JP4196185B2 - Nucleic acid separation method - Google Patents

Description

  The present invention relates to a method for separating nucleic acid from a sample containing nucleic acid and a reagent used for carrying out the method. More specifically, a nucleic acid purification method for separating a target nucleic acid by solid-liquid separation by subjecting a sample containing nucleated cells or the like to enzymatic treatment of a cell lysis solution and depositing the nucleic acid on a solid phase carrier, and a method thereof It is related with the kit for implementing.

In recent years, the use of nucleic acid analysis has been spreading to various fields such as scientific research, medical care, and industry, and a method capable of extracting and purifying a large amount of high-purity nucleic acid from various samples in an efficient manner is required.
As a method for obtaining nucleic acid from a sample containing 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 sample components such as proteins and lipids. Utilizing the difference in solubility that a highly hydrophilic nucleic acid is dissolved in an aqueous phase. Although this method can extract high-purity nucleic acid, phenol / chloroform is toxic, so it is not suitable for nucleic acid extraction from a large amount of specimens due to limitations of working environment.
As an alternative method that does not use such a toxic solvent, a chaotropic solution is used to solubilize proteins, lipids, and other contaminants in the aqueous phase, the nucleic acid is adsorbed on silica beads and recovered in the solid phase, and then the nucleic acid is re-watered. There is a Boom method (Boom et al. J. Clin. Microbiol. 28: 495-503 (1990)) that dissolves in a phase. In this method, the extracted nucleic acid is often short and fragmented. In particular, it is not suitable for DNA analysis methods such as Southern plot.
In addition to these, reagents that coprecipitate with DNA using cationic polymers have also been developed. However, the separation of DNA involves fine adjustment of the reagent, and a centrifugal operation is required for liquid-liquid separation. Since it is indispensable from the beginning to the end and the efficiency of routine processing of a large number of samples is low, utilization in industry does not progress.

  Further, in the method using a chaotropic solution and a silica carrier, the amount of specimen that can be processed is less than 1 mL, and the purity of the recovered nucleic acid tends to be significantly lowered as the specimen volume increases. This is due to the adsorption ability of the silica support used. If the amount of silica carrier used is increased, the remaining amount of chaotropic salt in the solid-liquid separation will also increase due to the dead volume proportional to the amount of carrier used. This can be dealt with by increasing the number of times of cleaning, but as a result, the operation time increases and the operation process becomes complicated, which is not an effective solution.

  On the other hand, a method of precipitating a nucleic acid-containing solution with an ethanol solution and winding it with a glass rod has been known for a long time (for example, Koichi Anan, “Basic Biochemical Experimental Method 2” Maruzen, 1974). Although there are factors based on the affinity between glass and nucleic acid, this technique has some practical applications due to the contamination of macromolecules other than nucleic acid and the low purity of recovered nucleic acid.

  Thus, a method that can easily and quickly extract and purify nucleic acids from various biological samples containing nucleic acids with high purity without using toxic solvents or corrosive reagents, and is open to automation. Has been. In view of such circumstances, as a result of intensive research, the present inventors have clarified nucleic acid in a sample by lysing nucleated cells and subjecting the water-soluble organic solvent to a certain treatment with an enzyme or the like. In addition, by utilizing the fact that a coprecipitation state is formed together with the fine particles, simple solid-liquid separation is realized, and the problems of the conventional method are solved at a stretch and the present invention is completed. Based on the method according to the present invention, an operation for separating nucleic acid from a blood sample with high purity can be automatically processed.

  The present invention does not use conventional toxic solvents such as phenol and chloroform, and does not use corrosive reagents such as chaotropic substances. The object is to provide a new means for quickly extracting.

The present invention relates to a method for separating and purifying nucleic acid from a sample containing nucleated cells, and to provide a reagent therefor. Specifically 1) A step of contacting a sample containing nucleated cells with a lysis solution containing at least a cell component degrading enzyme and a surfactant,
2) A sample containing nucleated cells is brought into contact with a water-insoluble solid phase carrier having an average particle size of 0.01 to 1000 μm in the presence of a water-soluble organic solvent, and the nucleic acid released from the nucleated cells is immobilized on the solid phase. Adsorbing and bonding to the surface of the phase carrier; and
3) It relates to a nucleic acid separation method comprising a step of separating the solid phase carrier from a sample.
The present invention provides a reagent for separating and purifying nucleic acid from a sample containing nucleated cells, which comprises at least the cell component degrading enzyme, a water-insoluble solid phase carrier, and a water-soluble organic solvent.

