JP2005130806A - Method for separating and refining nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating and refining a nucleic acid by using a porous film excellent in separation performance, having good washing efficiency, capable of simply and rapidly carrying out separation and refining of the nucleic acid, excellent in automatic and miniaturizing suitability and capable of producing the film having substantially same separation performance in large amounts and to provide an apparatus for separating and refining the nucleic acid in which treatment can efficiently be carried out in a short time without causing contamination and miniaturization is made possible. <P>SOLUTION: The method for separating and refining the nucleic acid comprises a step for absorbing the nucleic acid into the porous film by passing a sample solution containing the nucleic acid through a nucleic aid-absorbing porous film, a step for passing a washing liquid through the nucleic aid-absorbing porous film and washing the porous film in a state in which the nucleic acid is absorbed and a step for desorbing the nucleic acid from the interior of the porous film by passing a recovering liquid through the nucleic acid-absorbing porous film. In the method for separating and refining the nucleic acid, the nucleic acid-absorbing porous film is a porous film in which the nucleic acid is absorbed by interaction in which ion bond is not substantially participated and pH of the recovering liquid is 2-11. A kit and an apparatus for separating and refining the nucleic acid are also provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for separating and purifying nucleic acid. More specifically, 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 sample solution containing the obtained nucleic acid is used to contain the nucleic acid using a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane in a container having at least two openings and a pressure generator. The present invention relates to a method for separating and purifying nucleic acid from a sample.

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

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

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

As one of widely known separation and purification methods, there is a method in which a nucleic acid is adsorbed on a solid phase such as silicon dioxide, silica polymer, magnesium silicate, and the like, followed by separation and purification by operations such as washing and desorption (for example, Patent Document 1). ). Although this method is excellent in separation performance, it is not sufficient in terms of simplicity, speed, automation, and suitability for miniaturization, industrial mass production of adsorption media having the same performance is difficult, and handling is inconvenient. There are problems such as difficulty in processing into various shapes.
In the conventional solid phase method, a method is known in which nucleic acid in a sample is adsorbed to a solid phase and then desorbed through washing or the like to separate and purify the nucleic acid. For example, a QIAamp DNA Purification Kit (QIAamp DNA Blood Mini Kit) is often used. When this kit is used, the length of DNA that can be recovered is about 20 kbp, which is relatively short.
For extraction of nucleic acids, the phenol / chloroform method is generally used (the phenol / chloroform method for DNA, the acid guanidine phenol / chloroform method for RNA). Yes, the purity of the nucleic acid is low.

  In addition, as one method for separating and purifying nucleic acid simply and efficiently, a solution consisting of an organic polymer having a hydroxyl group on the surface is used by using a solution for adsorbing nucleic acid on the solid phase and a solution for desorbing nucleic acid from the solid phase, respectively. Although a method for separating and purifying nucleic acid by adsorbing and desorbing nucleic acid to a phase is described (Patent Document 2), further improvement is desired.

  On the other hand, conventional nucleic acid separation and purification methods include those using a centrifugal method, those using magnetic beads, and those using a filter. For example, as a nucleic acid separation apparatus using a filter, a large number of filter tubes containing filters are set in a rack, a sample solution containing nucleic acid is dispensed into the rack, and the periphery of the bottom of the rack is placed through a sealing material. The inside is sealed with an air chamber, the inside is depressurized, and all filter tubes are sucked from the discharge side at the same time to allow the sample liquid to pass through to adsorb the nucleic acid to the filter. Thus, a mechanism for cleaning and desorption has been proposed (see, for example, Patent Document 3).

  However, these automatic devices are large and suitable for analyzing a large amount of samples, but they are not suitable and expensive when the number of samples is small and the analysis frequency is low, and the processing efficiency is low. Have. In particular, when the characteristics of each sample solution are different as in the case of collected whole blood, as in Patent Document 3, when the entire suction is completed and the resistance disappears, There are cases where the reduced pressure acting on the filter tube becomes small and the processing of the sample liquid having a high viscosity is not completed. Increasing the reduced pressure capacity becomes an obstacle to downsizing the apparatus, and since the reduced pressure volume is large, it takes time until the reduced pressure is applied, and it is difficult to detect that all the liquid has been discharged, Long time setting is an obstacle to improvement of processing efficiency. In addition, in the case of a sample solution having a low viscosity, there is a problem that the liquid is discharged from the filter tube more vigorously, and foam droplets adhere to adjacent filter tubes and racks to cause contamination, resulting in a decrease in accuracy.

Japanese Patent Publication No. 7-51065 JP 2003-128691 A Japanese Patent No. 2832586

An object of the present invention is to provide a method for separating and purifying nucleic acid by adsorbing a nucleic acid in a specimen to a nucleic acid-adsorbing porous membrane and then desorbing it through washing or the like. Nucleic acid separation using a porous membrane that is efficient, can be efficiently recovered, simple, rapid, excellent in automation and miniaturization, and capable of mass production of substantially the same separation performance It is to provide a purification method.
A further object of the present invention is to provide a nucleic acid separation and purification apparatus that can be processed in a short time and efficiently to prevent contamination, and that can be miniaturized.

  As a result of intensive studies to solve the above problems, the present inventor, as a result of a method for separating and purifying nucleic acid including a process of adsorbing and desorbing a nucleic acid on a porous membrane, the porous membrane does not substantially involve ionic bonds. It was found that nucleic acids can be efficiently recovered by using a porous membrane that adsorbs nucleic acids by action and using a recovery solution having a pH of 2 to 11.

That is, the present invention provides the following method for separating and purifying nucleic acids.
(1) (i) a step of allowing a sample solution containing nucleic acid to pass through a nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid in the porous membrane;
(Ii) passing the washing solution through the nucleic acid-adsorbing porous membrane and washing the porous membrane with the nucleic acid adsorbed; and (iii) passing the recovered solution through the nucleic acid-adsorbing porous membrane. In the method for separating and purifying nucleic acid comprising the step of desorbing nucleic acid from the porous membrane, the nucleic acid-adsorbing porous membrane is a porous membrane that adsorbs nucleic acid by an interaction that does not substantially involve ionic bonds. A method for separating and purifying nucleic acid, wherein the pH of the recovered liquid is 2 to 11.

(2) In each step of (i), (ii) and (iii) described in (1) above, the sample solution, washing solution or recovery solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane under pressure. The method for separating and purifying nucleic acid as described in (1) above, wherein

(3) A nucleic acid separation and purification cartridge in which the nucleic acid-adsorbing porous membrane is housed in a container having at least two openings in each of the steps (i), (ii), and (iii) described in (1) above. A sample solution containing nucleic acid, a washing solution or a recovery solution is injected into one opening of the cartridge, and the inside of the cartridge is pressurized using a pressure difference generator coupled to the one opening of the cartridge. The method for separating and purifying nucleic acid according to (1) above, wherein the solution is allowed to pass through and discharged from another opening.

(4) The method for separating and purifying nucleic acid according to any one of (1) to (3) above, wherein the ionic strength of the recovered liquid is 290 mmol / L or less.
(5) The method for separating and purifying nucleic acid according to any one of (1) to (4) above, wherein the salt concentration of the recovered solution is 90 mmol / L or less.

(6) A nucleic acid solubilizing reagent comprising a nucleic acid separation and purification cartridge, a proteolytic enzyme, a chaotropic salt, and a surfactant for performing the method for separating and purifying nucleic acid according to any one of (1) to (5) above A kit comprising a solution, a cleaning solution, and a reagent for a recovery solution.

(7) An apparatus for automatically performing the method for separating and purifying nucleic acid according to any one of (1) to (5) above.

  By the method for separating and purifying nucleic acid of the present invention using a recovery solution having a pH of 2 or more and 11 or less, the nucleic acid can be separated and purified from a sample containing the nucleic acid with good separation performance, simply, rapidly and in an automated manner.

In the method for separating and purifying nucleic acid of the present invention, the pH of the recovered solution is pH 2-11. Furthermore, it is preferable that it is pH 5-9.
In particular, the ionic strength and the salt concentration are preferably low because they affect the elution of adsorbed nucleic acid. The recovered liquid preferably has an ionic strength of 290 mmol / L or less, and more preferably has a salt concentration of 90 mmol / L or less.
By these, the recovery rate of nucleic acid can be improved, and more nucleic acid can be recovered. The recovered nucleic acid may be single-stranded or double-stranded.

