JPH10127285A - Isolation of plasmid dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject DNA without using specific device by suspending calcium phosphate-based particles in a buffer solution containing specific amounts of (HOCH2 )3 C-NH2 and a water soluble Ca salt, adding a lytic solution thereto and recovering a supernatant component. SOLUTION: Calcium phosphate-based compound particles having 1.0-2.0 Ca/P ratio, 1μm to 1mm average particle diameter and 1-300m<2> /g specific surface area by BET method are suspended in a buffer solution containing 1-100mM tris(hydroxymethyl)aminomethane and 1-100mM water-soluble calcium salt (e.g. calcium chloride) and having pH6.0-9.0 and a solution containing RNA and plasmid DNA obtained after lysis operation is added to the suspension to absorb RNA in the calcium phosphate-based compound particles and the residual component is recovered from the supernatant of the suspension. Thereby, the objective plasmid DNA is isolated by a simple operation without using specific device from a nucleic acid which is one of the most basic operation in genetic engineering field.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for isolating plasmid DNA from nucleic acid, which is one of the most basic operations in the field of genetic engineering.

[0002]

2. Description of the Related Art Conventionally, a method of incorporating a target DNA into an Escherichia coli plasmid vector and culturing the same to prepare the target DNA in a large amount is one of the most frequently performed operations in the field of genetic engineering. At present, plasmid vectors are recovered from E. coli by genomic DNA after lysis.
This is performed by removing a mixture of proteins and the like to obtain a mixture of RNA and plasmid DNA, and several methods are known, including the alkali-SDS method. In order to further obtain purified plasmid DNA from a mixture of RNA and plasmid DNA, a column separation method using an ion exchange resin, a cesium chloride density gradient ultracentrifugation method, and the like are generally used. However, in the column separation method using an ion-exchange resin, since the target plasmid DNA is diluted with a large amount of eluent, concentration of the eluted plasmid DNA is complicated. Further, in the cesium chloride density gradient ultracentrifugation method, since the centrifugal treatment is performed for a long time at a high rotation speed with a large-scale device, it requires careful attention, is complicated, and is not economical.

[0003]

An object of the present invention is to provide a method for obtaining purified plasmid DNA from a solution containing RNA and plasmid DNA by a simple operation without requiring any special equipment.

[0004]

According to the present invention, RNA is adsorbed on calcium phosphate compound particles when a buffer having a specific pH containing a specific concentration of tris (hydroxymethyl) aminomethane and a specific concentration of calcium ions is allowed to act. On the other hand, they found that plasmid DNA was not adsorbed, and were completed based on this finding. That is, the method for isolating plasmid DNA according to the present invention is 1 m
A Ca / P ratio of 1.0 to 1.0 in a buffer solution containing M to 100 mM tris (hydroxymethyl) aminomethane and 1 to 100 mM of a water-soluble calcium salt at pH 6.0 to 9.0.
2.0 calcium phosphate compound particles are suspended therein, and a solution containing RNA and plasmid DNA obtained after the bacteriolysis operation is added to the suspension, whereby RNA is adsorbed to the calcium phosphate compound particles and suspended. The method is characterized in that the plasmid DNA is recovered from the supernatant of the suspension.

[0005] The present invention also relates to a method for isolating plasmid DNA by liquid chromatography. This method comprises packing calcium phosphate compound particles having a Ca / P ratio of 1.0 to 2.0 into a chromatography column. ,
After equilibration with a buffer solution containing 1 mM to 100 mM tris (hydroxymethyl) aminomethane and 1 to 100 mM of a water-soluble calcium salt at pH 6.0 to 9.0, the RNA and plasmid DNA obtained after the bacteriolysis procedure are separated. And a buffer solution containing 1 mM to 100 mM tris (hydroxymethyl) aminomethane and 1 to 100 mM of a water-soluble calcium salt at pH 6.0 to 9.0 are successively passed through, and the plasmid DNA is recovered from the effluent. It is characterized by the following.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The calcium phosphate compound used in the present invention is not particularly limited as long as it has a Ca / P ratio of 1.0 to 2.0. For example, Ca ₁₀ (PO ₄ ) ₆ (O
H) ₂ , Ca ₁₀ (PO ₄ ) ₆ F ₂ , Ca ₁₀ (PO ₄ ) ₆ Cl ₂ ,
Ca ₃ (PO ₄ ) ₂ , Ca ₂ P ₂ O ₇ and Ca ₄ O (PO ₄ )
It can be used one or more selected from among _2. Of these, from the viewpoint of stability and adsorption performance, Ca
Those having a / P ratio of 1.5 to 1.8 are preferred, and those having a hydroxyapatite having a Ca / P ratio of 1.67 as a main component are most preferred. When using fluorapatite, the content of fluorine in the total calcium phosphate compound is 5% by weight.
It is preferred that: If the fluorine content exceeds 5% by weight, fluorine is eluted, which is not preferable. These calcium phosphate compounds can be synthesized by a known method.

