JP2002212198A - Method for separating nucleic acid and carrier for separating nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for rapidly and simply separating a high-purity nucleic acid in a large amount from a sample containing the nucleic acid and to obtain a solid phase used for the separation. SOLUTION: This method for separating the nucleic acid from the sample is characterized by bringing the sample containing the nucleic acid into contact with a water-insoluble solid phase having unevennesses on the surface in the presence of a water-soluble organic solvent, adsorbing the nucleic acid on the solid phase and thereby separating the nucleic acid from the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for rapidly and simply separating a large amount of high-purity nucleic acid from a nucleic acid-containing sample and a solid phase used for the separation.

[0002]

2. Description of the Related Art As a method for obtaining a nucleic acid from a sample containing the nucleic acid, a phenol / chloroform extraction method has been used for a long time. This method uses phenol / chloroform to denature, dissolve, or precipitate poorly water-soluble analyte components such as proteins and lipids, and uses the difference in solubility to dissolve nucleic acids in an aqueous phase. Also, without using toxic organic solvents, solutes such as proteins and lipids are solubilized in the aqueous phase using a chaotropic solution, the nucleic acids are adsorbed on silica beads, and the nucleic acids are collected in a solid phase. The Boom method of recovering in the aqueous phase is known. However, in these methods, when the extracted nucleic acid is used as a template for polymerase chain reaction (PCR) or when digested with a restriction enzyme, the enzyme reaction is inhibited by phenol / chloroform or chaotropic ions remaining in the nucleic acid solution. May be done. In that case, it was necessary to purify the nucleic acid again by ethanol precipitation, ultrafiltration, column chromatography or the like. Also,
Since phenol and chloroform are toxic, there are restrictions on the place of use and experimental equipment, and centrifugation, which requires several times, has made it difficult to cope with mechanization. Further, in the method using a chaotropic solution and a silica solid phase, the amount of a specimen that can be treated is less than 1 ml, and as the volume of the specimen increases, the purity of the recovered nucleic acid tends to be significantly reduced. This is due to the adsorption capacity of the silica solid phase used. Increasing the surface area of the silica solid phase is not an effective workaround because the residual amount of chaotropic ions increases with the capacity of the solid phase. As shown in JP-A-8-173159, a cationic polymer, polyethyleneimine, is added to a nucleic acid-containing lysate to form a water-insoluble salt with the nucleic acid, and the precipitate is recovered. In this method, as a method for recovering nucleic acid from the precipitate, an operation of adding a strongly basic solution such as sodium hydroxide solution to the precipitate to dissociate the polyethyleneimine and the nucleic acid, An operation of adding a solution of an anionic surfactant and an operation of neutralizing the pH of the solution are required in order to prevent recombination between the solution and the nucleic acid, and it is hardly simple. Also, the addition of an alkali such as a sodium hydroxide solution may cause hydrolysis of nucleic acids, and furthermore, mixed polyethyleneimine and anionic surfactants inhibit enzymatic reactions such as PCR, but are difficult to remove. When used in a PCR reaction, dilution must be performed until its inhibitory effect can be neglected. However, when the nucleic acid content is low, its detection may be hindered. It has long been known that a nucleic acid-containing solution is precipitated with an ethanol solution and wound up with a glass rod (for example, Koichi Anan, Basic Biochemistry Experimental Method-2, Maruzen 1974).
This method also has factors based on the affinity of the nucleic acid for glass and nucleic acid, but the more critical factor is the strong physical and geometric entanglement of the nucleic acid precipitate with the glass rod. That is, when the amount of precipitated nucleic acid is small, or when the amount of nucleic acid is not enough to precipitate, even if the nucleic acid is present in the same ethanol solution, the nucleic acid cannot be wound by the glass rod. Since the affinity between the nucleic acid and the glass rod is low, the purity of the wound nucleic acid is low, and contamination of proteins and the like other than the nucleic acid is observed.

[0003]

SUMMARY OF THE INVENTION The present invention does not use a toxic solvent such as conventional phenol / chloroform and does not use a corrosive reagent such as chaotropic agent. Blood, cells, tissues,
A means for extracting and purifying a large amount from a source such as a plant is provided.

[0004]

According to the present invention, a nucleic acid-containing sample is brought into contact with a solid phase having irregularities on its surface (hereinafter simply referred to as "solid phase") in the presence of a water-soluble organic solvent to convert the nucleic acid. An object of the present invention is to provide a method for separating nucleic acids, comprising separating nucleic acids from a sample by causing the nucleic acids to adsorb to the solid phase.