Hereinafter, this method will be described together with means for carrying out the method.
In the present specification, the “nucleic acid” means DNA and / or RNA as a double strand (ds), a single strand (ss), or a combination thereof (partial ds or ss).

Nucleated cells that are the subject of the present invention are widely present in nature and are not limited to animal species. Specifically, blood, bone marrow, umbilical cord blood, tissue, cultured cells, urine, saliva and the like are the target samples of the present invention. Depending on the type of these specimens, a physical operation may be applied before the present invention is applied so that the nucleated component is easily released. For example, a specimen collected from an organ or tissue can be applied to the present invention after the tissue structure is crushed with a mixer or the like.
Further, the shape of the specimen, the specimen amount, etc. that can be applied to the present invention are not particularly limited. As long as the tissue is crushed, it may be in the form of pellets, dissolved in an aqueous solution, or dispersed.

  In the sense of minimizing the degradation of the nucleic acid contained in the target specimen of the present invention, the collected specimen is preferably left as it is, and preferably applied in a fresh state, but when it must be stored unavoidably, It may be refrigerated or frozen. For example, in the case of a blood sample, one that has been refrigerated for about one week can be applied to the present invention.

  The present invention is particularly suitable for nucleic acid extraction from a blood sample. The blood sample may be normal peripheral blood, arterial blood, venous blood, or a tangible (blood cell) component collected after centrifugation or the like, buffy coat (blood Inflammatory scab, leukocyte fraction). When targeting whole blood and blood components, the following steps of hemolysis and subsequent leukocyte separation are required. In the present invention, the type of blood collection tube is not particularly limited, and any of heparin blood collection tubes, EDTA blood collection tubes, citric acid blood collection tubes and the like may be used. Furthermore, the blood volume that can be processed in the present invention is not particularly limited, and can correspond to a very wide range of blood volume of several μL to several hundred mL.

  In the operations of cell lysis and nucleic acid adsorption (and elution), it is desirable that the temperature, time, and the like be in accordance with predetermined conditions from the viewpoint of preventing unnecessary side reactions and leukocyte or nucleic acid damage. Hereinafter, each process of the present invention will be described.

-Process of hemolysis and leukocyte separation The amount of blood nucleic acid depends on the number of white blood cells. The hemolysis operation can be separated from white blood cells by centrifugation by destroying red blood cells that do not have nuclei but are present in large amounts in the blood (ie, hemolysis). The hemolysis method is not particularly limited, and examples thereof include a method using osmotic pressure such as isotonicity and hypotonicity, a physical destruction method, and a heat shock method by rapid heating. In the present invention, a method combining the heat shock method is particularly preferable.
A method by allowing a hemolytic agent to act on a blood sample is preferred. Known hemolytic agents include ammonium chloride, ammonium oxalate, saponin and the like. Of these, those containing at least 0.01 to 0.5 M ammonium chloride are suitable as hemolytic agents that exhibit a remarkable hemolytic effect under mild conditions. In addition, a buffer for controlling pH, such as Tris buffer or phosphate buffer, sodium chloride for adjusting osmotic pressure, salts such as potassium chloride, magnesium for preventing destruction of leukocytes as necessary. A salt, a chelating agent such as EDTA for inhibiting DNA degradation, or a saccharide such as saccharose may be added. In addition, when using a buffer together, the density | concentration can be suitably selected in the range of several mM to several hundred mM.

  In general, in the case of whole blood, the hemolytic agent is preferably 0.1 to 30 times the volume, more preferably 2 to 10 times the volume of one blood sample obtained by separating blood cells and plasma or serum by centrifugation. Use minutes. When the amount used is less than 0.1 volume, the hemolytic effect is dilute and the effectiveness is poor. On the other hand, when the volume exceeds 30 times, the operation capacity becomes large, and the entire processing efficiency including the efficiency of the centrifugation operation is remarkably lowered. For effective red blood cell lysis, it is desirable to use a hemolytic agent containing 0.01-0.5 M ammonium chloride that has been warmed to 30-85 ° C, preferably 40-70 ° C. If the temperature is lower than 30 ° C, the hemolytic effect is low, and if the temperature exceeds 85 ° C, the amount of denatured protein in the blood increases rapidly and precipitates together with the target white blood cells during centrifugation, so the purity of white blood cells tends to decrease. Become prominent. When the hemolysis operation is performed under the above conditions, only red blood cells are completely destroyed, and the white blood cells can be recovered without being damaged.