The recovered liquid is preferably purified distilled water, Tris / EDTA buffer or the like. When the recovered nucleic acid is subjected to PCR (polymerase chain reaction), a buffer solution used in the PCR reaction (for example, KCl 50 mmol / l, Tris-Cl 10 mmol / l, MgCl ₂ 1.5 mmol / l in an aqueous solution having a final concentration) ) Can also be used.

  In the process of desorbing and recovering nucleic acid from the nucleic acid-adsorbing polyporous membrane, the recovery solution can be collected using a tube, pipette, automatic injection device, or a supply means having the same function as the nucleic acid-adsorbing porous material. It is supplied to a nucleic acid separation / purification cartridge equipped with a membrane. The recovery liquid is supplied from one opening of the cartridge for separating and purifying nucleic acid (opening into which a sample solution containing nucleic acid is injected), and a pressure difference generator (for example, a spoid, a syringe, a pump, a power pipette, etc.) coupled to the opening is connected to the recovery liquid. By using it, the inside of the cartridge for separating and purifying nucleic acid can be pressurized and passed through the nucleic acid-adsorbing porous membrane and discharged from an opening different from the one opening. Further, the recovery liquid can be supplied from one opening and discharged from the same opening. Furthermore, it is also possible to supply and discharge the recovery liquid from an opening different from the one opening to which the sample solution containing the nucleic acid in the nucleic acid separation and purification cartridge is supplied. However, a method in which the nucleic acid separation and purification cartridge is supplied from one opening, passed through the nucleic acid-adsorbing porous membrane, and discharged from an opening different from the one opening is more preferable because of excellent recovery efficiency.

  Nucleic acid can be desorbed by adjusting the volume of the recovered liquid relative to the volume of the sample solution containing the nucleic acid prepared from the specimen. The amount of the recovered liquid containing the separated and purified nucleic acid depends on the amount of sample used at that time. In general, the amount of the recovered liquid is several tens to several hundreds of μl. However, when the amount of the sample is extremely small, or when it is desired to separate and purify a large amount of nucleic acid, the amount of the recovered liquid is 1 μl to several tens of ml. You can change the range.

  By reducing the volume of the recovery solution compared to the volume of the sample solution containing the original nucleic acid, a recovery solution containing concentrated nucleic acid can be obtained. Preferably, (collected liquid volume) :( sample solution volume) = 1: 100 to 99: 100, more preferably (collected liquid volume) :( sample solution volume) = 1: 10 to 9:10. it can. Thus, the nucleic acid can be easily concentrated without performing an operation for concentration in the step after the separation and purification of the nucleic acid. By these methods, it is possible to provide a method for obtaining a nucleic acid solution in which the nucleic acid is more concentrated than the specimen.

  As another method, nucleic acid is desorbed under the condition that the volume of the recovery solution is larger than that of the sample solution containing the initial nucleic acid, thereby obtaining a recovery solution containing the nucleic acid of a desired concentration. A recovery solution containing nucleic acid at a concentration suitable for PCR and the like can be obtained. Preferably, (recovered liquid volume) :( sample solution volume) = 1: 1 to 50: 1, more preferably (recovered liquid volume) :( sample solution volume) = 1: 1 to 5: 1. it can. As a result, there is an advantage that there is no need to adjust the concentration after separation and purification of nucleic acid. Furthermore, by using a sufficient amount of the recovery liquid, it is possible to increase the nucleic acid recovery rate from the porous membrane.

  In addition, nucleic acids can be easily recovered by changing the temperature of the recovery solution according to the purpose. For example, by desorbing nucleic acids from the porous membrane at a temperature of the recovered solution of 0 to 10 ° C., the function of the nucleolytic enzyme is suppressed without adding any reagent or special operation to prevent degradation by the enzyme. Thus, nucleic acid solution can be easily and efficiently obtained by preventing nucleic acid degradation.

  In addition, when the temperature of the recovered solution is 10 to 35 ° C., the nucleic acid can be recovered at a general room temperature, and the nucleic acid can be desorbed and separated and purified without requiring a complicated process.

  As another method, the temperature of the recovery liquid is set to a high temperature, for example, 35 to 70 ° C., so that desorption of nucleic acids from the porous membrane can be easily performed at a high recovery rate without complicated operations.

  The number of injections of the recovery liquid is not limited and may be one time or multiple times. Usually, when the nucleic acid is separated and purified quickly and easily, it is carried out by one collection, but the collection liquid may be injected several times, such as when collecting a large amount of nucleic acid.

  In the recovery step, the nucleic acid recovery solution can be made into a composition that can be used in the subsequent subsequent steps. The separated and purified nucleic acid is often amplified by a PCR (polymerase chain reaction) method. In this case, the separated and purified nucleic acid solution needs to be diluted with a buffer solution suitable for the PCR method. In the recovery step according to this method, a buffer solution suitable for the PCR method is used as the recovery solution, so that the subsequent PCR step can be easily and rapidly transferred.

  In the recovery step, a stabilizer for preventing degradation of the recovered nucleic acid can be added to the nucleic acid recovery solution. As a stabilizer, an antibacterial agent, an anti-fouling agent, a nucleic acid degradation inhibitor and the like can be added. Examples of the nucleolytic enzyme inhibitor include EDTA. As another embodiment, a stabilizer may be added to the collection container in advance.

The collection container used in the collection process is not particularly limited, but a collection container made of a material that does not absorb 260 nm can be used. In this case, the concentration of the recovered nucleic acid solution can be measured without transferring to another container. A material that does not absorb at 260 nm is, for example, quartz glass, but is not limited thereto.

  The method for separating and purifying nucleic acid of the present invention comprises (i) a step of passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid in the porous membrane; Passing the porous membrane through the porous membrane and washing the porous membrane with the nucleic acid adsorbed; and (iii) passing the recovered solution through the nucleic acid adsorbing porous membrane to remove the nucleic acid from the porous membrane. At least a step of desorption is included.

  More specifically, as the nucleic acid separation and purification step of the present invention, (a) a sample solution containing nucleic acid is housed in a container having at least two openings, and a nucleic acid-adsorbing porous membrane through which the solution can pass is housed. (B) injecting the nucleic acid separation / purification cartridge into a pressurized state using a pressure difference generator connected to the one opening of the nucleic acid separation / purification cartridge; Passing the sample solution containing the nucleic acid through the nucleic acid-adsorbing porous membrane and discharging the sample solution from the other opening of the nucleic acid separation and purification cartridge, thereby allowing the nucleic acid to be adsorbed in the nucleic acid-adsorbing porous membrane; (c) nucleic acid A step of injecting a washing solution into the one opening of the separation / purification cartridge; (d) the inside of the nucleic acid separation / purification cartridge is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation / purification cartridge; Washing the injected washing solution through the nucleic acid-adsorptive porous membrane and discharging it from other openings so that the nucleic acid-adsorptive porous membrane is adsorbed with nucleic acid; (e) nucleic acid separation A step of injecting the recovered liquid into the one opening of the purification cartridge; (f) injecting the nucleic acid separation and purification cartridge under pressure using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification cartridge; The recovered liquid is passed through the nucleic acid-adsorbing porous membrane and discharged from other openings, whereby the nucleic acid is desorbed from the nucleic acid-adsorbing porous membrane and discharged out of the nucleic acid separation and purification cartridge container. Can do.

  In the above-described nucleic acid separation and purification step, the steps from injection of the sample solution containing the first nucleic acid to obtaining the nucleic acid outside the nucleic acid separation and purification cartridge can be completed within 10 minutes, depending on conditions, within 2 minutes. . In the nucleic acid purification step, it is possible to obtain a nucleic acid in a yield of 50% or more and, depending on conditions, a yield of 90% or more.

  In the nucleic acid purification step, DNA having a molecular weight ranging from 1 kbp to 200 kbp, particularly 20 kbp to 140 kbp can be recovered. That is, long-chain DNA can be recovered as compared with the conventional spin column method using a glass filter (Qiagen).