[0005] The material for forming the solid phase of the present invention may be insoluble in water. The term water-insoluble as used herein specifically refers to water,
A solid phase that does not dissolve in an aqueous solution containing any other water-soluble composition is meant. Specifically, it includes an inorganic compound, a metal, a metal oxide, an organic compound, or a composite material combining these. Examples of the inorganic compound include glass, alumina, silicon nitride, and the like; examples of the metal include stainless steel and zirconia; and examples of the organic compound include polymers such as polystyrene, polypropylene, polyethylene, polyamide, and polymethyl methacrylate. Of these, organic polymers, especially polystyrene, are preferred. When the surface roughness of the solid phase of the present invention is represented by a normal maximum expression method of surface roughness, the maximum height is preferably 0.005 to 100 μm. If the maximum height is less than 0.005 μm or exceeds 100 μm, the size does not match the size of the initial precipitate of nucleic acid, and efficient nucleic acid extraction cannot be performed. The maximum height is a method of expressing surface roughness defined in the JIS standard (B0601). Specifically, for example, the roughness of the solid phase surface is measured by a stylus method, a part of the obtained irregular surface curve is selected as a reference length, and the parallel two-dimensional length including the maximum irregular part of the part is selected.
A straight line is drawn, and the interval between the two straight lines is used as the maximum height value. In the solid phase of the present invention, the method of making the surface uneven is different depending on the material used. For example, in the case of an inorganic material such as glass, hydrofluoric acid treatment, which is a method for producing frosted glass, can be diverted as it is. In the case of an organic polymer, a method in which the surface of the formed polymer is polished with a sandper or a method in which the surface is polished is used. Providing the surface of the solid phase with irregularities also increases the specific surface area of the solid phase. The shape of the solid phase of the present invention includes particles, tubes, plates, tubes, containers and the like,
Particles are particularly preferred. As the shape of the particles, for example, a spherical shape, a cubic shape, an elliptical shape, a rectangular shape, or the like can be used. When the solid phase of the present invention is particles, the Stokes diameter of the particles is usually 0.01 mm to 50 mm, particularly 0.05 to 15 mm.
Is preferred. When the Stokes diameter of the particles is less than 0.01 mm, the dispersion stability of the solid phase becomes difficult. On the other hand, when the Stokes diameter exceeds 50 mm, handling of the solid phase, particularly in automation, becomes difficult with current equipment. The Stokes diameter in the present invention is an effective diameter when the solid phase is a particle, and the diameter of the solid phase having a shape other than spherical is the diameter of a spherical carrier having the same Stokes diameter as the diameter determined according to Stokes law. Was considered.

In the present invention, the water-soluble organic solvent is selected from the group consisting of methanol, ethanol, propanol, propanol, isopropanol, butanol and 2-butanol. Among them, ethanol and isopropanol are particularly preferred. The concentration of these water-soluble organic solvents at the time of nucleic acid extraction is 20 to 99% by weight, preferably 40 to 95% by weight.
If necessary, a salt, a surfactant and the like may be added to the water-soluble organic solvent. For example, a sodium salt, a potassium salt, a magnesium salt, or sodium dotesylsulfonate is used. The concentration of the salt to be added is 0.1-50m
M, particularly preferably 0.5 to 10 mM. Salt concentration
When the concentration is less than 0.1 mM, the effect of the added salt to precipitate nucleic acids is reduced, and when the concentration exceeds 0.50 mM, precipitation of proteins and the like having high water affinity other than nucleic acids is not preferred. Further, the concentration of the surfactant is preferably 0.01 to 1% by weight, more preferably 0.05 to 0.5% by weight. Surfactant concentration of 0.01% by weight
When it is less than the above, the micelle effect of the hydrophobic protein is weakened,
If it exceeds 1% by weight, precipitation of the surfactant is caused, which is not preferable. The concentrations of the salt and the surfactant are based on the total amount of the sample and the water-soluble organic solvent.

[0007] The sample containing the nucleic acid of the present invention is obtained by lysing cells of a sample selected from the group consisting of blood, tissue, cell culture solution, and bacteria-containing solution. Nucleic acid-containing cells are separated by the method described above. For example, in the case of blood, the whole blood is centrifuged, the collected puffy coat (layer formed of lymphocytes) is hemolyzed with ammonia chloride, and the collected lymphocyte cells are lysed before being used as a sample of the present invention. use. In the case of targeting tissue cells, a known method or a commercially available kit suitable for each tissue can also be used.

