JP2000035423A - Separation method of nucleic acid by liquid chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To separate a larger quantity of long chain nucleic acid such as furosemide or DNA in a short time by using hydrophobic chromatography. SOLUTION: For a compound used for synthesis (quantization) of base material constituting hydrophobic chromatography filler, there is no particular limit, when after synthesis of the base material various functional groups or various ion exchange groups can be added to it after reaction, but because influence on an object to be separated due to hydrophobic function shown by the base material itself and swelling/shrinkage of the base material itself against change of salt concentration or pH are made minimum, it is enough to be monomer showing comparative hydrophilic property. Then single functional monomer and multiple funcional monomer are gathered at proper rate, and after diluent and polymerization initiator are added to them and stirred, this solution is added to water solution of suspension stabilizer and heated, and polymerized to make the base material. In this case, the grain size of the base material is set to be 2-500 μm so as to meet with the using purpose, and the pore diameter of the base material is favorable in the range of 500-4000 angstrom.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating and purifying nucleic acids used for genomic analysis and genetic manipulation. More specifically, the present invention relates to the treatment of genetic diseases and the like caused by DNA abnormalities using liquid chromatography. Specific D used in cells of animals, humans, etc., which is effective for gene therapy to be used
The present invention relates to a method for separating long-chain nucleic acids represented by NA, for example, DNA such as plasmids, organelles and phages in bacteria.

[0002]

2. Description of the Related Art In recent years, gene therapy has attracted attention, and a method for separating and purifying a large amount of a long-chain nucleic acid such as a plasmid or a specific DNA used for gene therapy more simply and in a shorter time has been eagerly desired.

Here, in order to use the nucleic acid for human therapy, it is desirable that the nucleic acid can be separated and purified with the same structure (higher-order structure) as that existing in the living body. Here, since an enzymatic reaction is used for genetic recombination of a nucleic acid, the nucleic acid needs to be separated and purified until it can be used as a substrate for the reaction. Further, in order to prevent impurities from adversely affecting the human body, it is necessary that nucleic acids be separated and purified to high purity. BACKGROUND ART Conventionally, chemical treatment methods have been frequently used for separation and purification of long-chain nucleic acids such as DNA and plasmids contained in cells and bacteria.

[0004] Among various long-chain nucleic acids currently used for gene therapy, plasmids are limited in their cleavage sites due to specific restriction enzymes, and are relatively easily used for gene recombination. I have. The following is an example of general purification of a plasmid from E. coli.

[0005] First, RNase is added to lysozyme for a short time to break the cell wall and degrade Escherichia coli RNA. Next, a mixed solution of NaOH and sodium dodecyl sulfate (SDS) is added for the purpose of dissolving the cell membrane. NaO
H partially changes DNA and partially degrades RNA, and SDS functions to dissolve membranes, separate and denature proteins. Subsequently, SDS-protein complex and cell residue were
Precipitate by adding potassium N acetate (pH 4.8).
At this time, pH is important, and NaO used in the above-mentioned operation is used.
It has the function of neutralizing H and the function of recombining the plasmid. Thereafter, the precipitate is removed by centrifugation, and the desired plasmid is obtained in the supernatant.

[0006] In these series of operations (hereinafter, referred to as a pretreatment step), it is important to perform gentle and firm mixing. If vigorous vibration is applied during this operation, the chromosome of the bacterium will be cut into fragments so small that they do not aggregate, which may cause contamination as a contaminant during plasmid purification.

Subsequently, isopropanol is added to the supernatant obtained by centrifugation to precipitate and concentrate the plasmid. Finally, the protein is removed from the plasmid fraction using phenol and chloroform, and the plasmid is precipitated with alcohol.

[0008] By the above-described series of operations, a relatively pure plasmid can be obtained (hereinafter, the above-described nucleic acid separation and purification method is referred to as a chemical separation method). However, in the chemical separation method, the separation and purification steps are complicated, and a large amount of an organic solvent must be used.

In addition to the chemical separation and purification method, there is a plasmid separation method by electrophoresis. This method is currently the highest resolution technology. There are two types of electrophoresis, filter paper electrophoresis and gel electrophoresis, but gel electrophoresis is currently common. The electrophoresis method has an advantage that a very high-purity plasmid can be obtained. However, on the other hand, there are many problems such as a long separation time, difficult collection, and a small sample load. As a result, electrophoretic separation is currently used only when it is desired to further improve the purity of the plasmid fraction purified by the above-described chemical separation and purification method.

[0010] In order to solve the problems of the chemical separation and purification method and the separation by electrophoresis described above, a nucleic acid separation and purification method using liquid chromatography has been performed. So far, there have been examples in which long-chain nucleic acids such as plasmids have been separated and purified using ion exchange chromatography or reversed phase chromatography.

[0011]

The method for separating and purifying nucleic acids using liquid chromatography is simpler in operation than a chemical separation method, can easily separate nucleic acids, and uses an organic solvent and the like. There is an advantage that there is no need. However, ion exchange chromatography, or
The conventional separation method using only the reversed-phase chromatography has a problem that nucleic acids with sufficiently high purity, particularly long-chain nucleic acids such as plasmids, cannot be obtained in large quantities.