In order to separate white blood cells from the hemolyzed sample, a commonly used centrifugation is suitable. Centrifugation conditions such as the number of rotations, time, and temperature may be appropriately selected according to the state of the sample.
-Step of cell lysis The lysis operation of leukocytes can use a known method for decomposing cell membranes, nuclear membranes, proteins and the like to release nucleic acids from the cell nuclei into the lysate.
For this purpose, for example, osmotic pressure, shear stress, freeze crushing, cell membrane destruction method using mechanical force by grinding agent, ultrasonic method using physical energy, various surfactants, modification Chemistry using bioagents or enzymes, biochemical methods, or a combination of these.

A preferred operation in the method of the present invention is to enzymatically digest leukocytes in order to obtain cell contents containing the target nucleic acid from the separated leukocytes. The enzymatic digestion is performed by adding a lysis solution containing a cell component degrading enzyme and a surfactant to a sample containing leukocytes.
Lysis solution It is preferable that the cell lysis solution, ie, the lysis solution, contains at least one kind of cell component degrading enzyme and the concentration of each enzyme is 0.01 to 50 mg / ml. The enzyme concentration here is a concentration when a reagent having an enzyme purity of 80% or more is used.
In the present invention, the cell component degrading enzyme is at least one selected from the group consisting of amylase, lipase, protease, and nuclease. On the other hand, an enzyme concentration of 0.01 mg / ml or less is not preferable because the efficiency for decomposing components other than nucleic acids contained in cells is extremely low. If it is 50 mg / ml or more, the possibility that the enzyme itself is mixed with the recovered nucleic acid is not preferable.

  An amylase as used herein is an enzyme that degrades a glycosyl compound belonging to 3.2 according to an enzyme classification method. Specifically, α amylase, β amylase, glucoamylase, isoamylase and the like are exemplified. These may be used alone or in combination. Lipases are esterolytic enzymes belonging to 3.1 according to the enzyme classification method.

  Protease belongs to 3.4 according to the enzyme classification method, and is a general term for enzymes that specifically hydrolyze peptide bonds, and generally includes proteinases, peptide hydronases, peptidases, pronases and the like. By including such a proteolytic enzyme, the degree of separation between the nucleic acid and the protein can be increased. Specific examples of protease include trypsin, chymotrypsin, pepsin, protease K, pronase, papain and their similar types, subsystems, and the like. These may be used alone or in combination.

Nucleases are classified into 3.1 according to the enzyme classification method, and are a general term for enzymes that hydrolyze DNA and RNA polynucleotide chains. In the method according to the present invention, both DNA and RNA can be extracted. When the nucleic acid to be recovered is DNA, RNA nuclease is used. When RNA is recovered, DNA nuclease is used to promote extraction and separation of these nucleic acids.
These cell component degrading enzymes may be those extracted from living organisms, or may be enzymes prepared by gene recombination techniques. Many of these enzymes are also commercially available.
By adding these cell component degrading enzymes, components other than the target nucleic acid can be decomposed by an enzymatic reaction, so that high-purity nucleic acids can be separated. Fragmentation of components other than nucleic acids amplifies solubility in organic solvents. In addition, even when components other than nucleic acids precipitate due to the addition of an organic solvent, unlike the target nucleic acid, which is a macromolecule, it becomes a fine mass, so that it does not adhere to, deposit on, or adsorb to the solid phase carrier of the present invention. It can be easily removed by solid-liquid separation and washing.
On the other hand, since the amount of the added enzyme is very small compared to the cell component, the solid phase surface is blocked by the cell component, so that the added enzyme component is not strongly adsorbed on the solid phase surface. It is easily removed by ordinary solid-liquid separation and washing, is released into the nucleic acid solution, and is not extracted by mixing with the target nucleic acid.