[0008] The operation of lysing the cells varies depending on the type of the specimen cells, but a known adaptation method can be used. For example, a cell membrane destruction method using osmotic pressure, an ultrasonic method using physical energy, a chemical method using a surfactant, or a combination thereof may be used. In particular, depending on the chemical method, some reagents may affect the affinity between nucleic acid and glass.
Anionic surfactants, especially sodium dodecyl sulfonate, are preferred. The concentration of the surfactant is preferably 0.1 to 10% by weight, particularly preferably 0.25 to 5% by weight. Further, in order to increase the degree of separation between nucleic acid and protein, the cell lysate can be treated with a protease. Also, in order to make the distinction between DNA and RNA more complete, digest the DNA or RNA with a DNAse or RNase and then use the remaining RNA.
Alternatively, DNA extraction may be performed.

In the present invention, the nucleic acid is adsorbed on the solid phase by bringing the sample containing the nucleic acid into contact with the solid phase in the presence of the water-soluble organic solvent. The mixing order of the sample containing the nucleic acid, the solid phase, and the water-soluble organic solvent is not limited. The solid phase varies depending on its shape and surface roughness, but in the case of beads, generally about 1 to 20 particles per 5 ml of sample can be used. The time required for adsorption of the nucleic acid to the solid phase is usually 0.5 to 20 minutes, preferably 5 to 15 minutes after mixing the water-soluble organic solvent, the sample containing the nucleic acid and the solid phase. The adsorption temperature is 10 to 60 ° C, preferably 15 to 40 ° C.
It is. Especially when extracting long nucleic acids such as genomes
It is preferable to lengthen the adsorption step time. When stirring the reaction solution in the adsorption step, usually 0.5 to 20 rotations / minute,
Preferably, the speed is 1 to 5 revolutions / minute.

After the step of adsorbing the nucleic acid to the solid phase is completed, the solid phase is separated from the reaction solution, and the separated solid phase is washed. The washing liquid may be the same solvent as the water-soluble organic solvent used in the adsorption step, or may be another water-soluble organic solvent. Also, if the adsorption of nucleic acids to the solid phase can be maintained,
If necessary, the concentration of the water-soluble organic solvent may be reduced.

The nucleic acid adsorbed on the solid phase is dried after the washing. The drying temperature is usually preferably from room temperature to 60 ° C., and the drying time is preferably from 5 to 20 minutes. Here, air drying is preferable as the drying method. The nucleic acid adsorbed on the solid phase can be eluted into the liquid layer by adding sterile distilled water Tris-HCl / EDTA buffer to the dried solid phase and stirring.

In the practice of the present invention, when the amount of nucleic acid is small, it is preferable to carry out the batch method.
To increase the nucleic acid extraction rate, a column method is preferable. The particle size of the solid phase used can be selected according to the embodiment.

[0013]

Hereinafter, embodiments of the present invention will be described.
The present invention is not limited to these examples. In the examples, parts and% are by weight. Example 1 Sample preparation of a sample of whole blood 10 ml of heparin-containing whole blood was centrifuged at 3000 rpm for 10 minutes to separate plasma and blood cells. After removing the plasma, 2 ml of puffy coat was collected. To 2 ml of puffy coat, 8 ml of 0.2 M amonium chloride was added, and the mixture was slowly stirred 2-3 times, and then left standing in ice water for 10 minutes to perform hemolysis. Subsequently lymphocytes precipitate was collected at 3000 rpm x5 minutes, after recovered lymphocytes were washed twice with PBS, and were counted cells was 10 ⁷ / ml. Lysis of lymphocytes Take 200ul of lymphocytes collected above, and add 20mM Tris-HCl
(pH8) /0.2MNaCl/0.2%SDS/10mM CaCl2 / 0.1% proteinase K
After adding 200 ul of the same volume to the solution and gently stirring, the mixture was incubated at 50 ° C. for 1 hour to obtain a nucleic acid-containing sample. When confirmed by an optical microscope, this operation resulted in almost no debris of the lymphocytes and a transparent homogeneous solution. Separation of nucleic acid To a tube containing 200 μl of the nucleic acid-containing sample obtained above, 500 μl of an ethanol solution was added, and three polystyrene beads having a Stokes diameter of 6 mm (manufactured by Immunochemical Co., Ltd., maximum surface roughness of 5 μm) were added. The mixture was slowly rotated and stirred for 10 minutes. Subsequently, the supernatant was discarded, 0.5 ml of 70% ethanol was added, and the mixture was stirred up and down three times, and then the supernatant was discarded. The polystyrene beads were allowed to stand in the tube for 10 minutes, and after confirming that the ethanol solution around the polystyrene beads had evaporated, 100 μl of TE buffer was added, and the mixture was incubated at room temperature for 10 minutes to elute nucleic acids. The amount of recovered DNA was calculated from the ultraviolet absorbance at 260 nm (A260), and the purity was determined to be 260 nm.
The ratio of absorbance at 280 nm (A260 / A280) and 0.8% agarose gel electrophoresis were confirmed.
It was digested with 2 units of restriction enzyme HindIII, and the digestion results after the enzyme treatment were also confirmed by electrophoresis.