Accordingly, an object of the present invention is to provide a separation method utilizing liquid chromatography which can separate a larger amount of long-chain nucleic acids such as plasmids and DNA in a shorter time.

[0013]

Means for Solving the Problems The invention of claim 1 of the present invention made in view of the above object is a method for separating nucleic acids, characterized by using hydrophobic chromatography. The invention of claim 7 of the present application is a method for separating nucleic acids, characterized by using a combination of hydrophobic chromatography and ion exchange chromatography. Hereinafter, the present invention will be described in detail by taking as an example a case where a plasmid that is a long-chain nucleic acid is separated and purified from Escherichia coli cultured in a large amount.In such a case, when separating and purifying a nucleic acid such as a plasmid or DNA as a target product, It is a useful method.

The compounds used for the synthesis of the base material constituting the hydrophobic chromatographic or ion-exchange chromatographic filler used in the present invention are various compounds which exhibit hydrophobicity after the synthesis of the base material (in other words, the formation of particles). Any compound may be used as long as it can add a functional group or various ion exchange groups in a post-reaction. For example, as a monofunctional monomer, styrene, o-halomethylstyrene, m-halomethylstyrene, p-halomethylstyrene, o-haloalkylstyrene, m-haloalkylstyrene, p-haloalkylstyrene, α-methylstyrene, α-methylo -Halomethylstyrene, α-methyl m-halomethylstyrene, α-methyl p-halomethylstyrene, α-methyl o
-Haloalkylstyrene, α-methyl m-haloalkylstyrene, α-methyl p-haloalkylstyrene, o-
Hydroxymethylstyrene, m-hydroxymethylstyrene, p-hydroxymethylstyrene, o-hydroxyalkylstyrene, m-hydroxyalkylstyrene,
p-hydroxyalkylstyrene, α-methyl o-hydroxymethylstyrene, α-methyl m-hydroxymethylstyrene, α-methyl p-hydroxymethylstyrene,
α-methyl o-hydroxyalkylstyrene, α-methylm-hydroxyalkylstyrene, α-methylp-hydroxyalkylstyrene, glycidyl methacrylate,
Glycidyl acrylate, hydroxyethyl acrylate, hydroxymethacrylate, vinyl acetate and the like can be exemplified.

Here, examples of the haloalkyl group which is substituted on the aromatic ring include halogens such as CL, Br, I, F, etc., which are substituted with straight-chain and / or branched saturated carbon having 2 to 15 carbon atoms. Hydrogen.

Examples of polyfunctional monomers include divinylbenzene, trivinylbenzene, divinyltoluene, trivinyltoluene, divinylnaphthalene, trivinylnaphthalene, ethylene glycol dimethacrylate, ethylene glycol diacrylate, diethylene glycol dimethacrylate, diethylene glycol diacrylate, and methylene bis. Examples thereof include methacrylamide and methylenebisacrylamide.

As described above, the compound used in the present invention is not particularly limited as long as various functional groups exhibiting hydrophobicity or various ion exchange groups can be introduced in a post-reaction. The target substance to be separated is affected by the hydrophobic function exhibited by itself, or a change in salt concentration, in order to minimize the swelling and shrinkage of the base material itself in response to a change in pH, a monomer having a relatively hydrophilic property, for example, Glycidyl methacrylate,
It is particularly preferable to prepare a substrate using glycidyl acrylate, hydroxyethyl acrylate, hydroxymethacrylate, vinyl acetate, or the like.

The general method of producing a substrate using the above-described monomer is as follows (the method of producing the substrate is not limited to the method shown here). First, a monofunctional monomer and a polyfunctional monomer are weighed in an appropriate ratio, and a precisely weighed diluent (a solvent used for the purpose of adjusting the pores of formed particles) and a precisely weighed polymerization initiator are added, followed by thorough stirring. . Then, after being added to an aqueous solution in which a previously weighed suspension stabilizer is dissolved, stirring is performed with a stirrer to form oil droplets of a desired size, and the mixed solution is gradually heated to polymerize, so-called water. Oil droplet suspension polymerization may be performed.

The ratio of the monofunctional monomer to the polyfunctional monomer is not particularly limited. When relatively soft particles (substrate) are prepared per 1 mol of the monofunctional monomer, the polyfunctional monomer is used in an amount of 0.01 to 0.2. About 0.2 to 0.5 moles may be used for producing hard particles, or about 0.2 to 0.5 moles. For producing harder particles, only polyfunctional monomers may be used. The polymerization initiator is not particularly limited, and generally used azobis-based and / or peroxide-based ones are used. There is no particular limitation on the suspension stabilizer. If it is possible to prevent the droplets from agglomerating, an ionic surfactant, a nonionic surfactant,
Any of the amphiphilic polymers or mixtures thereof can be used.

The particle size of the formed particles is not particularly limited, and particles having an appropriate particle size of 2 to 500 μm may be selected according to the purpose of use. For example, for analytical purposes, the particle size is 2
The thickness is preferably about 30 μm, more preferably about 2 to 10 μm.
10 to 100 for large-scale purification of high-purity nucleic acid
about 100 μm when separating the target substance from the stock solution
The thickness may be about 00 μm, more preferably about 200 to 400 μm. The particle size can be adjusted by adjusting the rotation speed of the stirrer during polymerization. If particles having a small particle size are required, the rotation speed should be increased, and if large particles should be obtained, the rotation speed should be reduced. Here, the selection of the diluent is particularly important because the diluent used is used for controlling the pores in the produced particles.
The basic idea is to adjust the solvent used for the polymerization by a combination of a solvent that is a poor solvent for the monomer and a solvent that is a good solvent for the monomer.