  Examples of the surfactant used in the lysis solution include an anion, a cation, and a nonionic surfactant. Among these, it is desirable to use an anionic surfactant from the viewpoint of promoting the destruction efficiency of the leukocyte membrane and the subsequent enzyme reaction efficiency. Specifically, sodium dodecyl sulfate, sodium laurate, sodium dodecylbenzenesulfonate, sodium sulfate, higher alcohol sodium sulfate, ammonium lauryl sulfate, and the like are preferable. These may be used alone or in combination. The surfactant concentration in the lysis solution is preferably 0.01 to 15% (w / v), more preferably 0.25 to 10% (w / v).

Furthermore, if necessary, a buffer solution (pH 5-9) for controlling pH such as Tris buffer, phosphate buffer, citrate buffer, salts such as sodium chloride, potassium chloride, lithium chloride, sodium acetate, etc. May be added. In addition, when using a buffer together, the density | concentration can be suitably selected in the range of several mM to several hundred mM.
Cell Lysis Operation by Enzymatic Digestion In the present invention, the above-mentioned multiple types of enzymes may coexist in the lysis solution and may be added at the same time to treat white blood cells, or the enzymes may be added and processed in order before and after. May be. When using a plurality of enzymes, it is preferable to adjust the concentration of each enzyme in the range of 0.01 to 50 mg / ml, preferably 0.1 to 10 mg / ml. When using these enzymes, it goes without saying that the buffer solution, salt, ionic species, surfactant and the like are appropriately adjusted according to the type of enzyme used to maximize the enzyme activity.

In the present invention, the enzyme treatment conditions may be 30 to 85 ° C., preferably 40 to 60 ° C., and heated for 0.1 to 10 hours with stirring. In particular, for a specimen with a lot of white blood cells, stronger stirring is preferable in order to reduce the viscosity of the reaction system. In the present invention, it is preferable to add a solid phase carrier to the reaction system because a stronger stirring effect can be obtained than with a normal vortex. In addition, if the temperature of the enzyme treatment is less than 30 ° C., the treatment time is undesirably long. Exceeding 85 ° C. is not preferable because the enzyme itself tends to denature.
-Adsorption process to a solid phase carrier Usually, by contacting a nucleic acid from a sample containing cell contents after the cell lysis treatment with a solution containing a water-soluble organic solvent containing a solid phase carrier disclosed below. It is adsorbed on the solid support and separated from other polymer contaminants.

Solid phase carrier The composition of the solid phase carrier of the present invention is not particularly limited. Fine particles composed of organic, inorganic, or organic / inorganic composites that are generally commercially available, or fine particles composed of glass, ceramics, metal or metal oxide, or composites thereof. It may be.

The shape of the solid phase carrier of the present invention is not particularly limited. Usually, particles are desirable. Examples of the shape of the particle include a sphere, an ellipsoid, a cone, a cube, and a rectangular parallelepiped. Of these, the carrier of spherical particles is preferable because it is easy to produce and is easy to rotate and stir the solid support at the time of use. For a carrier having a shape other than a spherical shape, the Stokes diameter is preferably in the range of 0.01 to 1000 μm, and more preferably in the range of 0.1 to 100 μm. When the diameter is less than 0.01 μm, it takes time to recover the particles, which is not preferable. On the other hand, when the diameter exceeds 1000 μm, it tends to be affected by factors other than the particle size, such as the particle surface condition.
The “Stokes diameter” in the present invention is an effective diameter when the solid phase carrier is a particle, and the size of the solid phase carrier having a shape other than a spherical shape is the same as the Stokes law. It shall be regarded as the diameter of a spherical carrier having a diameter. Specifically, the sedimentation velocity is obtained from a Stokes equation in which a carrier having a shape other than a spherical shape is dispersed in an aqueous solution and centrifuged. For spherical particles made of the same material, the sedimentation rate is similarly determined, and the particle size of the spherical carrier having the same sedimentation rate is defined as the Stokes particle size of the carrier having a shape other than the spherical shape.

In addition to the case where the entire carrier including the surface thereof is composed of the same material, the solid phase carrier may be a hybrid body composed of a plurality of materials as necessary. In this case, it is desirable that the material constituting the surface of the solid support is selected from the group consisting of synthetic polymers, inorganic substances, and glass. More specifically, a magnetically responsive material such as iron oxide or chromium oxide may be introduced into the particles in order to be compatible with automated equipment.
Specifically, these solid phase carriers can be prepared by an organic synthesis reaction. For example, it is a polymer of a monomer selected from the group consisting of an aromatic vinyl compound, polyacrylic acid ester, and polymethacrylic acid ester (hereinafter referred to as “specific polymer”). By copolymerizing a saturated carboxylic acid monomer, a carboxylic acid group can be introduced on the surface of the organic polymer particle.