  In the nucleic acid fraction purification step, the spectral absorbance ratio (260 nm / 280 nm) with an ultraviolet-visible spectrophotometer is 1.6 to 2.0 in the case of DNA, and 1.8 to 2 in the case of RNA. A nucleic acid having a purity of 2 can be recovered, and a high-purity nucleic acid with a small amount of impurities can be steadily obtained. Furthermore, a nucleic acid having a purity of about 1.8 when the spectral absorbance ratio (260 nm / 280 nm) in an ultraviolet-visible spectrophotometer is about DNA and about 2.0 when RNA is used can be recovered.

  In the above process, examples of the pressure difference generator include a pump capable of pressurization or decompression, 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 nucleic acid separation / purification cartridge.

  There are no limitations on the specimens that can be used in the present invention. For example, in the diagnostic field, whole blood collected as specimens, 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), bacteria, viruses, etc., or lysates and homogenates thereof.

  First, these specimens are treated with an aqueous solution (nucleic acid solubilizing reagent) containing a reagent for solubilizing nucleic acid by dissolving cell membranes and nuclear membranes. As a result, the cell membrane and the nuclear membrane are dissolved, and the nucleic acid is dispersed in the aqueous solution to obtain a sample solution containing the nucleic acid.

  In order to solubilize cell membrane and nuclear membrane and solubilize nucleic acid, for example, when the target sample is whole blood, (1) removal of red blood cells, (2) removal of various proteins, and (3) white blood cells And dissolution of the nuclear membrane is required. (1) Removal of red blood cells and (2) removal of various proteins are performed in order to prevent non-specific adsorption to the membrane and clogging of the porous membrane. (3) Lysis of leukocytes and lysis of the nuclear membrane are subject to extraction. Each is required to solubilize certain nucleic acids. In particular, (3) lysis of leukocytes and nuclear membrane lysis are important steps, and in the method of the present invention, it is necessary to solubilize nucleic acids by this step.

  The specimen containing the nucleic acid may be a specimen containing a single nucleic acid or a specimen containing a plurality of different types of nucleic acids. The type of nucleic acid to be collected is not particularly limited, such as DNA or RNA. The number of specimens may be one or more. The length of the nucleic acid to be recovered is not particularly limited, and for example, a nucleic acid having an arbitrary length of several bp to several Mbp can be used. From the viewpoint of handling, the length of the nucleic acid to be recovered is generally about several bp to several hundreds kbp. The nucleic acid separation and purification method of the present invention can rapidly extract a nucleic acid that is relatively longer than the conventional simple nucleic acid separation and purification method, and recovers a nucleic acid of 50 kbp or more, preferably 70 kbp or more, more preferably 100 kbp or more. Can be used. Gently stirring and pipetting is preferred in terms of recovering longer DNA.

  Hereinafter, a process of dissolving a cell membrane and a nuclear membrane, solubilizing a nucleic acid, and obtaining a sample solution containing the nucleic acid from a specimen will be described. In the present invention, a nucleic acid solubilizing reagent is used to solubilize nucleic acid by dissolving the cell membrane and the nuclear membrane. Examples of the nucleic acid solubilizing reagent include a solution containing a chaotropic salt, a surfactant, and a proteolytic enzyme.

  A method for obtaining a sample solution containing a nucleic acid from a specimen by dissolving a cell membrane and a nuclear membrane and solubilizing a nucleic acid includes: (I) a step of injecting a specimen containing cells or viruses into a container; , A step of adding a nucleic acid solubilizing reagent solution containing a chaotropic salt, a surfactant and a proteolytic enzyme, and mixing the sample and the nucleic acid solubilizing reagent solution; (III) a step of incubating the mixed solution obtained above (IV) A method including a step of adding a water-soluble organic solvent to the incubated mixed solution can be mentioned.

  In the step of dissolving the cell membrane and the nuclear membrane, solubilizing the nucleic acid, and obtaining a sample solution containing the nucleic acid from the specimen, homogenizing the specimen improves the appropriateness of the automation process. The homogenizing treatment can be performed by, for example, ultrasonic treatment, using sharp protrusions, using high-speed stirring treatment, processing for extruding from fine voids, processing using glass beads, or the like.

  Further, in the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the amount of nucleic acid recovered can be obtained by using a nucleic acid solubilizing reagent containing a proteolytic enzyme. In addition, the recovery efficiency is improved, and it is possible to reduce the amount and speed of the specimen containing the necessary nucleic acid.

As the proteolytic enzyme, at least one proteolytic enzyme can be preferably used from serine protease, cysteine protease, metal protease and the like. As the proteolytic enzyme, a mixture of a plurality of proteolytic enzymes can be preferably used.
The serine protease is not particularly limited, and for example, protease K can be preferably used.
The cysteine protease is not particularly limited, and for example, papain and cathepsins can be preferably used.
The metal protease is not particularly limited, and for example, carboxypeptidase can be preferably used.
The proteolytic enzyme can be used at a concentration of preferably 0.001 IU to 10 IU, more preferably 0.01 IU to 1 IU per 1 ml final volume of the nucleic acid solubilizing reagent solution.

  As the proteolytic enzyme, a proteolytic enzyme that does not contain a nucleolytic enzyme can be preferably used. A proteolytic enzyme containing a stabilizer can be preferably used. As the stabilizer, metal ions can be preferably used. By including a proteolytic enzyme stabilizer, the amount of proteolytic enzyme required for nucleic acid recovery can be reduced, and the cost required for nucleic acid recovery can be reduced.

The proteolytic enzyme may be mixed with other reagents such as chaotropic salts and surfactants in advance and used for nucleic acid recovery as one reagent.
The proteolytic enzyme may be provided as two or more reagents separate from other reagents such as chaotropic salts and surfactants. In the latter case, a reagent containing a proteolytic enzyme is first mixed with a sample, and then mixed with a reagent containing a chaotropic salt and a surfactant. Moreover, after mixing the reagent containing a chaotropic salt and a surfactant previously, you may mix a proteolytic enzyme.
In addition, the proteolytic enzyme can be dropped directly into a specimen or a mixed solution of the specimen and a reagent containing a chaotropic salt and a surfactant directly from the proteolytic enzyme storage container. In this case, the operation can be simplified.

It is also preferable that the nucleic acid solubilizing reagent is supplied in a dried state. Moreover, the container previously containing the proteolytic enzyme of the dried state like freeze-drying can be used. A sample solution containing nucleic acid can also be obtained by using both the nucleic acid solubilizing reagent supplied in a dried state and the container previously containing the proteolytic enzyme in a dried state.
When a sample solution containing nucleic acid is obtained by the above method, the storage stability of the nucleic acid solubilizing reagent and proteolytic enzyme is good, and the operation can be simplified without changing the nucleic acid yield.

A method for mixing the specimen with a nucleic acid solubilizing reagent solution containing a chaotropic salt, a surfactant, and a proteolytic enzyme is not particularly limited.
When mixing, it is preferable to mix at 30 to 3000 rpm for 1 second to 3 minutes with a stirrer. Thereby, the yield of nucleic acid to be separated and purified can be increased. Or it is also preferable to mix by inversion mixing 5 to 30 times. In addition, mixing can also be performed by performing pipetting operations 10 to 50 times. In this case, the yield of nucleic acid separated and purified by a simple operation can be increased.

  By incubating a mixture of a sample and a nucleic acid solubilizing reagent solution containing a chaotropic salt, a surfactant, and a proteolytic enzyme at the optimal temperature and reaction time of the proteolytic enzyme, The yield can be increased. The incubation temperature is usually 20 ° C. to 70 ° C., preferably the optimum temperature for the proteolytic enzyme, and the incubation time is usually 1 to 90 minutes, preferably the optimum reaction time for the proteolytic enzyme. The incubation method is not particularly limited, and can be performed by putting it in a hot water bath or a warmer.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the nucleic acid solubilizing reagent solution is preferably pH 5-10, more preferably pH 6-9, Those having a pH of 7 to 8 are preferably used.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the final concentration of the chaotropic salt in the nucleic acid solubilizing reagent solution is 0.5 mol / L or more. More preferably, it is 0.5 mol / L-4 mol / L, More preferably, it is 1 mol / L-3 mol / L. As the chaotropic salt, guanidine hydrochloride is preferable, but other chaotropic salts (guanidine isothiocyanate, guanidine thiocyanate) can also be used. These salts may be used alone or in combination.

