containing nucleic acid from a specimen, which is performed prior to the separation and purification of nucleic acid including the process of adsorbing and desorbing nucleic acid on a porous membrane, the inventor reduces the radius of nucleic acid particles in the sample solution to 1 μm or less It has been found that the purification operation can be performed promptly without causing clogging of the filter by adjusting.
Therefore, the present invention is particularly characterized in that the radius of the nucleic acid particles in the sample solution containing the nucleic acid is adjusted to 1 μm or less.

[1]
(1) preparing a sample solution containing nucleic acid;
(2) contacting the sample solution with a solid phase to adsorb nucleic acid to the solid phase;
(3) bringing the washing solution into contact with the solid phase and washing the solid phase with the nucleic acid adsorbed; and (4) bringing the recovery solution into contact with the solid phase to desorb the nucleic acid from the solid phase. In a method for separating and purifying nucleic acid comprising a step,
A method for separating and purifying nucleic acid, characterized in that, in step (1) of preparing a sample solution containing nucleic acid, the diameter of the nucleic acid in the sample solution is adjusted to 1 μm or less.
[2]
The method for separating and purifying nucleic acid according to [1], wherein the particle diameter of the nucleic acid in the sample solution is 0.5 μm or less.
[3]
The method for separating and purifying nucleic acid according to [1], wherein the particle diameter of the nucleic acid in the sample solution is 0.13 μm or less.
[4]
In the step (1) of preparing the sample solution containing the nucleic acid, a pretreatment liquid containing at least one selected from a chaotropic salt, a surfactant, an antifoaming agent, a nucleic acid stabilizer and a buffer is added to the sample containing the nucleic acid. After the addition, the method for separating and purifying nucleic acid according to any one of [1] to [3], wherein a water-soluble organic solvent is added for adjustment.
[5]
The method for separating and purifying nucleic acid according to any one of [1] to [4], wherein the solid phase is in the form of a membrane.
[6]
The method for separating and purifying nucleic acid according to any one of [1] to [5], wherein the water-soluble organic solvent contains at least one selected from methanol, ethanol, propanol and butanol.
[7]
The method for separating and purifying nucleic acid according to any one of [1] to [6], wherein the solid phase contains silica or a derivative thereof, diatomaceous earth, or alumina.
[8]
The method for separating and purifying nucleic acid according to any one of [1] to [6], wherein the solid phase contains an organic polymer.
[9]
The solid phase is a film containing any one or more of Teflon, polyester, polyethersulfone, polycarbonate, polyacrylate copolymer, polyurethane, polybenzimidazole, polyolefin, polyvinyl chloride, and polyvinylidene fluoride. The method for separating and purifying nucleic acid according to any one of [1] to [6].
[10]
The method for separating and purifying nucleic acid according to any one of [1] to [6], wherein the solid phase is a membrane containing a nylon membrane having a positive or negative charge.
[11]
The method for separating and purifying nucleic acid according to [8], wherein the organic polymer is an organic polymer having a polysaccharide structure.
[12]
The method for separating and purifying nucleic acid according to [8], wherein the organic polymer is a membrane containing any one or more of cellulose, cellulose mixed ester, cellulose nitrate, and cellulose acetate nitrocellulose.
[13]
An apparatus for adjusting the diameter of a nucleic acid in a sample solution containing nucleic acid to 1 μm or less, a solid phase composed of an organic polymer having a hydroxyl group on the surface, a container having at least two openings for accommodating the solid phase, and the container A nucleic acid separation and purification unit comprising a pressure difference generating device coupled to one opening of the first.

  According to the present invention, by adjusting the radius of nucleic acid particles in a sample solution containing nucleic acid to 1 μm or less, nucleic acid is separated and purified from a sample containing nucleic acid with good separation performance, in a simple, rapid and automatable manner. be able to.