The material forming the water-insoluble solid phase carrier may be insoluble in water. The term “water-insoluble” as used herein means a solid phase that does not specifically dissolve in water or an aqueous solution containing any other water-soluble composition. Specifically, an inorganic compound, a metal, a metal oxide, an organic compound, or a composite material combining these is included.
The material specifically used as the solid phase carrier is not particularly limited, but is generally a synthetic organic polymer such as polystyrene, polypropylene, polyacrylate, polymethyl methacrylate, polyethylene, polyamide, glass, silica, It may be an inorganic substance such as silicon dioxide, silicon nitride, zirconium oxide, aluminum oxide, sodium oxide, calcium oxide, magnesium oxide or zinc oxide, or a metal such as stainless steel.

The solid support formed from these complexes is suitable for the present invention because it can have various specific properties. For example, by impregnating with iron oxide, the solid phase carrier can be provided with magnetic responsiveness, and solid-liquid separation can be realized more easily. In addition, by introducing a ceramic layer on the surface of the organic solid phase carrier, the solid phase hardness is extremely improved, and the physical destruction of the cells can be easily achieved by vortexing, increasing the reaction efficiency of the subsequent enzyme reaction, uniform It is useful for the realization of sex.

Water-soluble organic solvent Examples of the water-soluble organic solvent used in the present invention include a solvent soluble in water containing a hydroxyl group, and alcohol is particularly preferable. Specifically, at least one or more alcohols are selected from the group consisting of butanol, 2-butanol, pentanol, 2-pentanol, methanol, ethanol, propanol, and isopropanol. As the water-soluble organic solvent, one type of solvent may be used alone, or two or more types may be used in combination. Of these, ethanol and isopropanol are particularly preferable.

When these water-soluble organic solvents are used, the concentration of the water-soluble organic solvent at the time of nucleic acid extraction is 25 to 100% by volume, preferably 40 to 100% by volume. In general, nucleic acids released from cells and hosts are precipitated and adsorbed on the surface of the solid support at the concentration of the organic solvent.
If necessary, a salt, a water-soluble polymer, a polysaccharide, or a surfactant may be added before or simultaneously with the addition of the water-soluble organic solvent. For example, sodium salt, potassium salt, magnesium salt, polyvinyl alcohol, agarose, dextran, dextran sulfate, sodium dodecylsulfonate, and the like can be mentioned. By adding these additives, nucleic acids are often captured on a solid phase carrier with higher efficiency and easily re-dissolved.
The concentration of the salt to be added in the solvent solution is preferably 0.1 to 50 mM, more preferably 0.5 to 10 mM. A salt concentration of less than 0.1 mM is not preferred because the effect of nucleic acid precipitation by the added salt is small. On the other hand, when the salt concentration exceeds 50 mM, precipitation of hydrophilic proteins other than nucleic acids also occurs, so that the salting-out effect of nucleic acids alone is diminished. Similarly, the water-soluble polymer and polysaccharide concentration in the solvent solution is preferably 0.0001-10% (w / v), and the surfactant concentration is preferably 0.01-15% (w / v), particularly 0.05-0.5. % (W / v) is preferred. If the concentration of the surfactant is less than 0.01 w / v%, the micelle effect of the hydrophobic protein is weak, and if it exceeds 15 w / v%, the surfactant is precipitated.

Adsorption Operation In the present invention, a nucleic acid is adsorbed to the solid phase of the solid phase carrier by bringing the sample containing the nucleic acid into contact with the solid phase carrier in the presence of a water-soluble organic solvent.
Here, the contacting order of the lysis solution, the solid phase carrier and the water-soluble organic solvent is not limited.
That is, (a) a method in which a solid phase carrier is added in advance during cell lysis treatment, (b) a method in which a solid phase is added after cell lysis treatment, or (c) a solid phase carrier and a sample are first added. Any of the methods of lysing cells after direct contact may be used.
However, (c) when the solid phase carrier and the sample are contacted first, the lysis solution should be added immediately after the solid phase carrier and the sample are brought into contact with each other.
Among these methods, (b) a method in which a solid phase carrier is added after the dissolution treatment is preferable. In this case, the solid phase carrier may be added together with the water-soluble organic solvent or separately.