In the present invention, the calcium phosphate compound particles have an average particle size in the range of 1 μm to 1 mm and a specific surface area of 1 to 300 m ² / BET method.
g, preferably in the range of 5-300 m ² / g. When the average particle size is smaller than 1 μm, handling is difficult. When the specific surface area is smaller than 1 m ² / g, sufficient adsorption performance cannot be obtained due to the low specific surface area. On the other hand, when the average particle size is larger than 1 mm and the specific surface area is larger than 300 m ² / g, the performance is deteriorated due to the reduction of the packing density and the generation of uneven aggregates. The calcium phosphate compound as described above can be produced by synthesizing various calcium compounds and phosphate compounds as starting materials, granulating and drying, and then classifying. In order to obtain particles meeting the above conditions, it may be necessary to form porous granules, but the porous granules can be produced by a conventional method.

In the method of the present invention, the calcium phosphate-based compound particles adsorb RNA in the nucleic acid component and do not adsorb plasmid DNA.
In a buffer of pH 6.0 to 9.0 containing M to 100 mM tris (hydroxymethyl) aminomethane and 1 mM to 100 mM of a water-soluble calcium salt (hereinafter sometimes referred to as a calcium salt-containing tris buffer). A calcium phosphate compound particle having a Ca / P ratio of 1.0 to 2.0 is suspended, and RNA obtained after the lysis operation is suspended in the suspension.
And a solution containing plasmid DNA. The lysis operation can be performed by any known method such as the alkali-SDS method. Genomic DNA in E. coli by lysis
Then, a solution containing mainly plasmid DNA and RNA is used after removing proteins and lipids as components of bacteria and cells.

[0009] When the concentration of tris (hydroxymethyl) aminomethane is less than 1 mM, there is no buffering effect, and the pH of the solution is not compensated. If it exceeds 100 mM, the plasmid will be adsorbed on calcium phosphate compound particles. The buffer used in the present invention may be 1 mM to 1 in addition to tris (hydroxymethyl) aminomethane.
Contains 00 mM water soluble calcium salt. Examples of water-soluble calcium salts include calcium chloride, calcium acetate,
Calcium nitrate or the like is used. If the concentration of these calcium salts is less than 1 mM, none of the nucleic acids in the sample will adsorb to the calcium phosphate compound particles, and if it exceeds 100 mM, the plasmid will be adsorbed to the calcium phosphate compound particles.

In the present invention, a buffer having a pH of 6.0 to 9.0 containing tris (hydroxymethyl) aminomethane and a water-soluble calcium salt at the above concentrations is used. If the pH of this buffer is less than 6.0, it becomes difficult for RNA to be adsorbed on calcium phosphate compound particles, and p
If H exceeds 9.0, the plasmid DNA will be adsorbed to the calcium phosphate compound particles. PH of this solution
Is adjusted to fall within the above range, it is preferable to use, for example, an acid such as hydrochloric acid in the buffer containing tris (hydroxymethyl) aminomethane and the water-soluble calcium salt.

According to the method of the present invention, RN in nucleic acid components
Since A is adsorbed on the calcium phosphate compound particles and only the plasmid DNA remains in the tris (hydroxymethyl) aminomethane buffer containing calcium ions, the plasmid DNA is recovered from this solution by alcohol precipitation using ethanol or the like. It can be easily performed by such a method. The calcium phosphate-based compound particles as described above are packed in a chromatography column, and plasmid DNA can be easily isolated from nucleic acid components by liquid chromatography.
A is not adsorbed by the filler and is contained in the effluent. When performing chromatography, a column is filled with calcium phosphate compound particles, equilibrated in advance, and then a sample solution containing RNA and plasmid DNA is injected to perform a separation operation. In this case, both the equilibration and the separation operation are performed. It is preferable to use a calcium salt-containing Tris buffer having the same concentration and pH as the above.