The pore diameter of the substrate may be appropriately selected depending on the molecular size of the nucleic acid to be separated. For a packing material for hydrophobic chromatography, the packing material is in the range of 500 to 4000 angstroms. For agents, it is preferred to be in the range of 1500-4000 angstroms.

In hydrophobic chromatography, in order to separate nucleic acids having different hydrophobicities, preferably using packing materials having different hydrophobicities, the surface modification of the substrate is important. Applicable hydrophobic groups are not particularly limited as long as they do not deviate from the purpose of separating nucleic acids having different hydrophobicities with fillers having different hydrophobicities, and include the following compounds (a) to (c). Hydrophobic groups containing one or more compounds selected from the group as main components are particularly preferred.

Compound (a): a saturated or unsaturated hydrocarbon group having 2 to 20 carbon atoms which may be long-chain or branched (however, the hydrocarbon group may contain an aromatic ring. ).

Compound (b): The following structural formula 1 (where n is
= 0 to 20, the methylene group may be linear or branched,
m = 0 to 3 and may be linear or branched, and A is a C ＝ O group or an ether group, but there is no A and the substrate and the methylene group may be directly bonded. .

Compound (c): an ether group of an alkylene glycol having 2 to 20 carbon atoms comprising a repeating unit of 0 to 10 (provided that the terminal opposite to the functional group reacted with the substrate remains an OH group) , May be capped with an alkyl group having 1 to 4 carbon atoms).

[0026]

Embedded image

When the surface is modified by using the compounds classified into the above three categories, they may be used alone or in combination. An example of the proper use of these will be described below with reference to the alkyl group included in the compound (a). For example, RNA from Escherichia coli and RNA in human and animal cells
In order to separate a compound having high isohydrophobicity, an alkyl group having 2 to 15 carbon atoms is particularly suitable. In the case of a compound having relatively low hydrophobicity such as DNA derived from Escherichia coli and DNA in cells of humans and animals, an alkyl group having 4 to 18 carbon atoms is particularly suitable. Further, in the case of a compound having low hydrophobicity such as a plasmid, an alkyl group having 6 to 20 carbon atoms is preferable. The separation of these compounds is not limited to the above examples, and the compounds may be appropriately selected and the surface may be modified. The reason is that the hydrophobicity of the filler varies depending on the salt concentration in the culture solution or the salt concentration of the eluent for adsorption. Further, even with the same functional group, the hydrophobicity of the filler varies depending on the ratio of addition.

The pore size of the hydrophobic chromatography substrate is particularly preferably in the range of 500 to 4000 Å, but can be appropriately selected from the above range depending on the molecular size of the nucleic acid to be separated. In general,
Since the holding power and adsorption capacity (sample load) of nucleic acid to the packing material differ depending on the pore size, a base material with a large pore size is used for a nucleic acid with a large molecular size, and a base material with a small pore size is used for a nucleic acid with a small molecular size. It is preferable to use

Next, an example of a method of reacting these hydrophobic groups with a substrate will be described. The base material is styrene base,
For reactions with compounds of the first and second categories,
A halogen-containing compound B and / or carbonyl halide C is directly added to an aromatic ring in a substrate as a dehalogenated compound B and / or an acylated compound C by utilizing a Friedel-Crafts reaction using a catalyst such as FeCL3, SnCL2, or AlCL3. I can do it. When the base material is a particle containing a halogen group, for example, NaOH using a compound containing OH in a functional group to be added, such as butanol,
A functional group can be introduced through an ether bond by using a Williamson reaction with an alkali catalyst such as KOH or KOH. When the functional group to be added is an amino group-containing compound like hexylamine, it can be added using a dehalogenation reaction using an alkali catalyst such as NaOH or KOH. If the base material contains an OH group, conversely, if an epoxy group, a halogen group or a carbonyl halide group is introduced into the functional group to be added, the functional group can be introduced through an ether or ester bond. . When the base material contains an epoxy group, the functional group can be introduced through an ether or amino bond by reacting with an OH group or amino group-containing compound in the functional group to be added. When the functional group to be added contains a halogen group, the functional group can be added through an ether bond by using an acid catalyst. Since the ratio of introducing a functional group into the substrate is affected by the hydrophobicity of the object to be separated,
Although not limited, generally 1 g of dried substrate
A filler to which a functional group is added in an amount of about 0.05 to 4.0 mmol per one layer is preferable.

In the above description, a method of forming a substrate (particles) and then adding a functional group by a post-reaction is described above.
Even if a method of forming a base material after polymerization using the above-mentioned monomer to which a functional group is added before polymerization is used, there is no particular problem because there is no difference in function. Further, porous silica gel may be used as the base material. As an example of a method for producing silica gel, alkyltrimethoxy is directly applied to particles produced according to the method described in “Latest High-Performance Liquid Chromatography” on page 289 or less (author: Toshio Minamihara, Nobuo Ikegawa, published by Tokyo Hirokawa Shoten, 1988). Silane coupling may be performed using a compound such as silane. Alternatively, after performing a silane coupling using an epoxy group-containing silane coupling agent, a functional group may be added according to the above-described method. As for the introduction ratio of the functional group, a filler to which about 0.05 to 4.0 mmol of the functional group is added per 1 g of the dried substrate is preferable.