In the present invention, the solid phase carrier varies depending on the particle size, but it is generally preferable to use about 0.01 to 100 mg of the particle weight per 1 ml of the sample sample in terms of dry matter.
The time required for adsorption of the nucleic acid to the solid phase carrier is usually 0.1 to 20 minutes, preferably 3 to 15 minutes after mixing the water-soluble organic solvent, the nucleic acid-containing sample and the solid phase carrier. In particular, when extracting a long nucleic acid such as genomic DNA, it is preferable to lengthen the adsorption process time. Moreover, adsorption temperature is 0-60 degreeC, Preferably it is 4-25 degreeC.

When the reaction solution is stirred in the adsorption step, the speed is usually 0.5 to 20 revolutions / minute, preferably 1 to 5 revolutions / minute.
-Washing and elution step After completion of the nucleic acid adsorption step on the solid support, the solid support is separated from the reaction solution and washed. In addition, before washing the nucleic acid as necessary, the residual protein component may be dissolved and removed by soaking in a protein solution such as guanidine hydrochloride, granidine thiocyanate, or urea. The washing liquid may be the same solvent as the water-soluble organic solvent used in the adsorption step or another water-soluble solvent as long as it does not elute the nucleic acid bound to the solid support. In addition, the concentration of the water-soluble organic solvent may be lowered as necessary if the adsorption of the nucleic acid to the solid phase carrier can be maintained.

The nucleic acid adsorbed on the solid support may be dried and used in the next step after the washing. The drying method is not limited, and vacuum drying, heat drying, ventilation drying and the like are used. Air drying is particularly desirable for drying. The drying temperature is preferably room temperature to 60 ° C., and the drying time is preferably 5 to 20 minutes.
The solid-phase carrier to which the nucleic acid thus obtained is adsorbed can be used for various nucleic acid analyzes such as amplification by PCR or restriction enzyme treatment as it is depending on the purpose of use. Alternatively, the nucleic acid adsorbed on the solid phase carrier may be eluted into the liquid layer by adding sterile distilled water and Tris-HCl / EDTA buffer (TE) to the dried solid phase carrier and stirring.

  In order to elute the nucleic acid from the solid phase carrier, a low ionic strength solvent or buffer is allowed to act on the solid phase carrier, and heated if necessary. The temperature for elution of nucleic acid is preferably 50 to 90 ° C, more preferably 70 to 80 ° C. Examples of such an eluent include water, Tris-HCl buffer, and phosphate buffer. The eluted nucleic acid is recovered by removing the carrier by operations such as centrifugation, filtration, and decantation.

Embodiment of Nucleic Acid Separation Method The method according to the present invention is not particularly limited, and various embodiments according to the sample and the purpose can be taken.

  Since the present invention extracts and purifies nucleic acid from a blood sample that can be collected very easily from a subject, the present invention can be particularly preferably applied to the purpose of using data on nucleic acid in the clinical field. This is because both the DNA and RNA can be separated by the method of the present invention, and the obtained nucleic acid does not need to be further purified, and can be directly used for the PCR method and various analyses.

The nucleic acid separation method of the present invention can also carry out the entire procedure in a single reaction vessel, which provides the following advantages.
(1) In one stage of the method, the nucleic acid released from the nucleic acid-containing sample binds to at least a large part of the solid phase in the subsequent purification process, so that the risk of contamination can be greatly reduced. Contamination with pathogens is largely limited to the first stage of the separation stage, where the sample is placed in a reaction vessel, with respect to the human body associated with the processing of a sample that may be infected with a pathogen such as a virus or bacteria. In the initial treatment, the virus or bacteria is effectively inactivated. Another contamination is a problem of cross contamination when a large number of specimens are processed simultaneously. By using a single reaction vessel, contamination risk can be avoided or reduced to a minimum.

(2) It is convenient to realize automation of processing.
The automation of nucleic acid extraction and preparation makes it possible to rapidly process a large amount of specimens without the need for skill in operation and advanced knowledge. Therefore, automation of nucleic acid preparation is extremely important because it has a wide range of uses and uses in various fields that require nucleic acid analysis including genetic engineering and genetic diagnosis.