[0012]

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Examples 1 to 11 and Comparative Example 1 pBulescriptSK (+) having an ampicillin resistance gene
The vector (trade name of Stratagene) was transformed into JM109 competent cells, and cultured on an LB-agar medium containing 50 μg / ml ampicillin at 37 ° C. overnight to obtain a single colony strain. This was cultured with shaking at 37 ° C. for 24 hours in an LB culture solution containing 50 μg / ml ampicillin to obtain bacterial cells. After the cells were centrifuged, a plasmid-RNA mixed solution (hereinafter, referred to as a sample solution) having a nucleic acid concentration of 200 μg / ml was obtained by an alkali-SDS method. In 70 μl of a buffer solution containing the tris (hydroxymethyl) aminomethane-hydrochloride at the concentration shown in Table 1 and the calcium chloride at the concentration shown in Table 1 at the pH shown in Table 1, the average particle size is 20 μm and the specific surface area is 50 m ² / A suspension of 10 g (w / v) of hydroxyapatite was prepared, and 10 μl of a sample solution was added to 0.2 ml of the suspension, followed by shaking at room temperature for 5 minutes. Thereafter, the supernatant of the suspension was collected, subjected to 0.8% agarose gel electrophoresis, and analyzed for nucleic acid components by ethidium bromide staining. The results shown in Table 1 were obtained.

[0014]

[Table 1]

As is evident from the results shown in Table 1, highly purified purified plasmid DNA could be obtained using the calcium salt-containing Tris buffer.

Example 12 Hydroxyapatite having an average particle size of 20 μm and a specific surface area of 50 m ² / g was packed in a chromatography column.
The column was washed with a calcium salt-containing Tris buffer having the same concentration and pH as in Example 2, and the plasmid-RNA mixed solution after the lysis operation in Example 1 was used as a sample solution. The buffer was passed through three times the column volume and used as an eluent. The eluate was collected, subjected to 0.8% agarose gel electrophoresis, and analyzed for nucleic acid components by ethidium bromide staining. As a result, the presence of plasmid DNA was confirmed.

[0017]

According to the method of the present invention, a purified plasmid DNA can be efficiently obtained from a solution containing RNA and plasmid DNA obtained by a lysis operation by a simple operation without requiring a special apparatus. In addition, compared with a column separation method using an ion exchange resin, an elution operation is not required, so that the number of operation steps can be reduced, and plasmid D
NA can be easily concentrated and purified by an alcohol precipitation method or the like.

Claims (9)

[Claims] 1. The method according to claim 1, wherein said buffer is a buffer containing 1 mM to 100 mM tris (hydroxymethyl) aminomethane and 1 to 100 mM of a water-soluble calcium salt at pH 6.0 to 9.0.
By suspending calcium phosphate compound particles having an a / P ratio of 1.0 to 2.0, and adding a solution containing RNA and plasmid DNA obtained after the bacteriolysis operation to the suspension, the calcium phosphate compound particles are suspended. A method for isolating plasmid DNA, comprising adsorbing RNA to compound particles and collecting plasmid DNA from a supernatant of the suspension. 2. The method according to claim 1, wherein the pH of the buffer is adjusted by adding an acid. 3. The particle size of the calcium phosphate compound is 1 μm.
The average particle diameter and 1~300m method for isolating plasmid DNA according to claim 1, wherein the particles having a BET specific surface area of ² / g of ~ 1 mm. 4. The plasmid DN according to claim 1, wherein the water-soluble calcium salt is calcium chloride or calcium nitrate.
A method for isolating A. 5. A chromatography column filled with calcium phosphate compound particles having a Ca / P ratio of 1.0 to 2.0, and 1 to 100 mM tris (hydroxymethyl) aminomethane and 1 to 100 mM water-soluble calcium. RNA and plasmid DNA obtained after lysis, after equilibration with a buffer containing pH 6.0-9.0 containing salts
And a buffer solution containing 1 mM to 100 mM of tris (hydroxymethyl) aminomethane and 1 to 100 mM of a water-soluble calcium salt at a pH of 6.0 to 9.0 are sequentially passed through, and plasmid DNA is recovered from the effluent. A method for isolating a plasmid DNA. 6. The method according to claim 5, wherein the pH of the buffer is adjusted by adding an acid. 7. The same concentration and p
The method for isolating a plasmid DNA according to claim 5, wherein an H buffer is used. 8. The calcium phosphate compound particles having a particle size of 1 μm
The method for isolating plasmid DNA according to claim 5, which is a particle having an average particle size of 1 to ¹ mm and a specific surface area by a BET method of 1 to 300 m2 / g. 9. The plasmid DN according to claim 5, wherein the water-soluble calcium salt is calcium chloride or calcium nitrate.
A method for isolating A.