  The nucleic acid solubilizing reagent solution may contain a water-soluble organic solvent. As this water-soluble organic solvent, alcohol is preferred. The alcohol may be any of primary alcohol, secondary alcohol, and tertiary alcohol. As the alcohol, methyl alcohol, ethyl alcohol, propyl alcohol and its isomer, butyl alcohol and its isomer can be preferably used. These water-soluble organic solvents may be used alone or in combination. The final concentration of these water-soluble organic solvents in the nucleic acid solubilizing reagent solution is preferably 1 to 20%.

In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, the surfactant mixed with the chaotropic salt and the proteolytic enzyme in the specimen is, for example, nonionic A surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant.
In the present invention, a nonionic surfactant can be preferably used. As the nonionic surfactant, a polyoxyethylene alkylphenyl ether surfactant, a polyoxyethylene alkyl ether surfactant, or a fatty acid alkanolamide can be used. Preferably, a polyoxyethylene alkyl ether surfactant is used. More preferably, the polyoxyethylene alkyl ether surfactant that can be used is POE decyl ether, POE lauryl ether, POE tridecyl ether, POE alkylene decyl ether, POE sorbitan monolaurate, Polyoxyethylene alkyl ether selected from POE sorbitan monooleate, POE sorbitan monostearate, polyoxyethylene sorbitol tetraoleate, POE alkylamine, POE acetylene glycol A surfactant.

  A cationic surfactant can also be preferably used. More preferably, the cationic surfactant is a cationic surfactant selected from cetyltrimethylammonium promide, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, cetylpyridinium chloride. These surfactants may be used alone or in combination.

When recovering nucleic acids other than RNA, such as DNA, in the step of dissolving the cell membrane and nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, an RNase is added to the nucleic acid solubilizing reagent solution. It is preferable to add. In this case, interference by RNA coexisting with the recovered nucleic acid can be reduced. It is also preferable to add a DNA-degrading enzyme inhibitor.
On the other hand, when recovering nucleic acids other than DNA such as RNA, it is preferable to add a DNA degrading enzyme to the nucleic acid solubilizing reagent solution. In this case, interference by DNA coexisting with the recovered nucleic acid can be reduced. It is also preferable to add an RNase inhibitor. As the RNase inhibitor, those that specifically inhibit RNase are preferable.
The RNA degrading enzyme is not particularly limited, and for example, an RNA-specific degrading enzyme such as ribonuclease H (RNase H) can be preferably used.
The DNA degrading enzyme is not particularly limited, and for example, a DNA-specific degrading enzyme such as DNase I can be preferably used.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain a sample solution containing the nucleic acid from the specimen, an alcohol is preferably used as the water-soluble organic solvent to be added to the incubated mixture. Can do. The alcohol may be any of primary alcohol, secondary alcohol, and tertiary alcohol, and methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol and isomers thereof can be preferably used. The final concentration of these water-soluble organic solvents in the nucleic acid solubilizing reagent solution is preferably 5 to 90%.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain the sample solution containing the nucleic acid from the specimen, the sample solution containing the nucleic acid preferably contains an antifoaming agent. The antifoaming agent preferably includes two components, a silicon-based antifoaming agent and an alcohol-based antifoaming agent, and the alcohol-based antifoaming agent is preferably an acetylene glycol surfactant.

  Specific examples of antifoaming agents include silicon-based antifoaming agents (eg, silicone oil, dimethylpolysiloxane, silicone emulsion, modified polysiloxane, silicone compound, etc.), alcohol-based antifoaming agents (eg, acetylene glycol, heptanol, ethyl). Exanol, higher alcohol, polyoxyalkylene glycol, etc.), ether type antifoaming agents (eg, heptylcellosolve, nonylcellosolv-3-heptylcorbitol, etc.), oil-based antifoaming agents (eg, animal and vegetable oils, etc.), fatty acid type Antifoaming agents (eg, stearic acid, oleic acid, palmitic acid, etc.), metal soap antifoaming agents (eg, aluminum stearate, calcium stearate, etc.), fatty acid ester antifoaming agents (eg, natural wax, tributyl phosphate, etc.) ) Phosphate-based antifoaming agents (for example, sodium octyl phosphate), amine-based antifoaming agents (for example, diamylamine), amide-based antifoaming agents (for example, stearamide), and other antifoaming agents (for example, Ferric sulfate, bauxite, etc.). Particularly preferably, the antifoaming agent can be used in combination of two components, a silicon antifoaming agent and an alcohol antifoaming agent. Moreover, it is also preferable to use an acetylene glycol surfactant as the alcohol-based antifoaming agent.

  In the step of dissolving the cell membrane and the nuclear membrane and solubilizing the nucleic acid to obtain the sample solution containing the nucleic acid from the specimen, the sample solution containing the nucleic acid has a surface tension of 50 mPa · m or less. The viscosity is preferably 1 to 10000 mPa · S, and the specific gravity is preferably 0.8 to 1.2.

  Hereinafter, the nucleic acid-adsorbing porous membrane used in the present invention and the adsorption process will be described. The nucleic acid-adsorptive porous membrane of the present invention is a solution through which a solution can pass. Here, “solution can pass through” means that when a pressure difference is generated between the space where one surface of the membrane contacts and the space where the other surface of the membrane contacts, To the side, it means that the solution can pass through the inside of the membrane. Or, when a centrifugal force is applied to the membrane, it means that the solution can pass through the membrane in the direction of the centrifugal force.

  The nucleic acid-adsorbing porous membrane of the present invention is characterized in that it is a porous membrane that adsorbs nucleic acids by an interaction that does not substantially involve ionic bonds, thereby providing excellent separation performance and washing efficiency. It can be isolated and purified. More preferably, the nucleic acid-adsorptive porous membrane is a porous membrane having a hydrophilic group, and the material for forming the porous membrane itself is a porous membrane having a hydrophilic group or a material for forming a porous membrane. The porous membrane which introduce | transduced the hydrophilic group by processing or coating is meant. The material for forming the porous film may be either organic or inorganic. For example, a porous film in which the material forming the porous film itself is an organic material having a hydrophilic group, a porous film in which a hydrophilic group is introduced by treating a porous film of an organic material having no hydrophilic group, A porous film in which a hydrophilic group is introduced by coating a porous film made of an organic material that does not have a hydrophilic group, a porous film in which the material forming the porous film itself is an inorganic material having a hydrophilic group, hydrophilic Porous membranes with hydrophilic groups introduced by treating porous membranes of inorganic materials that do not have groups, and inorganic groups without inorganic groups coated with materials having hydrophilic groups to introduce hydrophilic groups A porous film or the like can be used, but an organic material such as an organic polymer is preferably used as a material for forming the porous film from the viewpoint of ease of processing.

The hydrophilic group refers to a polar group (atomic group) capable of interacting with water, and all groups (atomic groups) involved in nucleic acid adsorption are applicable. The hydrophilic group preferably has a moderate interaction strength with water, and examples thereof include a hydroxyl group, a carboxyl group, a cyano group, an oxyethylene group, an amino group, and —NHCONH _2. It is.

Examples of the porous film having a hydrophilic group include a porous film made of an organic material having a hydroxyl group. Examples of the organic material having a hydroxyl group include surface saponified products of acetylcellulose described in JP-A No. 2003-128691. The acetyl cellulose may be any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose, but triacetyl cellulose is particularly preferable. In this case, the amount (density) of hydroxyl groups on the solid surface can be controlled by the degree of saponification treatment (saponification rate). In order to increase the nucleic acid separation efficiency, it is preferable that the amount (density) of hydroxyl groups is large. For example, in the case of acetylcellulose such as triacetylcellulose, the saponification rate is preferably about 5% or more, and more preferably 10% or more. In order to increase the surface area of the organic polymer having a hydroxyl group, the porous membrane of acetylcellulose is saponified.
The amount (density) of spatial hydroxyl groups can be controlled by a combination of the degree of saponification treatment (saponification rate) and the pore diameter of the porous membrane. In this case, the porous membrane may be a front-back symmetrical porous membrane, but a back-asymmetric porous membrane can be preferably used.

  As porous membranes of organic materials having hydroxyl groups, polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, acetylcellulose, different acetyl values Examples of the porous film formed include a mixture of acetylcellulose, and a porous film of an organic material having a polysaccharide structure can be preferably used.