The method of the present invention is also extremely suitable for the purpose of rapidly separating nucleic acids from a large number of samples, that is, automatic processing because of the above-mentioned characteristics. This is because the method of the present invention does not require water / phenol two-phase extraction, and the centrifugation operation is basically unnecessary except for the step of separating leukocytes, so that there is no particular obstacle to mechanization.
Nucleic Acid Separation Kit The present invention also provides a reagent kit for separating and purifying nucleic acid from a sample sample containing nucleated cells. The reagent of the present invention includes at least hemolyzed blood, lysis solution, enzyme solution, water-insoluble solid phase carrier, and water-soluble organic solvent.

  By using the nucleic acid extraction method and its reagent of the present invention, nucleic acid can be purified from blood in high purity and in large quantities in a short time, simply and at low cost. In addition, by establishing a method that is friendly to the working environment and workers without using toxic solvents and corrosive solvents, it can be widely applied in fields such as genetic engineering, gene diagnosis, gene therapy, genome chemistry, and genome drug discovery. In addition, the method can be automated.

Each component which comprises these kits may be prepared separately, or may be together as long as there is no trouble.
Further, if necessary, the kit may contain a washing solution, an eluent, an auxiliary agent, a dedicated container, and other necessary accessories.

As the “lysis solution”, “water-insoluble solid phase carrier”, and “water-soluble organic solvent”, any of those described above is preferably used.
Examples of the “washing solution” and “eluent” include the same solvents as the water-soluble organic solvent used in the adsorption step, or other water-soluble solvents, phosphate buffers, Tris-HCl buffers, and the like. Both may be the same type of buffer solution, and the action of washing and elution can be realized by changing the liquidity of the buffer solution, specifically by changing the ionic strength, pH, and type of salts contained. Can be made.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited by these Examples.

  1 ml of whole blood containing heparin anticoagulant in a 12 ml test tube containing hemolytic agent heated to 70 ° C., ie, 0.15M NH 4 Cl, 20 mM MgCl 2 and 20 mM CaCl 2 in 50 mM Tris-HCl buffer (pH 7.6) 4 ml of the solution to be added was added, and the mixture was stirred up and down, allowed to stand still for 5 minutes, and then centrifuged (3000 rpm × 5 minutes) to remove the supernatant.

  Trypsin (5 mg / ml), RNA nuclease (2 mg / ml), lipase (2 mg / ml), amylase (1 mg / ml), 0.5% (w / v) sodium laurate on the pellet at the bottom of the tube 0.1 ml of 200 mM potassium chloride solution was added and heated at 50 ° C. for 10 minutes. After completion of the reaction, 10 μl of a magnetic particle dispersion (10 wt%) composed of polystyrene having an average diameter of 20 μm and a surface of polystyrene and the inside of iron oxide and polystyrene was added and vortexed. Subsequently, 2 ml of a 50 volume% IPA / 50 volume% ethanol mixed solution was added, and the mixture was stirred with a vortex mixer to adsorb the DNA to the particles. The magnet was then moved to the side of the test tube to fix the particles and remove the supernatant. This washing operation was repeated twice. Subsequently, after washing twice with 1 ml of 20 mM Tris buffer (pH 7.6), TE and 0.5 ml were added and heated with stirring at 60 ° C. for 10 minutes, and then the polystyrene magnetic beads were magnetically separated to obtain a liquid layer portion. Was recovered.

DNA contained in the collected supernatant was quantified by absorbance measurement. The recovered DNA amount was calculated from the ultraviolet absorbance (A260) at 260 nm, and the purity was confirmed by the absorbance ratio A260 / A280, A260 / A230), and both were confirmed to be 1.9 or more. The DNA length was examined by pulse electrophoresis on a 1% by weight agarose gel. As a result, it was found that it was distributed in the 100K to 200K region. Digested DNA, 1 μg after digestion with 0.2 units of restriction enzyme, HindIII, EcoRI, confirmed by electrophoresis, and as a result, confirmed to be at the same level as the DNA recovered with phenol / chloroform did. Furthermore, the globulin gene was amplified using 1fg DNA as a template, and it was confirmed that the PCR reaction could be performed without any problem.
In addition, the time required when this experiment was carried out simultaneously with 10 samples excluding the time for reagent preparation took about 1 hour.

Instead of the enzyme trypsin used in Example 1, the same operation as in Example 1 was performed except that pepsin having the same concentration as trypsin was used.