The present invention includes (1) a step of preparing a sample solution containing nucleic acid, (2) a step of bringing the sample solution into contact with a solid phase and adsorbing the nucleic acid to the solid phase, and (3) a washing solution on the solid phase. In a method for separating and purifying nucleic acid, comprising the steps of washing the solid phase with the nucleic acid adsorbed thereon, and (4) contacting the recovered liquid with the solid phase to desorb the nucleic acid from the solid phase. In the step (1) of preparing a sample solution containing nucleic acid, the nucleic acid abduct diameter in the sample solution is adjusted to 1 μm or less.
The nucleic acid particle diameter can be measured by measuring the particle diameter with a dynamic light scattering photometer.
The nucleic acid particle diameter in the sample solution is more preferably 0.5 μm or less, and still more preferably 0.13 μm or less.
In the step (1) of preparing the sample solution containing the nucleic acid, a pretreatment liquid containing at least one selected from a chaotropic salt, a surfactant, an antifoaming agent, a nucleic acid stabilizer and a buffer in the sample containing the nucleic acid. After adding, adjust by adding a water-soluble organic solvent.
As the water-soluble organic solvent, methanol, ethanol, propanol and butanol can be used.
In the present invention, the nucleic acid abduct diameter in the sample solution containing nucleic acid can be adjusted to 1 μm or less, for example, by appropriately adjusting the concentration of the water-soluble organic solvent added to the sample solution.

As the solid phase, for example, one containing silica or a derivative thereof, diatomaceous earth, or alumina can be used, and one containing an organic polymer can be used. Further, a film shape is suitable, and specifically, Teflon, polyester, polyethersulfone, polycarbonate, polyacrylate copolymer, polyurethane, polybenzimidazole, polyolefin, polyvinyl chloride, polyvinylidene fluoride, A film containing any one or more can be used as appropriate. It is also preferable that the solid phase is a membrane including a nylon membrane having a positive or negative charge.
As the organic polymer, an organic polymer having a polysaccharide structure can be used. For example, it is also preferable that the film contains at least one of cellulose, cellulose mixed ester, cellulose nitrate, and cellulose acetate nitrocellulose.
Hereinafter, (1) a method for separating and purifying nucleic acid and (2) a unit for separating and purifying nucleic acid as preferred forms for carrying out the present invention will be described in more detail.

(1) Nucleic Acid Separation / Purification Method The following nucleic acid separation / purification method suitable for the present invention includes a step of adsorbing and desorbing nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface. In the present invention, the “nucleic acid” may be either single-stranded or double-stranded, and there is no molecular weight limitation.

  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. It is preferred to use surface saponified acetylcellulose 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).

  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 to introduce 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.

  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. 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.

  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.

  In the method for separating and purifying nucleic acid, 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. Can do.

  More preferably, (a) a solid phase composed 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.

The first embodiment of the method for separating and purifying nucleic acid in this case can include the following steps.
(a) a step of inserting one opening of the nucleic acid separation and purification unit into a sample solution containing nucleic acid; (b) a reduced pressure inside the container using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit A step of aspirating a sample solution containing a nucleic acid and bringing the sample solution into contact with a solid phase comprising an organic polymer having a hydroxyl group on the surface; (c) using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit A step of pressurizing the container and discharging the sample solution containing the aspirated nucleic acid out of the container; (d) a step of inserting one opening of the nucleic acid separation and purification unit into the nucleic acid washing buffer solution; and (e) a nucleic acid. Using a pressure difference generator connected to the other opening of the separation and purification unit to reduce the pressure in the container and sucking the nucleic acid washing buffer solution into contact with a solid phase comprising an organic polymer having a hydroxyl group on the surface; (f) Nucleic acid separation and purification unit A step of bringing the container into a pressurized state using a pressure difference generator coupled to another opening and discharging the aspirated nucleic acid washing buffer solution to the outside of the container; (g) one opening of the nucleic acid separation and purification unit; A step of inserting a nucleic acid adsorbed on a solid phase comprising an organic polymer having a hydroxyl group on the surface into a liquid capable of desorption, (h) using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit A step of depressurizing the inside of the container and sucking a liquid capable of desorbing a nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface and bringing it into contact with the solid phase; and (i) a nucleic acid separation and purification unit Using a pressure difference generator connected to another opening, the inside of the container is pressurized, and a liquid capable of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface is discharged out of the container. Process.