In particular, as a porous film of an organic material having a hydroxyl group, an organic polymer porous film made of a mixture of acetylcelluloses having different acetyl values can be preferably used. As a mixture of acetyl cellulose having different acetyl values, a mixture of triacetyl cellulose and diacetyl cellulose, a mixture of triacetyl cellulose and monoacetyl cellulose, a mixture of triacetyl cellulose, diacetyl cellulose and monoacetyl cellulose, a mixture of diacetyl cellulose and monoacetyl cellulose Can be preferably used. In particular, a mixture of triacetyl cellulose and diacetyl cellulose can be preferably used.
The mixing ratio (mass ratio) of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99. Furthermore, the mixing ratio (mass ratio) of triacetyl cellulose and diacetyl cellulose is preferably 90:10 to 50:50.

Moreover, as the porous film of the organic material having a hydroxyl group, a porous film made of an organic material obtained by saponifying a mixture of acetyl cellulose having different acetyl values can be preferably used. Organic materials obtained by saponifying a mixture of acetyl celluloses having different acetyl values include saponified products of a mixture of triacetyl cellulose and diacetyl cellulose, saponified products of a mixture of triacetyl cellulose and monoacetyl cellulose, triacetyl cellulose, diacetyl cellulose and mono A saponified product of a mixture of acetylcellulose and a saponified product of a mixture of diacetylcellulose and monoacetylcellulose can also be preferably used. In particular, a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose can be preferably used.
The mixing ratio (mass ratio) of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99. Furthermore, the mixing ratio (mass ratio) of triacetyl cellulose and diacetyl cellulose is preferably 90:10 to 50:50.

The saponification treatment refers to bringing acetyl cellulose into contact with a saponification treatment solution (for example, sodium hydroxide aqueous solution). Thereby, it becomes a regenerated cellulose and a hydroxyl group is introduced into the portion of acetylcellulose that has come into contact with the saponification solution.
In order to increase the nucleic acid separation efficiency, it is preferable that the number of introduced hydroxyl groups is larger. For example, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.
In order to change the saponification rate, the saponification treatment may be performed by changing the concentration of sodium hydroxide. The saponification rate can be determined by NMR or IR.

  Moreover, as a porous film of an organic material having a hydroxyl group, a porous film of cellulose can be preferably used. As the porous membrane of cellulose, a porous membrane of regenerated cellulose can be preferably used. Regenerated cellulose is obtained by saponifying the solid surface or the whole of acetyl cellulose by saponification treatment, and is different from the original cellulose in terms of crystal state and the like.

As a method for introducing a hydrophilic group into a porous film made of an organic material having no hydrophilic group, a graft polymer chain having a hydrophilic group in a polymer chain or in a side chain can be bonded to the porous film.
As a method of bonding the graft polymer chain to the porous film of the organic material, a method of chemically bonding the porous film and the graft polymer chain, and a compound having a double bond that can be polymerized starting from the porous film are polymerized. There are two ways to make the graft polymer chain.

  First, in the method of attaching the porous membrane and the graft polymer chain by chemical bonding, a polymer having a functional group that reacts with the porous membrane is used at the terminal or side chain of the polymer, and this functional group and the porous It can be grafted by chemically reacting with a functional group of the membrane. The functional group that reacts with the porous membrane is not particularly limited as long as it can react with the functional group of the porous membrane. For example, a silane coupling group such as alkoxysilane, an isocyanate group, an amino group, and a hydroxyl group. And carboxyl group, sulfonic acid group, phosphoric acid group, epoxy group, allyl group, methacryloyl group, acryloyl group and the like.

  Particularly useful compounds as a polymer having a reactive functional group at the end of the polymer or in the side chain are a polymer having a trialkoxysilyl group at the polymer end, a polymer having an amino group at the polymer end, and a polymer having a carboxyl group at the polymer end. , A polymer having an epoxy group at the polymer end, and a polymer having an isocyanate group at the polymer end. The polymer used at this time is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption. Specifically, polyhydroxyethylacrylic acid, polyhydroxyethylmethacrylic acid and salts thereof , Polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, and polyoxyethylene.

A method of polymerizing a compound having a polymerizable double bond based on a porous membrane to form a graft polymer chain is generally called surface graft polymerization. The surface graft polymerization method is a method of polymerizing a compound having a polymerizable double bond disposed so as to be in contact with the porous film by giving active species on the surface of the substrate by a method such as plasma irradiation, light irradiation, and heating. It refers to the method of bonding with a porous membrane.
A compound useful for forming a graft polymer chain bound to a substrate has a double bond that can be polymerized and has a hydrophilic group that participates in nucleic acid adsorption. It is necessary to be. As these compounds, any polymer, oligomer, or monomer compound having a hydrophilic group can be used as long as it has a double bond in the molecule. Particularly useful compounds are monomers having hydrophilic groups.

  Specific examples of the particularly useful monomer having a hydrophilic group include the following monomers. For example, a hydroxyl group-containing monomer such as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and glycerol monomethacrylate can be particularly preferably used. Also, carboxyl group-containing monomers such as acrylic acid and methacrylic acid, or alkali metal salts and amine salts thereof can be preferably used.

  As another method for introducing a hydrophilic group into a porous film made of an organic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of ease of work. Examples of polymers include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, acetylcellulose, and different acetyl values. Examples thereof include a mixture of acetylcellulose, and a polymer having a polysaccharide structure is preferable.

  In addition, after coating a porous film of an organic material having no hydrophilic group with acetyl cellulose or a mixture of acetyl celluloses having different acetyl values, the coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values is saponified. You can also. In this case, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.

  Examples of the porous film that is an inorganic material having a hydrophilic group include a porous film containing a silica compound. A glass filter can be mentioned as a porous film containing a silica compound. Further, a porous silica thin film as described in Japanese Patent Publication No. 3058342 can be given. This porous silica thin film is an amphiphilic substance by spreading a developing solution of a cationic type amphiphilic substance having a bilayer-forming ability on a substrate and then removing the solvent from the liquid film on the substrate. This multilayer bilayer thin film can be prepared by bringing the multilayer bilayer thin film into contact with a solution containing a silica compound, and then extracting and removing the multilayer bilayer thin film.

As a method for introducing a hydrophilic group into a porous membrane of an inorganic material having no hydrophilic group, a method of chemically bonding the porous membrane and a graft polymer chain, and a hydrophilic group having a double bond in the molecule are used. There are two methods for polymerizing a graft polymer chain using a monomer having a porous membrane as a starting point.
When the porous membrane and the graft polymer chain are attached by chemical bonding, a functional group that reacts with the functional group at the terminal of the graft polymer chain is introduced into the inorganic material, and the graft polymer is chemically bonded thereto. In addition, when a graft polymer chain is polymerized starting from a porous membrane using a monomer having a hydrophilic group having a double bond in the molecule, a compound having a double bond is polymerized. The starting functional group is introduced into the inorganic material.
As a graft polymer having a hydrophilic group and a monomer having a hydrophilic group having a double bond in the molecule, the porous film of the organic material having no hydrophilic group is chemically bonded to the graft polymer chain. In the method, the described graft polymers having hydrophilic groups and monomers having a hydrophilic group having a double bond in the molecule can be preferably used.

  As another method for introducing a hydrophilic group into a porous film of an inorganic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of ease of work. Examples of polymers include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, acetylcellulose, and different acetyl values. Examples thereof include a mixture of acetylcellulose.

  In addition, after coating acetyl cellulose or a mixture of acetyl celluloses having different acetyl values on a porous film of an inorganic material having no hydrophilic group, the coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values is coated. Can also be saponified. In this case, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.

  Examples of the porous film made of an inorganic material having no hydrophilic group include a porous film produced by processing a metal such as aluminum, ceramics such as glass, cement and ceramics, or new ceramics, silicon and activated carbon. .

  As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a thickness of 10 μm to 500 μm can be used. More preferably, a porous film having a thickness of 50 μm to 250 μm can be used.

  In addition, as the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a minimum pore diameter of 0.22 μm or more can be used. More preferably, a porous membrane having a minimum pore diameter of 0.5 μm or more can be used. As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a ratio of the maximum pore diameter to the minimum pore diameter of 2 or more can be used. More preferably, a porous membrane having a maximum pore size / minimum pore size ratio of 5 or more can be used.