Next, an example of a method for separating and purifying nucleic acids using these fillers will be described. First, as the eluent used in the hydrophobic chromatography of the present invention, at least two types of eluents, that is, a solution A containing a high-concentration salt and a solution B containing a low-concentration salt are used. From liquid A to B
An elution method in which the solution is switched stepwise to a solution, and a gradient elution method in which the salt concentration is continuously changed from the solution A to the solution B can be used. Buffers and salts used in these eluents can be those usually used in hydrophobic chromatography. The solution A containing a high concentration of salt is particularly preferably an aqueous solution having a salt concentration of 1.0 to 4.5 M and a pH of 6 to 8. Solution B containing a low concentration of salt has a salt concentration of 0.01
An aqueous solution having a pH of 0.5 to 0.5 M and a pH of 6 to 8 is particularly preferred. Examples of the salt include ammonium sulfate and sodium sulfate.

In the present invention, it is particularly preferable to carry out hydrophobic chromatography by a combination of a filler into which a functional group having weak hydrophobicity is introduced and a filler into which a functional group having strong hydrophobicity is sequentially introduced. This method is particularly suitable for separating plasmids. For example, in a culture solution obtained by mass-cultivating Escherichia coli, various components having different hydrophobicities such as polysaccharides, Escherichia coli genome, RNA, plasmids and proteins are contained.
According to the findings of the present inventors, there is a difference in the hydrophobicity between nucleic acids, and since the protein serving as an impurity has a higher hydrophobicity as compared with the target plasmid, various fillers having different hydrophobicities are used. Plasmids can be efficiently separated and purified by connecting the columns packed with, in order of decreasing hydrophobicity. Specifically, after the components in the culture solution are successively adsorbed on a packing material whose hydrophobicity increases in the order of decreasing hydrophobicity, only the column on which the target component is adsorbed is separated and eluted. It is.

In the present invention, it is particularly preferable to separate and purify nucleic acids by combining hydrophobic chromatography and ion exchange chromatography in order to efficiently obtain a large amount of high-purity nucleic acids. Here, for the hydrophobic chromatography, the above-mentioned fillers and the like can be used. Here, as the hydrophobic chromatography, it is particularly preferable to use columns packed with various packing materials having different hydrophobicities by connecting them in order of decreasing hydrophobicity.

The packing material used for ion exchange chromatography for further purifying the target plasmid, which has been separated in advance by hydrophobic chromatography, with a higher purity, has a relatively large pore size, particularly from 1500 to 4000.
Those in the angstrom range are preferred. The general method of preparing a substrate used for ion exchange chromatography is as described above, and the surface modification for introducing an ion exchange group into these substrates can be performed by a known method.

As the eluent used in the ion exchange chromatography, at least two kinds of eluents comprising a solution C containing a low concentration of salt and a solution D containing a high concentration of salt are used. An elution method in which the solution is switched stepwise from solution C to solution D, and a gradient elution method in which the composition is continuously changed from solution C to solution D can be used. Buffers and salts used in these eluents may be those commonly used in ion exchange chromatography. Solution C containing a low concentration of salt has a buffer concentration of 10-50 mM, p
An aqueous solution in which H is 6 to 9 is particularly preferred. The solution D containing a high concentration of salt is particularly preferably an aqueous solution having 0.1 to 2 M sodium salt in the solution C. Examples of the sodium salt include sodium chloride and sodium sulfate.

In addition, any eluent may contain components other than the buffer solution. In particular, a chelating agent for divalent metal ions, for example, ethylenediaminetetraacetic acid may inhibit the degradation of the plasmid by the DNA degrading enzyme in the homogenate of Escherichia coli. Since it can be suppressed, it is particularly preferable when a plasmid is separated. The concentration of the chelating agent for divalent metal ions is preferably 0.1 to 100 mM.

In a particularly preferred embodiment of the present invention,
The eluent A having a high salt concentration adjusted according to the method described above is passed through a column of hydrophobic chromatography connected in the order of hydrophobicity. After the steady state is reached, a culture solution of Escherichia coli or the like from which the operation of decomposing RNA by a decomposing enzyme or the like is omitted is injected into the column. Subsequently, the eluent A is passed, and the compound not adsorbed to any column is allowed to flow out of the system. Then, only the column to which the target compound is adsorbed is separated,
The target substance is eluted by the stepwise method or the gradient method. Next, the eluent C is passed through the above-mentioned ion exchange column, and after a steady state, the eluate containing the target substance is injected as it is. Thereafter, using the eluent D, the target substance is eluted by a stepwise method or a gradient method to obtain a purified product.

[0038]

The present invention provides a method for separating and purifying nucleic acids by a simple operation. Specifically, in a preferred embodiment of the present invention in which columns each packed with a filler having a different hydrophobicity are connected in ascending order of hydrophobicity, the column is simply passed through the pretreatment step solution in the conventional operation. In addition, a target nucleic acid, particularly a long-chain nucleic acid such as a plasmid can be separated and purified in large quantities. In the present invention, in the conventional pretreatment step, the nucleic acid can be separated and purified by directly passing the pretreatment step solution through a hydrophobic chromatography column, in which the operation of decomposing RNA derived from Escherichia coli with a degrading enzyme is omitted. .