The second embodiment of the method for separating and purifying nucleic acid 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) passing through the one opening of the nucleic acid separation and purification unit; The container is pressurized using a combined pressure difference generator, and the sample solution containing the injected nucleic acid is discharged from the other opening to come into contact with a solid phase composed of an organic polymer having a hydroxyl group on the surface. (C) injecting a nucleic acid washing buffer into the one opening of the nucleic acid separation / purification unit; (d) using a pressure difference generator connected to the one opening of the nucleic acid separation / purification unit; A step of bringing the injected nucleic acid washing buffer into contact with a solid phase composed of an organic polymer having a hydroxyl group on the surface by discharging the injected nucleic acid washing buffer from the other opening, and (e) the one opening of the nucleic acid separation and purification unit On the surface A step of injecting a liquid capable of desorbing a nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group; (f) using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit; Is put under pressure, and the liquid that can desorb the injected nucleic acid is discharged from the other opening, so that the nucleic acid adsorbed on the solid phase composed of organic polymer having a hydroxyl group on the surface is desorbed and discharged out of the container. Process.

  The method for separating and purifying nucleic acid using an organic polymer having a hydroxyl group on the surface will be described more specifically. Preferably, the nucleic acid in the sample solution is adsorbed to the solid phase by bringing the sample solution containing the nucleic acid into contact with a solid phase composed of an organic polymer having a hydroxyl group on the surface. Desorb from the solid phase using a suitable solution as described in. More preferably, the sample solution containing a nucleic acid is a solution obtained by adding a water-soluble organic solvent to a solution in which a nucleic acid is dispersed in a solution by treating a specimen containing cells or viruses with a solution that dissolves a cell membrane and a nuclear membrane. is there.

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

  First, these specimens are treated with an aqueous solution containing a reagent that dissolves the cell membrane and solubilizes the nucleic acid. As a result, the cell membrane and the nuclear membrane are dissolved, and the nucleic acid is dispersed in the aqueous solution. 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 membranes Must be dissolved. (1) Removal of red blood cells and (2) removal of various proteins should be performed in order to prevent non-specific adsorption to the solid phase and clogging of the porous membrane. Each is required to solubilize certain nucleic acids. In particular, (3) lysis of leukocytes and nuclear membrane lysis are important steps, and it is necessary to solubilize nucleic acids in this step. The above (1), (2) and (3) can be achieved simultaneously by incubating at 60 ° C. for 10 minutes with guanidine hydrochloride, Triton X-100 and protease K (manufactured by SIGMA) added.

  Examples of the nucleic acid solubilizing reagent 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 0.5 mol / L or more and 6 mol / L or less, preferably 1 mol / L or more and 5 mol / L or less.

  As the surfactant, Triton X-100 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. It is preferable to use a nonionic surfactant such as polyoxyethylene octylphenyl ether (Triton X-100). The concentration of the surfactant in the solution is usually 0.05% by mass to 10% by mass, particularly preferably 0.1% by mass to 5% by mass.

  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.

An antifoaming agent, a nucleic acid stabilizer, a buffering agent and the like can also be used in the sample solution containing the nucleic acid.
Examples of the buffering agent include “Chemical Handbook Basic” edited by The Chemical Society of Japan (Maruzen Co., Ltd., 1966), pages 1312-1320, R.C. M.M. C. Dawson et al, “Data for Biochemical Research”, 2nd edition (Oxford at the Clarendon Press, 1969), “Biochemistry,” pages 467-477 (1966), “Analytical 104” There is a pH buffer system described in pages 300-310 (1980). Examples of pH buffer systems include buffers containing borate; buffers containing citric acid or citrate; buffers containing glycine; buffers containing bicine; buffers containing HEPES; Good buffering agents such as buffering agents.

  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 the present 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 mixed solution. It is necessary that a salt be present in the obtained nucleic acid mixture.

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

  Examples of the water-soluble organic solvent used here include methanol, ethanol, isopropanol, propanol, and butanol, and ethanol is preferable. The concentration of the water-soluble organic solvent is preferably 5% by mass to 90% by mass, and more preferably 20% by mass to 60% by mass. The concentration of ethanol added is particularly preferably as high as possible without causing pseudo-collects.