Example 2 Example 1 was the same as Example 1 except that isopropyl alcohol was used instead of ethanol.
And went exactly the same. The amount of the recovered DNA was confirmed in the same manner as in Example 1.

Example 3 In Example 1, the same nucleic acid-containing sample as in Example 1 was used.
Add 500ul propanol solution to the tube containing 200ul,
A nucleic acid was separated in exactly the same manner as in Experimental Example 1 except that one alumina ball having a diameter of 10 mm (manufactured by Takizawa Rika Co., Ltd., maximum height of surface roughness: 0.01 μm) was added and the mixture was slowly rotated and stirred at room temperature for 10 minutes. . The amount of the recovered DNA was confirmed in the same manner as in Example 1.

Example 4 In Example 1, 2000 μl of a methanol solution was added to the tube containing 200 μl of the nucleic acid-containing sample in Example 1, and one zirconia ball (Nikkado, maximum surface roughness 10 μm) having a diameter of 20 mm was added. In addition, nucleic acids were separated in exactly the same manner as in Experimental Example 1 except that the mixture was slowly rotated and stirred at room temperature for 10 minutes. The amount of the recovered DNA was confirmed in the same manner as in Example 1.

Example 5 In Example 1, 200 μl of the nucleic acid-containing sample was used in Example 1.
Add 200ul of pentanol solution to the tube containing
Nucleic acids were separated in exactly the same manner as in Experimental Example 1 except that ten 0.5-mm silicon nitride balls (Iguchi Seimeido, maximum surface roughness of 0.05 μm) were added, and the mixture was slowly rotated and stirred at room temperature for 10 minutes. . The amount of the recovered DNA was confirmed in the same manner as in Example 1.

Reference Example 200 ul of the same nucleic acid-containing sample as that obtained in Example 1 was extracted by the phenol / chloroform method. Extraction method is Molecule Clo
ning (J. Sambrook, CSH Publishing). The amount of the recovered DNA was confirmed in the same manner as in Example 1. Comparative Example In Example 1, 2000 μl of an ethanol solution was added to a tube containing 200 μl of the nucleic acid-containing sample of Example 1, and the diameter was 6.4 m.
m nylon pace (Iguchi Seikeido, maximum surface roughness 0.
005 μm), and nucleic acids were separated in exactly the same manner as in Experimental Example 1 except that the mixture was slowly rotated and stirred at room temperature for 10 minutes. The amount of the recovered DNA was confirmed in the same manner as in Example 1.

[0019]

[Table 1]

[0020]

The unevenness on the order of microns is particularly consistent in size with the initial precipitates of nucleic acids, so that the microscopic contact effect between the nucleic acids and the solid phase of the present invention is significantly enhanced. The present invention utilizes the difference in the deposition rates of nucleic acids and components other than nucleic acids in a water-soluble organic solvent, the difference in the rate of formation of precipitates, and allows the initial precipitates of nucleic acids to be adsorbed on the solid phase surface of the solid phase of the present invention, Separation from other components was enabled. By using the nucleic acid separation method of the present invention, a large amount of high-purity nucleic acid can be rapidly and easily purified from a sample, and is used in fields such as genetic engineering, genetic diagnosis, gene therapy, genomic chemistry, and genomic drug discovery. Can be widely applied.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Mikio Hijikata Within JAE S.R. Co., Ltd., 2-11-24 Tsukiji, Chuo-ku, Tokyo (72) Inventor Koei Sato Takatani 2965 −37 F term (reference) 4B024 AA11 AA20 CA02 HA11 HA19 HA20 4C057 AA05 BB02 BB05 DD01 MM02 MM04 4D017 AA03 BA07 CA01 CA05 CA13 CB01

Claims (5)

[Claims] 1. A method comprising separating a nucleic acid from a sample by bringing a sample containing nucleic acids into contact with a water-insoluble solid phase having irregularities on its surface in the presence of a water-soluble organic solvent, and adsorbing the nucleic acids to the solid phase. A method for separating nucleic acids. 2. The method for separating nucleic acids according to claim 1, wherein the unevenness of the solid phase has a surface roughness of 0.005 to 100 μm when expressed by the maximum height method. 3. The method according to claim 1, wherein the solid phase has a Stokes diameter of 0.01 mm to 50 mm.
2. The method for separating nucleic acids according to claim 1, wherein the particles are mm particles. 4. The method according to claim 1, wherein the water-soluble organic solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, 2-butanol, pentanol and 2-pentanol. A method for separating nucleic acids. 5. A carrier for nucleic acid separation comprising a water-insoluble solid phase having irregularities on the surface.