In the present invention, it is particularly preferable to use a combination of hydrophobic chromatography and ion exchange chromatography to separate and purify a large amount of a high-purity target nucleic acid, particularly a long-chain nucleic acid such as a plasmid, by a simple operation. can do.

As described above, in the method for separating nucleic acids according to the present invention, a large amount of a high-purity target product can be obtained by a simple operation as compared with the conventional method.

[0041]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of Packing Material for Hydrophobic Chromatography A packing material for gel filtration having an average particle size of 20 μm and an average pore size of 2000 Å, G6000PW (manufactured by Tosoh Corporation) was used as a base material. A packing material for chromatography was prepared. 20 g of G6000PW washed and replaced with 1,4-dioxane, 1,4-dioxane 20
g, and 1 g of 1,2-epoxybutane were stirred at 45 ° C. for 6 hours and then mixed to obtain a filler having a butyl group as a weakly hydrophobic functional group (hereinafter referred to as Butyl-6PW). Similarly, G6000PW 20 g, 1,4-dioxane 2
After stirring 0 g and 1 g of 1,2-epoxyoctane at 45 ° C. for 6 hours, they were mixed to obtain a filler having an octyl group as a strongly hydrophobic functional group (hereinafter, Octyl-6PW). Each filler has an inner diameter of 7.5 mm and a length of 7.5 c.
m stainless steel column.

(2) Isolation of Plasmid by Hydrophobic Chromatography Escherichia coli having pBR322 as a plasmid was cultured at 37 ° C.
After culturing for 16 hours at, the culture was centrifuged at 8000 rpm for 20 minutes at 4 ° C. 100 mg of precipitated E. coli
After suspending in 10 ml of 25 mM Tris-HCl buffer (pH 7.5) containing lysozyme, 50 mM glucose, and 10 mM ethylenediaminetetraacetic acid (hereinafter, EDTA), and stirring,
The cells were left at room temperature for 5 minutes to dissolve the cell walls. Then 1%
Add 20 ml of a 0.2 N sodium hydroxide solution containing sodium dodecyl sulfate, mix gently, and add
The cells were left for 0 minutes to dissolve the cell membrane.

Next, 15 ml of a 3M sodium acetate buffer (pH 5.4) was added, mixed gently, and cooled under ice-cooling.
Left for 0 minutes. Next, at 4 ° C and 10,000 rpm for 20 minutes.
After centrifugation for minutes, the supernatant was recovered to obtain a disrupted solution of Escherichia coli.
E. coli homogenate contains an equal volume of 4M ammonium sulfate.
After addition of 1M phosphate buffer (pH 7.0), the mixture was subjected to hydrophobic chromatography.

A crushed Escherichia coli solution containing ammonium sulfate was connected to a connected column in which a Butyl-6PW column and an Octyl-6PW column equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 2 M ammonium sulfate and 1 mM EDTA were connected in series. After injecting 3 ml, 0.1 M phosphate buffer containing 2 M ammonium sulfate and 1 mM EDTA (pH
7.0) was sent to the connected column at a flow rate of 1 ml / min for 20 minutes to elute impurities out of the column. Next, But
After removing the yl-6PW column out of the flow path system, Oc
Include 1 mM EDTA only in the tyl-6PW column.
1M phosphate buffer (pH 7.0) flow rate 1ml / min
For 15 minutes. As a result, a chromatogram as shown in FIG. 1 was obtained. In FIG. 1, 1 is the peak of the impurity, and 2 is the peak of the fraction containing the plasmid. The column eluate corresponding to peak 2 was collected and further purified by ion exchange chromatography as described below.

(3) Separation of Plasmid Combining Hydrophobic Chromatography and Ion Exchange Chromatography DEAE-5PW (trade name, manufactured by Tosoh Corporation, 7.5 mm inner diameter) was used as a packing column for packing for ion exchange chromatography. , 7.5 cm in length)). 0.6M
DEAE-5P equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing sodium chloride and 1 mM EDTA
After injecting 3 ml of the plasmid fraction into the W column, a 20 mM Tris-HCl buffer (pH 7.5) containing 0.6 M sodium chloride and 1 mM EDTA was sent to the column at a flow rate of 1 ml / min for 35 minutes to remove impurities from the column. Eluted. Next, a 20 mM Tris-HCl buffer containing 1 mM EDTA (p
H7.5) from 0.6M to 0.
Elution was performed by a gradient method in which the concentration was continuously changed to 8 M at a flow rate of 1 ml / min for 30 minutes. As a result, FIG.
As a result, a chromatogram was obtained as shown in FIG. In FIG. 2, 1 is the peak of the impurity, and 2 is the peak of the fraction containing the plasmid. The column eluate corresponding to the peak of No. 2 was collected, and the purity was examined by agarose gel electrophoresis. When the gel after electrophoresis was stained with ethidium bromide, an electrophoretic image as shown in FIG. 3 was obtained. In FIG. 3, 3 is a marker, 5 is a plasmid fraction obtained by the present purification method, and 6 is an electrophoresis image of commercially available purified pBR322. As is clear from the figure, a high-purity supercoiled plasmid could be obtained by a simple operation according to this purification method.