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

  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 nucleic acid washing buffer solution. This 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. 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 mass (preferably about 20 to 100% by mass, more preferably about 40 to 80% by mass) 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 Triton X-100 is preferable. Preferred concentrations of Tris and Triton X-100 are 10 to 100 mmol / L and 0.1 to 10% by mass, respectively.

  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 acid preferably has a low salt concentration, and particularly preferably a solution having a salt concentration of 0.5 mol / L or less. As this solution, purified distilled water, TE buffer or the like can be used.

(2) Nucleic acid separation / purification unit The nucleic acid separation / purification unit of the present invention is a nucleic acid separation / purification unit in which a solid phase composed of an organic polymer having a hydroxyl group on the surface is contained in a container having at least two openings. There is no particular limitation on the material of the container, and it is sufficient that an organic polymer having a hydroxyl group can be accommodated on the surface 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.

  A conceptual diagram of the container is shown in FIG. Basically, it has a solid phase container, the solid phase can be stored in the container, and the solid phase does not go out of the container when sucking and discharging sample liquid etc. For example, what is necessary is just to join a syringe. For this purpose, it is preferred that the container is initially divided into two parts and can be integrated after the solid phase is accommodated. Further, in order to prevent the solid phase from going out of the housing unit, a mesh made of a material that does not contaminate DNA can be placed above and below the solid phase.

  There is no particular limitation on the shape of the organic polymer having a hydroxyl group on the surface accommodated in the container, and in the case of a circle, a square, a rectangle, an ellipse, a membrane, a cylinder, a scroll, or an organic compound having a hydroxyl group on the surface. Although any shape such as a polymer-coated bead may be used, a highly symmetrical shape such as a circle, a square, a cylinder, or a scroll shape and a bead are preferable from the viewpoint of production suitability.

  One opening of the container is inserted into a sample solution containing nucleic acid, the sample solution is brought into contact with an organic polymer having a hydroxyl group on the surface by suction from the other opening, and then discharged, and then the nucleic acid washing buffer The target nucleic acid can be obtained by sucking and discharging the solution, and then sucking and discharging the solution capable of desorbing the nucleic acid adsorbed on the organic polymer having a hydroxyl group on the surface and collecting the discharged liquid. it can.

  A target nucleic acid can be obtained by sequentially immersing an organic polymer having a hydroxyl group on a surface in a sample solution containing a nucleic acid, a nucleic acid washing buffer solution, and a solution capable of desorbing a nucleic acid adsorbed on the organic polymer having a hydroxyl group on the surface. Can be obtained.

  The nucleic acid separation and purification unit of the present invention comprises (a) a solid phase comprising an organic polymer having a hydroxyl group on the surface, (b) a container having at least two openings for containing the solid phase, and (c) the container It is preferable that the pressure difference generator connected to one opening is included. Hereinafter, the nucleic acid separation and purification unit will be described.

  The container is usually prepared in a manner divided into a main body that contains a solid phase composed of an organic polymer having a hydroxyl group on the surface 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 solution capable of desorbing nucleic acid adsorbed on a solid phase (hereinafter referred to as “sample solution etc.”), and the other is in a container. Is connected to a pressure difference generator capable of causing the pressure to be reduced or increased. The shape of the main body is not particularly limited, but it is preferable to make the cross section circular in order to facilitate the manufacture and to facilitate the diffusion of the sample solution and the like over the entire surface of the solid phase. It is also preferable to make the cross section square in order not to generate solid-state cutting waste.

  The lid needs to be joined to the main body so that the inside of the container can be depressurized and pressurized by 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 solid phase accommodated. That is, it is preferable that the thickness is about 1 mm or less (for example, about 50 to 500 μm) and the size is such that about 1 to 6 solid phases having a diameter of about 2 mm to 20 mm can be accommodated.

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

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

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

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

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

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

  Next, a 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 solid phase, and the nucleic acid in the sample solution is adsorbed on the solid phase. At this time, it is preferable to suck an amount of the sample solution that comes into contact with almost the entire solid phase. However, if the sample solution is sucked into the pressure difference generator, the device is contaminated.