Moreover, as the nucleic acid-adsorptive porous membrane through which the solution can pass, a porous membrane having a porosity of 50 to 95% can be used. More preferably, a porous film having a porosity of 65 to 80% can be used. As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a bubble point of 0.1 to 10 kgf / cm ² can be used. More preferably, a porous film having a bubble point of 0.2 to 4 kgf / cm ² can be used.

  As the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a pressure loss of 0.1 to 100 kPa can be used. More preferably, a porous membrane having a pressure loss of 0.5 to 50 kPa can be used. Here, the pressure loss is the minimum pressure required to pass water per 100 μm thickness of the membrane.

Moreover, as a nucleic acid-adsorptive porous membrane through which the solution can pass, the water permeation amount when passing water at a pressure of 1 kg / cm ² at 25 ° C. is 1 to 1 per 1 cm ^{2 of} the membrane. A porous membrane that is 5000 mL can be used. More preferably, a porous membrane having a water permeation amount of 5 to 1000 mL per 1 cm ^{2 of} membrane when water is passed at 25 ° C. at a pressure of 1 kg / cm ² can be used.

  In addition, as the nucleic acid-adsorbing porous membrane through which the solution can pass, a porous membrane having a nucleic acid adsorption amount of 0.1 μg or more per 1 mg of the porous membrane can be preferably used. More preferably, a porous membrane having a nucleic acid adsorption amount of 0.9 μg or more per 1 mg of the porous membrane can be used.

  Moreover, as a nucleic acid-adsorbing porous membrane through which the solution can pass, when a square porous membrane with a side of 5 mm is immersed in 5 mL of trifluoroacetic acid, it does not dissolve within 1 hour but dissolves within 48 hours. A porous membrane made of a cellulose derivative can be preferably used. Further, a porous membrane made of a cellulose derivative which dissolves within 1 hour when immersed in 5 mL of trifluoroacetic acid, but does not dissolve within 24 hours when immersed in 5 mL of dichloromethane is preferable. Can be used.

  When passing a sample solution containing nucleic acid through the nucleic acid-adsorptive porous membrane, it is preferable to pass the sample solution from one surface to the other surface. When passing a sample solution containing nucleic acid through the nucleic acid-adsorbing porous membrane, it is preferable to pass the sample solution from the side having the larger pore diameter of the nucleic acid-adsorbing porous membrane to the smaller side.

The flow rate when the sample solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane is preferably ² to 1500 μL / sec per cm ^{2 of} the membrane area. Furthermore, it is preferable that it is 5-700 microliters / sec per area cm < ² > of a film | membrane.

  Further, the number of nucleic acid-adsorbing porous membranes through which the solution to be used can pass may be one, but a plurality of nucleic acid-adsorbing porous membranes can also be used. The plurality of nucleic acid-adsorbing porous membranes may be the same or different.

  A cartridge for separating and purifying nucleic acid containing a nucleic acid-adsorbing porous membrane through which the above solution can pass inside in a container having at least two openings can be preferably used. In addition, a nucleic acid separation and purification cartridge in which a plurality of nucleic acid-adsorbing porous membranes through which the above solution can pass is contained in a container having at least two openings. In this case, the plurality of nucleic acid-adsorbing porous membranes accommodated in a container having at least two openings may be the same or different.

  The plurality of nucleic acid-adsorptive porous membranes may be a combination of an inorganic material nucleic acid-adsorptive porous membrane and an organic material nucleic acid-adsorptive porous membrane. For example, a combination of a glass filter and a porous membrane of regenerated cellulose can be mentioned. The plurality of nucleic acid-adsorptive porous membranes may be a combination of an inorganic material nucleic acid-adsorptive porous membrane and an organic material non-nucleic acid-adsorptive porous membrane, for example, glass filter, nylon or A combination with a porous membrane of polysulfone can be mentioned.

  It is preferable that the nucleic acid separation and purification cartridge does not contain other members in a container having at least two openings other than containing a nucleic acid-adsorbing porous membrane through which the above solution can pass. . As a material for the container, plastics such as polypropylene, polystyrene, polycarbonate, and polyvinyl chloride can be used. Biodegradable materials can also be preferably used. In addition, the container may be transparent or colored.

  As the nucleic acid separation / purification cartridge, a nucleic acid separation / purification cartridge provided with means for identifying individual nucleic acid separation / purification cartridges can be used. Examples of means for identifying individual nucleic acid separation and purification cartridges include bar codes and magnetic tapes.

  In addition, a nucleic acid separation and purification cartridge having a structure in which the nucleic acid-adsorbing porous membrane can be easily taken out from a container having at least two openings can also be used.

  Using the nucleic acid separation / purification cartridge containing the nucleic acid-adsorbing porous membrane through which each solution can pass, the nucleic acid can be separated and purified in the following steps. That is, (a) a sample solution containing nucleic acid is injected into one opening of a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane that allows the solution to pass through a container having at least two openings. (B) Depressurizing the inside of the nucleic acid separation / purification cartridge using a pressure difference generator connected to the other opening of the nucleic acid separation / purification cartridge, and the sample solution containing the injected nucleic acid is converted into a nucleic acid-adsorbing porous material. A step of adsorbing nucleic acid in the nucleic acid adsorbing porous membrane by passing through the membrane and discharging from the other opening of the nucleic acid separation and purification cartridge; (c) injecting a washing solution into the one opening of the nucleic acid separation and purification cartridge (D) Using a pressure difference generator connected to the other opening of the nucleic acid separation / purification cartridge, the inside of the nucleic acid separation / purification cartridge is depressurized, and the injected washing solution is subjected to nucleic acid adsorption. A step of washing the nucleic acid-adsorbing porous membrane with the nucleic acid adsorbed by passing through the porous membrane and discharging from the other opening; (F) The nucleic acid separation / purification cartridge is depressurized using a pressure difference generator coupled to the other opening of the nucleic acid separation / purification cartridge, or a centrifugal force is applied to the nucleic acid separation / purification cartridge, Passing the injected recovery liquid through the nucleic acid-adsorbing porous membrane and discharging it from the other opening, thereby desorbing the nucleic acid from the nucleic acid-adsorbing porous membrane and discharging it out of the cartridge for nucleic acid separation and purification; Can be done.

  As another nucleic acid separation and purification step, (a) a nucleic acid containing a nucleic acid-adsorbing porous membrane in which a solution containing a nucleic acid can be passed through a sample solution containing the nucleic acid in a container having at least two openings. A step of injecting into one opening of the separation and purification cartridge, (b) applying a centrifugal force to the nucleic acid separation and purification cartridge, passing the sample solution containing the injected nucleic acid through the nucleic acid-adsorbing porous membrane, and A step of adsorbing nucleic acid in the nucleic acid-adsorbing porous membrane by discharging from another opening, (c) a step of injecting a washing solution into the one opening of the nucleic acid separation / purification cartridge, (d) A step of washing the nucleic acid-adsorbing porous membrane with nucleic acid adsorbed by applying centrifugal force and passing the injected cleaning solution through the nucleic acid-adsorbing porous membrane and discharging it from the other openings. e) Nucleic acid Step of injecting the recovered liquid into the one opening of the separation and purification cartridge (f) Centrifugal force is applied to the nucleic acid separation and purification cartridge, and the injected recovery liquid is passed through the nucleic acid-adsorbing porous membrane and discharged from the other opening. Thus, a step of desorbing the nucleic acid from the nucleic acid-adsorbing porous membrane and discharging it out of the cartridge for nucleic acid separation and purification can be performed.

  Hereinafter, the cleaning process will be described. By performing the washing, the amount and purity of the nucleic acid recovered can be improved, and the amount of the specimen containing the necessary nucleic acid can be made very small. Further, by automating the cleaning and recovery operation, the operation can be performed easily and quickly. The cleaning step may be completed only once for speeding up, and it is preferable to repeat the cleaning multiple times when purity is more important.