Comparative Example 1 For comparison, plasmid pBR322 was purified from a disrupted solution of Escherichia coli only by ion exchange chromatography.

The E. coli lysate was prepared in the same manner as in the Examples, and an equal volume of 20 mM Tris-HCl buffer (pH 7.5) was added to prepare a sample for ion exchange chromatography.
20m containing 0.6M sodium chloride, 1mM EDTA
After injecting 3 ml of the sample into the above DEAE-5PW column equilibrated with M Tris-HCl buffer (pH 7.5),
M sodium chloride, 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA at a flow rate of 1 ml / min.
The solution was sent to the column for 0 minutes to elute impurities from the column. Next, elution was performed by a gradient method in the same manner as in the example. As a result, a chromatogram as shown in FIG. 4 was obtained. In FIG. 4, 1 is the peak of the impurity, and 2 is the peak of the fraction containing the plasmid. The column eluate corresponding to the peak of No. 2 was collected, and the purity was examined by agarose gel electrophoresis. As a result, an electrophoretic image as shown in FIG. 3 was obtained. In FIG. 3, 3 is a marker, 4 is a plasmid fraction obtained in the comparative example, 5 is a plasmid fraction obtained in Example 1, and 6 is an electrophoresis image of commercially available purified pBR322. The plasmid fraction obtained only by ion exchange chromatography contained many impurities.

Example 2 (1) Preparation of Packing Material for Hydrophobic Chromatography G6000PW (manufactured by Tosoh Corporation) having a mean particle size of 20 μm and a mean pore size of 2000 Å was used as a base material for gel filtration. A packing material for chromatography was prepared. 20 g of G6000PW washed and replaced with 1,4-dioxane, 1,4-dioxane 20
g, 1 g of 1,2-epoxyoctane and 0.5 ml of boron trifluoride as a catalyst were added, stirred and mixed at 45 ° C. for 6 hours, and a hydrophobic chromatographic packing material for adsorption of an octyl group-containing plasmid (hereinafter referred to as “filler”). , Octyl-6PW)
I got Next, an average particle diameter of 20 μm and an average pore diameter of 950
Angstrom's gel filtration filler, G5000PW
(Tosoh Corporation) was used as a base material, and a filler having low hydrophobicity was prepared. 20 g of G5000PW, 20 g of 1,4-dioxane, 1 g of 1,2-epoxybutane and 0.5 ml of boron trifluoride as a catalyst are added and stirred at 45 ° C. for 6 hours, and mixed to adsorb RNA or protein having a butyl group. Hydrophobic chromatography filler (hereinafter referred to as Butyl-5P)
W) was obtained. Each filler was packed in a stainless steel column having an inner diameter of 7.5 mm and a length of 7.5 cm.

(2) Separation of Plasmid Combining Hydrophobic Chromatography and Ion Exchange Chromatography-Separation by Hydrophobic Chromatography Escherichia coli having pBR322 as a plasmid was subjected to 37 ° C.
After culturing for 16 hours at, the culture was centrifuged at 8000 rpm for 20 minutes at 4 ° C. 100 mg of precipitated E. coli
After suspending in 10 ml of 25 mM Tris-HCl buffer (pH 7.5) containing lysozyme, 50 mM glucose, and 10 mM ethylenediaminetetraacetic acid (hereinafter, EDTA), and stirring,
The cells were left at room temperature for 5 minutes to dissolve the cell walls. Then 1%
Add 20 ml of a 0.2 N sodium hydroxide solution containing sodium dodecyl sulfate, mix gently, and add
The cells were left for 0 minutes to dissolve the cell membrane.

Next, 15 ml of a 3M sodium acetate buffer (pH 5.4) was added, mixed gently, and cooled under ice-cooling.
Left for 0 minutes. Next, at 4 ° C and 10,000 rpm for 20 minutes.
After centrifugation for minutes, the supernatant was recovered to obtain a disrupted solution of Escherichia coli.
E. coli homogenate contains an equal volume of 4M ammonium sulfate.
After addition of 1M phosphate buffer (pH 7.0), the mixture was subjected to hydrophobic chromatography.

A crushed Escherichia coli solution containing ammonium sulfate was connected to a connected column in which a Butyl-5PW column and an Octyl-6PW column equilibrated with a 0.1 M phosphate buffer (pH 7.0) containing 2 M ammonium sulfate and 1 mM EDTA were connected in series. After injecting 3 ml, 0.1 M phosphate buffer containing 2 M ammonium sulfate and 1 mM EDTA (pH
7.0) was sent to the connected column at a flow rate of 1 ml / min for 20 minutes to elute impurities out of the column. Next, But
After removing the yl-5PW column out of the channel system,
Include 1 mM EDTA only in the tyl-6PW column.
1M phosphate buffer (pH 7.0) flow rate 1ml / min
For 15 minutes. As a result, a chromatogram as shown in FIG. 5 was obtained. In FIG. 5, 1 is the peak of the impurity, and 2 is the peak of the fraction containing the plasmid. The column eluate corresponding to peak 2 was collected and further purified by ion exchange chromatography as described below.