  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 solid phase together with the nucleic acid. Therefore, it is necessary to have a composition that does not desorb nucleic acids from the solid phase but desorbs impurities.

  Next, a solution capable of desorbing the nucleic acid adsorbed on the solid phase is introduced into the container by the same decompression-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).

  FIG. 2 is a cross-sectional view of an example of the nucleic acid separation and purification unit of the present invention. However, the pressure difference generator is not shown. A container 1 that contains a solid phase is composed of a main body 10 and a lid 20 and is made of transparent polystyrene. The main body 10 contains a surface saponified triacetyl cellulose film as a solid phase 30. Further, an opening 101 for sucking a sample solution or the like is provided. A bottom surface 102 continuing from the opening is formed in a funnel shape, and a space 121 is provided between the solid phase 30. In order to support the solid phase 30 and maintain the space 121, a frame 103 integrated with the bottom surface 102 is provided.

  The main body has an inner diameter of 20.1 mm, a depth of 5.9 mm, and a length from the bottom surface 102 to the opening 101 of about 70 mm. The built-in solid phase 30 has a diameter of 20.0 mm, a thickness of about 50 to 500 μm, and an example of the thickness is 100 μm.

  In FIG. 2, a funnel-shaped pressing member 13 is provided on the upper part of the solid phase. A hole 131 is provided at the center of the pressing member 13, a group of protrusions 132 are provided below, and a space 122 is provided between the solid member 30. The diameter of the upper portion of the wall 104 is made larger than the diameter of the solid phase so that the sample solution or the like is not easily leaked from between the solid phase 30 and the wall 104 of the main body 10, and the end of the pressing member 13 is placed on the step 105. Yes.

  The lid 20 is joined to the main body 10 by ultrasonic heating. An opening 21 that couples the pressure difference generator is provided in a substantially central portion of the lid 20. Between the lid 20 and the pressing member 13, a space 123 for holding a sample solution or the like flowing out from the hole 131 is provided. The volume of the space 123 is about 0.1 ml.

(1) Preparation of Nucleic Acid Separation / Purification Container A nucleic acid purification container having an inner diameter of 7 mm and containing a solid phase for nucleic acid adsorption and having two openings is made of polypropylene.

(2) Nucleic acid separation and purification unit As the nucleic acid-adsorbing porous membrane, a porous membrane of acetylcellulose is used and accommodated in the nucleic acid-adsorbing porous membrane housing part of the nucleic acid purification cartridge prepared in (1) above. A porous membrane having an average pore diameter of 2 μm is used.

(3) Preparation of DNA solubilizing reagent and washing solution A DNA solubilizing reagent and washing solution having the formulation shown in Table 1 are prepared.

(4) Nucleic acid purification operation 5 μg of λDNA (Clontech) was dissolved in 100 μl of TE buffer to obtain an aqueous DNA solution. To this, 100 μl of a DNA solubilizing reagent having the formulation shown in Table 1 was added and stirred.
After stirring, 800 μl of various concentrations of ethanol shown in Table 2 were added and stirred.

  Then, the particle diameter of the nucleic acid particles of the nucleic acid-containing reagent treated as described above was measured with a dynamic light scattering photometer (DLS7000). The results are shown in Table 3.

  As is apparent from Table 3, the particle diameter of the nucleic acid particles is increased by increasing the concentration of added ethanol.

  After the measurement, the nucleic acid-containing sample treated as described above is injected into one opening of a nucleic acid purification unit having a porous organic polymer membrane made of a mixture of ethyl cellulose prepared in (1) and (2) above. Subsequently, a pressure difference generator is coupled to the one opening, the inside of the nucleic acid separation and purification unit is pressurized, and the sample solution containing the injected nucleic acid-containing sample is passed through the porous membrane, thereby allowing the porous It is brought into contact with the membrane and discharged from the other opening of the nucleic acid separation and purification unit. Subsequently, a washing solution is injected into the one opening of the nucleic acid separation and purification unit, a pressure difference generator is coupled to the one opening, the inside of the nucleic acid separation and purification unit is pressurized, and the injected washing solution is added to the porous Pass through the membrane and discharge through other openings. Subsequently, the recovered liquid is injected into the one opening of the nucleic acid separation and purification unit, and a pressure difference generator is connected to the one opening of the nucleic acid separation and purification unit so that the inside of the nucleic acid separation and purification cartridge is pressurized and injected. The recovered liquid is passed through the porous membrane, discharged from the other opening, and the liquid is recovered.