In the washing step, the washing solution is supplied to the nucleic acid separation / purification cartridge equipped with the nucleic acid-adsorbing porous membrane using a tube, pipette, automatic injection device, or supply means having the same function. The supplied washing solution is supplied from one opening of the cartridge for separating and purifying nucleic acid (opening into which a sample solution containing nucleic acid is injected), and a pressure difference generator (for example, a spoid, a syringe, a pump, a power pipette, etc.) coupled to the opening. ), The inside of the cartridge for separating and purifying nucleic acid is pressurized and allowed to pass through the porous nucleic acid adsorbing porous membrane and discharged from an opening different from the one opening. Also, the cleaning liquid can be supplied from one opening and discharged from the same opening. Furthermore, it is also possible to supply and discharge the cleaning liquid from an opening different from the one opening to which the sample solution containing the nucleic acid in the cartridge for nucleic acid separation and purification is supplied. However, a method of supplying from one opening of the nucleic acid separation and purification cartridge, passing through the nucleic acid-adsorptive porous membrane, and discharging from an opening different from the one opening is more preferable because of excellent cleaning efficiency.
The amount of the cleaning liquid in the cleaning process is preferably ² μl / mm ² or more. If the amount of the cleaning liquid is large, the cleaning effect is improved, but 200 μl / mm ² or less is preferable in order to maintain the operability and suppress the outflow of the sample.

In the washing step, the flow rate when the washing solution is passed through the nucleic acid-adsorbing porous membrane is preferably 2 to 1500 μL / sec, preferably 5 to 700 μL / sec, per unit area (cm ² ) of the membrane. More preferred. If the passage speed is lowered and time is taken, the washing is sufficiently performed. However, since the speed of the separation and purification operation of the nucleic acid is important, the above range is selected.

In the washing step, the temperature of the washing solution is preferably 4 to 70 ° C. Furthermore, it is more preferable that the temperature of the cleaning liquid is room temperature.
In the washing step, the cartridge for separating and purifying nucleic acid can be washed while being subjected to mechanical vibration, stirring by ultrasonic waves, or by centrifugation.

  In the washing step, the washing solution generally does not contain an enzyme such as a nucleolytic enzyme, but can contain an enzyme that degrades contaminants such as proteins. In some cases, a DNA degrading enzyme, an RNA degrading enzyme, or the like can be included. By using a washing solution containing a DNA degrading enzyme, only RNA in the sample can be selectively recovered. Conversely, by using a washing solution containing an RNase, only DNA in the sample can be selectively recovered.

  In the washing step, the washing liquid is preferably a solution containing a water-soluble organic solvent and / or a water-soluble salt. The washing liquid needs to have a function of washing out impurities in the sample solution adsorbed together with the nucleic acid on the nucleic acid adsorbing porous membrane. For this purpose, it is necessary to have a composition in which nucleic acids are not desorbed from the nucleic acid-adsorbing porous membrane but impurities are desorbed. For this purpose, since a water-soluble organic solvent such as alcohol is hardly soluble in nucleic acid, it is suitable for desorbing components other than nucleic acid while retaining the nucleic acid. In addition, by adding a water-soluble salt, the effect of adsorbing nucleic acids is enhanced, so that the selective removal of unnecessary components is improved.

  As the water-soluble organic solvent contained in the cleaning liquid, methanol, ethanol, isopropanol, n-isopropanol, butanol, acetone and the like can be used, and ethanol is particularly preferable. The amount of the water-soluble organic solvent contained in the cleaning liquid is preferably 20 to 100% by weight, and more preferably 40 to 80% by weight.

On the other hand, the water-soluble salt contained in the cleaning liquid is preferably a halide salt, and chloride is particularly preferable. The water-soluble salt is preferably a monovalent or divalent cation, particularly preferably an alkali metal salt or an alkaline earth metal salt. Among them, a sodium salt and a potassium salt are preferable, and a sodium salt is most preferable.
When the water-soluble salt is contained in the cleaning liquid, the concentration is preferably 10 mmol / L or more, and the upper limit is not particularly limited as long as it does not impair the solubility of impurities, but it is 1 mol / L or less. Is preferable, and 0.1 mol / L or less is more preferable.
In particular, the water-soluble salt is sodium chloride, and it is particularly preferable that sodium chloride is contained in an amount of 20 mmol / L or more.

It is preferable that the cleaning liquid does not contain a chaotropic substance. Thereby, the possibility that the chaotropic substance is mixed in the recovery process subsequent to the cleaning process can be reduced. When a chaotropic substance is mixed during the recovery step, an enzyme reaction such as a PCR reaction is often inhibited. Therefore, it is ideal that the washing liquid does not contain a chaotropic substance in consideration of the subsequent enzyme reaction or the like. In addition, since chaotropic substances are corrosive and harmful, it is extremely advantageous for the experimenter from the viewpoint of safety of the test operation that the chaotropic substances need not be used.
Here, the chaotropic substance is urea, guanidine salt, sodium isothiocyanate, sodium iodide, potassium iodide or the like as described above.

  Conventionally, during the washing process in the nucleic acid separation and purification process, since the washing liquid has high wettability with respect to a container such as a cartridge, the washing liquid often remains in the container, and the washing liquid is mixed into the recovery process following the washing process. This causes a decrease in the purity of the nucleic acid and a decrease in reactivity in the next step. Therefore, when nucleic acid is adsorbed and desorbed using a container such as a cartridge, washing residual liquid remains in the cartridge so that the liquid used for adsorption and washing, particularly the washing liquid, does not affect the next step. It is important not to.

  Accordingly, the surface tension of the cleaning liquid may be less than 35 mPa · m in order to prevent the cleaning liquid in the cleaning process from being mixed into the recovered liquid in the next process and to keep the cleaning liquid remaining in the cartridge to a minimum. preferable. If the surface tension is lowered, the wettability between the cleaning liquid and the cartridge is improved, and the remaining liquid volume can be suppressed.

  On the contrary, in order to reduce the residue of the cleaning liquid in the cartridge in the cleaning process, the surface tension of the cleaning liquid is set to 35 mPa · m or more, the water repellency with the cartridge is increased to form droplets, and the droplets flow down. The amount of remaining liquid can also be suppressed. Either surface tension is selected depending on the combination of the porous membrane adsorbing nucleic acid, the recovery liquid, the cleaning liquid, and the like.

  The washing process can be simplified by using the nucleic acid-adsorbing porous membrane according to the present invention. (1) The number of times that the washing solution passes through the nucleic acid-adsorptive porous membrane may be one, and (2) the washing step can be performed at room temperature. (3) The recovered liquid can be poured into the cartridge immediately after washing. (4) Any one or more of (1), (2), and (3) are also possible. In the conventional method, in order to quickly remove the organic solvent contained in the cleaning solution, a drying step is often required. However, since the nucleic acid-adsorptive porous membrane according to the present invention is a thin film, it can be omitted. .

  Conventionally, in the nucleic acid separation and purification process, there is a problem that contamination of the sample occurs due to the washing liquid often scattering and adhering to the other during the washing process. This kind of contamination in the washing step can be suppressed by devising the shape of the nucleic acid separation / purification cartridge in which the nucleic acid adsorbing polyporous membrane is mounted in a container having two openings and the waste liquid container.

The above-described process of separating and purifying nucleic acid from a sample containing nucleic acid using a nucleic acid separation / purification cartridge containing a nucleic acid-adsorbing porous membrane in a container having at least two openings and a pressure generator is an automatic process. It is preferable to carry out using an automatic device carried out at This not only simplifies and speeds up the operation, but also makes it possible to obtain a certain level of nucleic acid regardless of the skill of the operator.
The following is an automatic method for automatically separating and purifying nucleic acid from a specimen containing nucleic acid using a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane in a container having at least two openings and a pressure generator. Although an example of the automatic device performed in the above is shown, the automatic device is not limited thereto.

  The automatic device uses a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing porous membrane through which the solution can pass, and injects and pressurizes a sample liquid containing nucleic acid into the nucleic acid separation and purification cartridge. After adsorbing nucleic acid to the nucleic acid-adsorptive porous membrane, the washing solution is dispensed to the nucleic acid separation and purification cartridge and pressurized to remove impurities, and then the recovered solution is dispensed to the nucleic acid separation and purification cartridge to adsorb the nucleic acid. A nucleic acid separation and purification device that automatically performs a separation and purification operation for desorbing and recovering nucleic acid adsorbed on a porous porous membrane, and containing the nucleic acid separation and purification cartridge, the sample liquid, and the washing liquid discharge liquid A waste liquid container and a mounting mechanism for holding a recovery container containing the recovery liquid containing the nucleic acid, a pressurized air supply mechanism for introducing pressurized air into the nucleic acid separation and purification cartridge, and the nucleus By comprising a dispensing mechanism for dispensing the washing solution and a recovering solution to the cartridge for separation and purification is characterized in.