(3) Preparation of Packing Material for Ion Exchange Chromatography A gel filtration packing material having an average particle diameter of 20 μm and an average pore diameter of 2,000 Å, G6000PW (manufactured by Tosoh Corporation), was prepared by the following method. did. 20 g of G6000PW thoroughly washed with pure water, 40 g of pure water,
Epichlorohydrin (10 g), diethylaminoethanol (10 g), and NaOH (5 g) were stirred and mixed at 40 ° C. for 24 hours, and the total ion exchange capacity was 0.05 meq / ml-ge.
1 of an anion exchanger for plasmid purification was obtained. (Less than,
DEAE-6PW) This packing material was packed in a stainless steel column having an inner diameter of 7.5 mm and a length of 7.5 cm to obtain a packing material column for ion exchange chromatography.

(4) Separation of plasmid by a combination of hydrophobic chromatography and ion exchange chromatography 20 m containing 0.6 M sodium chloride and 1 mM EDTA
DEA equilibrated with M Tris-HCl buffer (pH 7.5)
After injecting 3 ml of the plasmid fraction into the E-6PW column,
20m containing 0.6M sodium chloride, 1mM EDTA
M Tris-HCl buffer (pH 7.5) at a flow rate of 1 ml / mi
n for 35 minutes to elute the impurities out of the column. Next, the concentration of sodium chloride in a 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA was adjusted to 0.6.
Elution was performed by a gradient method in which the concentration was continuously changed from M to 0.8M at a flow rate of 1 ml / min for 30 minutes. As a result, a chromatogram as shown in FIG. 6 was obtained. In FIG. 6, 1 is the peak of the impurity, and 2 is the peak of the fraction containing the plasmid. The column eluate corresponding to the peak of No. 2 was collected, and the purity was examined by agarose gel electrophoresis. When the gel after electrophoresis was stained with ethidium bromide, a high-purity supercoiled plasmid was obtained in this example.

Comparative Example 2 For comparison, the plasmid pBR322 was purified from the disrupted Escherichia coli only by ion exchange chromatography.

After the E. coli disrupted solution was prepared in the same manner as in Example 2, an equal volume of 20 mM Tris-HCl buffer (pH 7.5) was added to prepare a sample for ion exchange chromatography.
20m containing 0.6M sodium chloride, 1mM EDTA
After injecting 3 ml of the sample into the above DEAE-6PW column equilibrated with M Tris-HCl buffer (pH 7.5),
M sodium chloride, 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA at a flow rate of 1 ml / min.
The solution was sent to the column for 0 minutes, and the impurities were eluted out of the column. Next, elution was performed by a gradient method in the same manner as in the example. As a result, a chromatogram as shown in FIG. 7 was obtained. In FIG. 7, 1 is the peak of the impurity, and 2 is the peak of the fraction containing the plasmid. The column eluate corresponding to the peak of No. 2 was collected, and the purity was examined by agarose gel electrophoresis. As a result, the plasmid fraction obtained only by ion exchange chromatography contained many impurities.

Example 3 Adsorption Capacity of Plasmid The packing material for hydrophobic chromatography used in Example 2,
The adsorption capacity of the Octyl-6PW plasmid was examined.
The gel was packed in a stainless steel column having an inner diameter of 6.0 mm and a length of 10 mm, and was charged with 2.0 M ammonium sulfate and 0.1 mM.
0.1 M phosphate buffer containing EDTA (pH 7.0)
At a flow rate of 0.64 ml / min for 20 minutes to equilibrate. Next, about 0.4 mg / ml of plasmid pUC1
A 9 ml (base pair number 2,686) solution was injected into a 4 ml column, and a non-adsorbed fraction that was not adsorbed on the column was collected. Next, the eluate was diluted with 0.1 mM EDTA containing 0.1 mM EDTA.
After switching to M phosphate buffer (pH 7.0), the plasmid adsorbed on the column was eluted and the adsorbed fraction was collected. Next, a gel filtration column for nucleic acid separation TSKgelDN
The amount of plasmid pUC19 contained in each fraction was measured from a calibration curve of plasmid pUC19 (a relational expression between the amount of plasmid pUC19 injected and the area of the chromatograph) prepared in advance with A-PW (manufactured by Tosoh Corporation). The amount of adsorption per 1 ml of the gel and the recovery were calculated. Filler for ion exchange chromatography used in Example 1, DEAE-6P
Similarly, for W, the adsorption capacity of the plasmid was examined. Except for the adsorption and elution eluents, all were performed under the same conditions. As a result, the adsorption capacity of Octyl-6PW was 1.1 mg /
ml, DEAE-6PW adsorption capacity is 2.4mg / ml
Met. The recovery rates were 90.3% and 7%, respectively.
7.3%.