(5) Confirmation of separation and purification of DNA The 260 nm absorption spectrum of the collected solution is measured to determine the yield of DNA.
The measurement results are shown in Table 4.

  Moreover, it shows in Table 5 about the liquid passage time at that time.

  As is clear from Tables 4 and 5, in the samples having a large nucleic acid particle diameter (Level 3 and Level 4), the liquid passage time becomes extremely long and the DNA yield is low.

It is a conceptual diagram of a nucleic acid separation and purification unit. It is an example of a nucleic acid separation and purification unit.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container 10 Main body 101 Opening 102 Bottom surface 103 Frame 104 Wall 105 Level difference 121,122,123 Space 13 Holding member 131 Hole 132 Protrusion 20 Lid 21 Opening 30 Solid phase

Claims (13)

(1) preparing a sample solution containing nucleic acid;
(2) contacting the sample solution with a solid phase to adsorb nucleic acid to the solid phase;
(3) bringing the washing solution into contact with the solid phase and washing the solid phase with the nucleic acid adsorbed; and (4) bringing the recovery solution into contact with the solid phase to desorb the nucleic acid from the solid phase. In a method for separating and purifying nucleic acid comprising a step,
A method for separating and purifying nucleic acid, characterized in that, in step (1) of preparing a sample solution containing nucleic acid, the diameter of the nucleic acid in the sample solution is adjusted to 1 μm or less.   The method for separating and purifying nucleic acid according to claim 1, wherein the nucleic acid particle diameter in the sample solution is 0.5 µm or less.   The method for separating and purifying nucleic acid according to claim 1, wherein the nucleic acid particle diameter in the sample solution is 0.13 µm or less.   In the step (1) of preparing the sample solution containing the nucleic acid, a pretreatment liquid containing at least one selected from a chaotropic salt, a surfactant, an antifoaming agent, a nucleic acid stabilizer and a buffer is added to the sample containing the nucleic acid. The method for separating and purifying nucleic acid according to any one of claims 1 to 3, wherein the method is adjusted by adding a water-soluble organic solvent after the addition.   The method for separating and purifying nucleic acid according to any one of claims 1 to 4, wherein the solid phase is in the form of a membrane.   The method for separating and purifying nucleic acid according to any one of claims 1 to 5, wherein the water-soluble organic solvent contains at least one selected from methanol, ethanol, propanol and butanol.   The method for separating and purifying nucleic acid according to any one of claims 1 to 6, wherein the solid phase contains silica or a derivative thereof, diatomaceous earth, or alumina.   The method for separating and purifying nucleic acid according to claim 1, wherein the solid phase contains an organic polymer.   The solid phase is a film containing any one or more of Teflon, polyester, polyethersulfone, polycarbonate, polyacrylate copolymer, polyurethane, polybenzimidazole, polyolefin, polyvinyl chloride, and polyvinylidene fluoride. The method for separating and purifying nucleic acid according to any one of claims 1 to 6.   The method for separating and purifying nucleic acid according to any one of claims 1 to 6, wherein the solid phase is a membrane containing a nylon membrane having a positive or negative charge.   The method for separating and purifying nucleic acid according to claim 8, wherein the organic polymer is an organic polymer having a polysaccharide structure.   9. The method for separating and purifying nucleic acid according to claim 8, wherein the organic polymer is a membrane containing at least one of cellulose, cellulose mixed ester, cellulose nitrate, and cellulose acetate nitrocellulose.   An apparatus for adjusting the diameter of a nucleic acid in a sample solution containing nucleic acid to 1 μm or less, a solid phase composed of an organic polymer having a hydroxyl group on the surface, a container having at least two openings for accommodating the solid phase, and the container A nucleic acid separation and purification unit comprising a pressure difference generating device coupled to one opening of the first.

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