  The same operation as in Example 1 was performed except that proteinase K was used at the same concentration instead of the enzyme trypsin used in Example 1.

  Instead of the enzyme trypsin used in Example 1, the same operation as in Example 1 was performed except that peptidase at the same concentration as trypsin was used.

  The same operation as in Example 1 was performed except that polystyrene cross-linked particles having a particle size of 30 μm were used instead of the solid phase carrier composed of polystyrene / iron oxide used in Example 1.

The same volume of 5M guanidine hydrochloride was added to the cell lysis solution that had been subjected to the enzyme treatment in the same manner as in Example 1, and the mixture was allowed to stand at room temperature for 5 minutes after stirring with vibration. Thereafter, as in Example 1, 10 μl of a magnetic particle dispersion (10 wt%) having an average diameter of 20 μm, a surface of polystyrene and an inside of iron oxide and polystyrene was added and vortexed. The subsequent operation was the same as in Example 1.

When the trypsin (5 mg / ml), RNA nuclease (2 mg / ml), lipase (2 mg / ml), and amylase (1 mg / ml) of the pellet, which is the cell component of Example 1, were treated with 0.5% (w / v) 0.1 ml of 200 mM potassium chloride solution containing sodium laurate and 20 μl of 0.3% dextran (molecular weight 50,000) were added and heated at 50 ° C. for 10 minutes. The subsequent operation was the same as that in Example 1.

60 ml of 0.15M NH4Cl heated to 40 ° C. was added to 10 ml of blood collected from the EDTA blood collection tube, and after stirring up and down, it was centrifuged at 3000 rpm × 5 minutes to remove the supernatant. To the pellet, 0.5 ml of 200 mM KCl solution containing peptidase (5 mg / ml), lipase (2 mg / ml) and 0.5% (w / v) sodium dodecyl sulfate was added and heated at 50 ° C. for 0.5 hour. After completion of the reaction, add 5 μl of 5 μm polyacrylate resin particle dispersion (5% fixed particle), vortex, add 1 ml ethanol, stir the test tube 3 times up and down, and separate the particles by magnetic separation. And the liquid layer portion was removed. Next, the particle pellet was washed twice with 50% isopropyl alcohol aqueous solution and finally magnetically separated, washed once with 1 ml of 20 mM Tris buffer (pH 7.6), added to 0.5 ml of TE, and then at 70 ° C. for 30 minutes. Heated with stirring. Finally, the particle dispersion was solid-liquid separated with a magnet, and the supernatant was recovered.
Comparative Example 1
As a reference experiment, the leukocyte precipitate obtained in Example 1 was extracted by the phenol / chloroform method. The extraction method followed “Molecular Cloning” (J. Sambrook, 1982, published by CSH). The time required for this reference experiment was about 3 hours when 10 samples were run in parallel except for reagent preparation.
The results of Examples and Comparative Examples are shown in Table 1 below.

Claims (6)

A method for separating and purifying nucleic acid from a sample containing nucleated cells,
(1) contacting a sample containing nucleated cells with a lysis solution containing at least a cell component degrading enzyme and a surfactant;
(2) A sample containing nucleated cells is contacted with a water-insoluble solid phase carrier having an average particle size of 0.01 to 1000 μM in the presence of a water-soluble organic solvent, and the nucleic acid released from the nucleated cells is removed. A method for separating nucleic acid, comprising the step of adsorbing and binding to the surface of a solid phase carrier; and (3) separating the solid phase carrier from a sample.   The method for separating nucleic acids according to claim 1, wherein the cell component degrading enzyme is at least one selected from the group consisting of amylase, lipase, protease and nuclease.   The method for separating nucleic acids according to claim 1, wherein the surfactant is an anionic surfactant.   The water-insoluble solid support is at least one selected from the group consisting of polystyrene, polypropylene, polyacrylate, polymethyl methacrylate, polyethylene, polyamide, glass, silica, silicon dioxide, silicon nitride, zirconium oxide, aluminum oxide, and zinc oxide. The nucleic acid separation method according to claim 1, wherein   The method for separating nucleic acids according to claim 1, further comprising the step of (4) washing the separated solid phase carrier.   The method for separating nucleic acids according to claim 1, further comprising the step of (5) eluting the nucleic acid adsorbed on the solid phase carrier.