Comparative Example 3 For comparison, a packing material for hydrophobic chromatography having a smaller pore size than Octyl-6PW was prepared, and the adsorption capacity was compared. Average particle diameter 20 μm, average pore diameter 950
Angstrom's gel filtration filler, G5000PW
(Manufactured by Tosoh Corporation) as a base material. G5000
PW 20 g, 1,4-dioxane 20 g, 1,2-epoxyoctane 1 g, and boron trifluoride 0.5 m as a catalyst
was added and stirred and mixed at 45 ° C. for 6 hours to obtain Octyl-5PW having an octyl group. As a filler for ion exchange chromatography, a commercially available DEAE-5PW (average particle diameter: 20 μm, average pore diameter: 880 angstroms, manufactured by Tosoh Corporation) having a smaller pore diameter than DEAE-6PW was used. The measurement of the adsorption capacity of the plasmid was performed in the same manner as in the above-mentioned Example. As a result, Oct
The adsorption capacity of yl-5PW is 0.6 mg / ml, DEA
The adsorption capacity of E-5PW was 1.2 mg / ml. The recovery rates were 89.9% and 60.6%, respectively.

As is clear from the results of Example 3 and Comparative Example 3 described above, for a long-chain nucleic acid such as a plasmid, a larger pore diameter can provide better results in both the adsorption capacity and the recovery.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a chromatogram showing the result of separating a disrupted Escherichia coli solution by hydrophobic chromatography in Example 1.

FIG. 2 is a chromatogram showing the result of separating an eluate of hydrophobic chromatography by ion exchange chromatography in Example 1.

FIG. 3 is a view showing a gel electrophoresis image showing the results of a purity assay of plasmid fractions obtained in Example 1 and Comparative Example 1.

FIG. 4 is a chromatogram showing the result of separating a disrupted Escherichia coli solution by ion exchange chromatography in Comparative Example 1.

FIG. 5 is a chromatogram showing the results of separating a disrupted Escherichia coli solution by hydrophobic chromatography in Example 2.

FIG. 6 is a chromatogram showing the result of separating an eluate of hydrophobic chromatography by ion exchange chromatography in Example 2.

FIG. 7 is a chromatogram showing the results of separating Escherichia coli crushed liquid by ion exchange chromatography in Comparative Example 2.

[Explanation of symbols]

 1 peak of impurities 2 peak of plasmid fraction 3 DNA size marker 4 plasmid fraction obtained in Comparative Example 1 5 plasmid fraction obtained in Example 1 6 commercially available purified plasmid

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) G01N 30/48 G01N 33/50 P 33/50 C12N 15/00 A

Claims (13)

[Claims] 1. A method for separating nucleic acids, wherein hydrophobic chromatography is used. 2. The separation method according to claim 1, wherein a packing material into which a functional group for adsorbing a nucleic acid exhibiting hydrophobicity in the packing material used for hydrophobic chromatography is introduced. 3. The nucleic acid according to claim 1, wherein the packing material used for hydrophobic chromatography is combined with a packing material into which a functional group capable of adsorbing nucleic acids having different hydrophobicities has been introduced. Separation method. 4. In a hydrophobic chromatographic method using a combination of fillers having different hydrophobicities, a filler having weakly hydrophobic functional groups introduced therein is successively replaced with a filler having strongly hydrophobic functional groups introduced thereinto. 4. The method according to claim 3, wherein the combination is used.
Separation method as described. 5. The method according to claim 1, wherein the average particle size of the filler used in the hydrophobic chromatography is in the range of 2 to 500 μm. 6. The separation method according to claim 1, wherein the average pore diameter of the packing material used in the hydrophobic chromatography is in the range of 500 to 4000 Å. 7. A method for separating nucleic acids, comprising using a combination of hydrophobic chromatography and ion exchange chromatography. 8. Separation using a packing material in which a functional group capable of adsorbing a nucleic acid exhibiting a certain hydrophobicity is introduced into a packing material used for hydrophobic chromatography, and then separating the nucleic acid using ion exchange chromatography. The separation method according to claim 7, characterized in that: 9. The packing material for hydrophobic chromatography is separated by using a packing material in which a functional group having a weak hydrophobicity is introduced, followed by a packing material having a weakest hydrophobicity and a hydrophobicity increasing sequentially. 9. The method according to claim 7, wherein the nucleic acid is subsequently separated using ion exchange chromatography. 10. The filler used in hydrophobic chromatography and ion exchange chromatography has an average particle size of 2 to 10.
The separation method according to any one of claims 7 to 9, wherein the distance is in the range of 500 µm. . 11. The separation method according to claim 7, wherein the packing material used in the ion exchange chromatography has an average pore diameter in the range of 1500 to 4000 angstroms. 12. The method according to claim 1, wherein the nucleic acid to be separated is a long nucleic acid. 13. A packing material used for hydrophobic chromatography, wherein a functional group provided for adsorbing a nucleic acid comprises at least one compound selected from the group consisting of the following compounds (a) to (c) as a main component: Claim 1 characterized by the fact that
12. The separation method according to 12. Compound (a): may be long-chain or branched, having 2 to 2 carbon atoms
20 saturated hydrocarbon groups or unsaturated hydrocarbon groups (provided that
An aromatic ring may be contained in the hydrocarbon group). Compound (b): The following structural formula 1 (where n = 0 to 20 and the methylene group may be linear or branched, m = 0 to 3 and linear or branched, and A is CＡO Which is a group or an ether group but does not have A and may be directly bonded to the base material). Compound (c): an ether group of an alkylene glycol having 2 to 20 carbon atoms, which is composed of 0 to 10 repeating units (provided that the terminal opposite to the functional group reacted with the substrate has an OH group, And may be capped with 1-4 alkyl groups). Embedded image