The mounting mechanism includes a stand mounted on the main body of the apparatus, a cartridge holder supported on the stand so as to be movable up and down, and holding the nucleic acid separation and purification cartridge, and the position relative to the nucleic acid separation and purification cartridge is exchanged below the cartridge holder. What comprises the said waste-liquid container and the container holder holding the said collection | recovery container as possible is suitable.
The pressurized air supply mechanism includes an air nozzle that ejects pressurized air from a lower end portion, and a pressure that moves the air nozzle up and down relative to the nucleic acid separation and purification cartridge supported by the air nozzle and held in the cartridge holder. A head provided with a head and positioning means for positioning the nucleic acid separation / purification cartridge in the rack of the mounting mechanism is suitable.

  The dispensing mechanism holds a cleaning liquid dispensing nozzle that dispenses the cleaning liquid, a recovered liquid dispensing nozzle that dispenses the recovered liquid, the cleaning liquid dispensing nozzle, and the recovered liquid dispensing nozzle. A nozzle moving table that can move in sequence on the cartridge for nucleic acid separation and purification held in the mounting mechanism, a cleaning liquid supply pump that draws the cleaning liquid from the cleaning liquid bottle that stores the cleaning liquid and supplies the cleaning liquid to the cleaning liquid dispensing nozzle, and a recovery liquid It is preferable to include a recovery liquid supply pump that sucks the recovery liquid from the recovery liquid bottle and supplies it to the recovery liquid dispensing nozzle.

  According to the above automatic apparatus, the nucleic acid separation and purification cartridge, the mounting mechanism that holds the waste liquid container and the recovery container, the pressurized air supply mechanism that introduces pressurized air into the nucleic acid separation and purification cartridge, and the nucleic acid separation and purification cartridge It is equipped with a dispensing mechanism that dispenses the cleaning solution and the recovery solution, and injects and pressurizes the sample solution containing the nucleic acid into the nucleic acid separation and purification cartridge equipped with the nucleic acid-adsorbing porous membrane member to adsorb the nucleic acid to the nucleic acid-adsorbing porous membrane member After that, after washing and dispensing impurities, the recovery solution is dispensed to separate and recover the nucleic acid adsorbed on the nucleic acid-adsorptive porous membrane member automatically. Thus, a mechanism capable of automatically separating and purifying nucleic acid in a sample solution efficiently in a short time can be configured in a compact manner.

  When the mounting mechanism includes a stand, a vertically movable cartridge holder that holds the nucleic acid separation and purification cartridge, and a container holder that holds the waste liquid container and the recovery container in an exchangeable manner, the nucleic acid separation and purification cartridge and The set of both containers and the exchange of the waste liquid container and the collection container can be easily performed.

  In addition, when the pressurized air supply mechanism includes an air nozzle, a pressure head for moving the air nozzle up and down, and a positioning means for positioning the nucleic acid separation / purification cartridge, the pressurized air can be reliably secured with a simple mechanism. Can be supplied.

  In addition, the dispensing mechanism includes a washing liquid dispensing nozzle, a recovery liquid dispensing nozzle, a nozzle moving table that can move in sequence on the nucleic acid separation and purification cartridge, and a washing liquid drawn from the washing liquid bottle and supplied to the washing liquid dispensing nozzle. If the cleaning liquid supply pump and the recovery liquid supply pump that sucks the recovery liquid from the recovery liquid bottle and supplies it to the recovery liquid dispensing nozzle are provided, the cleaning liquid and the recovery liquid can be sequentially dispensed by a simple mechanism.

[Example 1]
(1) Preparation of nucleic acid purification cartridge A nucleic acid purification cartridge having an inner diameter of 7 mm and a portion for accommodating a nucleic acid-adsorbing porous membrane is prepared from high impact polystyrene.

(2) As the nucleic acid-adsorbing porous membrane, a porous membrane obtained by saponifying a triacetyl cellulose porous membrane is used and accommodated in the nucleic acid-adsorbing porous membrane housing part of the nucleic acid purification cartridge prepared in (1) above.

(3) Preparation of Nucleic Acid Solubilizing Reagent Solution and Washing Solution A nucleic acid solubilizing reagent solution and a washing solution having the following formulation are prepared.

(Nucleic acid solubilizing reagent solution)
Guanidine hydrochloride (Life Technology) 382g
Tris (Life Technology) 12.1g
Triton-X100 (ICN) 10g
1000ml distilled water
(Cleaning solution)
10 mmol / L Tris-HCl 65% ethanol

  Table 1 shows the recovery solutions used for the purification of nucleic acids and the evaluation of the amount of adsorbed nucleic acids.

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

[Example 2]
The operation is performed under the same conditions as in Example 1 except that the recovered liquid 2 shown in Table 1 is used as the recovered liquid.
[Comparative Example 1]
The operation is performed under the same conditions as in Example 1 except that the recovered liquid 3 shown in Table 1 is used as the recovered liquid.

(5) Evaluation of nucleic acid recovery amount Experiments were carried out 10 times for each of Examples 1 and 2 and Comparative Example 1, and the average value of the amount of nucleic acid that could be recovered was taken and recovered in Example 1 using recovery solution 1. The relative values with the average value of the amount of nucleic acid taken as 1 are shown in Table 2.

  As is clear from the results in Table 2, it can be seen that nucleic acids can be efficiently recovered and purified by using the method of the present invention.

[Comparative Examples 2 to 4]
The operation is performed under the same conditions as in Example 1 except that the recovered liquid 4, the recovered liquid 5 or the recovered liquid 6 shown in Table 1 is used as the recovered liquid.

(6) Evaluation of nucleic acid recovery amount The experiments of Example 1 and Comparative Examples 2 to 4 are repeated 10 times. Table 3 shows the average value of the amount of nucleic acid recovered and the average value of the nucleic acid recovered in Example 1 using the recovery liquid 1 as 1.

  As is apparent from the results in Table 3, it can be seen that by using the method of the present invention, nucleic acids can be efficiently recovered and purified when the salt concentration is low or the ionic strength is low.

Claims (7)

(I) passing a sample solution containing nucleic acid through a nucleic acid-adsorbing porous membrane, and adsorbing the nucleic acid in the porous membrane;
(Ii) passing the washing solution through the nucleic acid-adsorbing porous membrane and washing the porous membrane with the nucleic acid adsorbed; and (iii) passing the recovered solution through the nucleic acid-adsorbing porous membrane. In the method for separating and purifying nucleic acid comprising the step of desorbing nucleic acid from the porous membrane, the nucleic acid-adsorbing porous membrane is a porous membrane that adsorbs nucleic acid by an interaction that does not substantially involve ionic bonds. The method for separating and purifying nucleic acid, wherein the pH of the recovered liquid is 2 to 11.   In each of the steps (i), (ii) and (iii) according to claim 1, the sample solution, the washing solution or the recovery solution containing the nucleic acid is passed through the nucleic acid-adsorbing porous membrane under pressure. The method for separating and purifying nucleic acid according to claim 1.   One opening of the nucleic acid separation and purification cartridge in which the nucleic acid-adsorbing porous membrane is accommodated in a container having at least two openings in each of the steps (i), (ii) and (iii) according to claim 1 A sample solution, a washing solution or a recovery solution containing nucleic acid is injected into the cartridge, and the inside of the cartridge is pressurized using a pressure difference generator coupled to the one opening of the cartridge, and the injected solutions are allowed to pass through. The method for separating and purifying nucleic acid according to claim 1, wherein the nucleic acid is discharged from another opening.   The method for separating and purifying nucleic acid according to any one of claims 1 to 3, wherein the ionic strength of the recovered liquid is 290 mmol / L or less.   The method for separating and purifying nucleic acid according to any one of claims 1 to 4, wherein the salt concentration of the recovered liquid is 90 mmol / L or less.   A nucleic acid solubilizing reagent solution containing a nucleic acid separation and purification cartridge, a proteolytic enzyme, a chaotropic salt and a surfactant, a washing solution, and a recovery for performing the method for separating and purifying nucleic acid according to any one of claims 1 to 5. A kit containing a liquid reagent.   An apparatus for automatically performing the method for separating and purifying nucleic acid according to any one of claims 1 to 5.