JPH10155481A - DNA recovery method - Google Patents

Abstract

(57) [Problem] To provide a method of recovering purified DNA by using a chaotropic ion method, using an instrument having a simpler structure, and performing fewer operations. A method of lysing microbial cells, adsorbing free DNA to a carrier, and recovering the DNA adsorbed to the carrier.
A step of lysing microbial cells in the presence of a carrier and adsorbing the DNA obtained by the lysis to the carrier, a step of separating the solution used for lysis and adsorption from the carrier, and a step of adsorbing the DNA adsorbed on the carrier Is recovered by eluting with a DNA elution solution. A step of supplying microbial cells to a column provided with a carrier on a membrane filter having a solution holding capacity and a solution permeating capacity at the time of suction, and then lysing the microbial cells in the column, and obtaining DNA obtained by lysis. A step of adsorbing to the carrier, a step of removing the solution used for lysis and adsorption in the previous step from the column by suction, supplying a DNA elution solution to the column, and collecting the DNA adsorbed to the carrier by suction The process.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for recovering DNA contained in a microorganism.

[0002]

2. Description of the Related Art In the field of genetic engineering, it is always practiced to transform a microorganism such as Escherichia coli, culture the resulting transformant, and recover a desired plasmid DNA from the replicate-amplified transformant. I have. However, multiple steps are required to recover and purify the plasmid DNA from the transformant, which is troublesome. Therefore, a method of recovering and purifying plasmid DNA more easily has been proposed. For example,
Japanese Patent Application Laid-Open No. 4-360686 discloses a plasmid DNA which removes insoluble substances in a solution from a lysate of microbial cells through a membrane filter, removes impurities by ultrafiltration, and concentrates DNA. A method for purifying cosmid DNA is disclosed.

[0003] Furthermore, Japanese Patent Application Laid-Open No. 8-23976 discloses a method for purifying a supercoiled plasmid DNA in which impurities are removed from a plasmid mixture by a filtration filter having an average pore size of 10 nm to 35 nm. However, in these methods, purified D
RNA contained in microbial cells along with DNA in NA
In order to collect only DNA, a separate RNA degradation step is required.

[0004]

By the way, RNA and D
As a method for separating NA, a method in which a DNA-adsorbing carrier and a chaotropic solution are used in combination (chaotropic ion method) is known [R. Room et al, J. Clin.
MicroBiol. Vol. 28, No. 3, p495-503]. Utilizing this method, RNA contained in microbial cells along with DNA
Japanese Patent Laid-Open No. 7-25 describes a method for purifying only DNA by removing DNA.
No. 0681. This method comprises the steps of collecting a transformant culture solution into a first cartridge for DNA extraction and purification, a step of separating lysate and unnecessary RNA, a step of first DN
Filtration of impurities by cartridge for A extraction and purification, 2nd
This is a method for extracting and purifying plasmid DNA, which comprises the steps of adsorbing, washing, and eluting DNA with the cartridge for DNA extraction and purification described above.

However, in this method, two cartridges must be used, and the first cartridge for DNA extraction and purification has at least a trap filter and a membrane filter, and the second cartridge for DNA extraction and purification is It has at least a glass fiber filter, a glass powder layer, and a membrane filter, and has a more complicated structure than a simple filter.
Further, in this method, it is necessary to repeatedly supply and discharge the solution (by suction) using the above two cartridges.

Accordingly, an object of the present invention is to provide a method for recovering purified DNA by using a chaotropic ion method, using a device having a simpler structure, and performing fewer operations.

[0007]

SUMMARY OF THE INVENTION The present invention provides a method for lysing microbial cells, adsorbing free DNA to a carrier, and recovering the DNA adsorbed on the carrier. Lysing the body and adsorbing the DNA obtained by lysis on the carrier, separating the solution used for lysis and adsorption from the carrier, and eluting the DNA adsorbed on the carrier with a DNA elution solution The present invention relates to a method for recovering DNA (first method), which comprises a step of recovering the DNA.

Further, the present invention relates to a method for lysing microbial cells, adsorbing free DNA on a carrier, and recovering the DNA adsorbed on the carrier, comprising a membrane having a solution holding ability and a solution permeating ability upon suction. A step of supplying microbial cells to a column provided with a carrier on a filter, and then lysing the microbial cells in the column;
A step of adsorbing NA to the carrier, a step of removing the solution used for lysis and adsorption in the previous step from the column by suction, supplying a DNA elution solution to the column, suctioning the DNA adsorbed to the carrier And a method of recovering DNA (second method).

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below. In the present invention, the first method and the second method are characterized by lysing microbial cells, adsorbing free DNA on a carrier, and adsorbing DN
This is a method for recovering A. Microbial cells to be subjected to the method of the present invention are not particularly limited. Any microbial cells containing the target DNA may be used. For example, it can be a transformant in which a target DNA has been introduced into a host microorganism.

In the method of the present invention, lysis of microbial cells, adsorption and elution of free DNA to a carrier are known methods. However, a feature of the method of the present invention is that the lysis of the above-mentioned microbial cells and the adsorption of free DNA to a carrier are one step.
That is to do in the pot. In the first method of the present invention, a lysis solution and a DNA adsorption solution are sequentially added to microbial cells in the presence of a carrier to adsorb DNA to the carrier, or lysed to microbial cells in the presence of a carrier. Solution and a solution for neutralization and DNA adsorption are sequentially added to the carrier,
Is adsorbed. Glass adsorbs DNA in the presence of a solution for DNA adsorption containing chaotropic ions,
NA does not adsorb [R. Room et al., J. Clin. MicroBi
ol. Vol.28, No.3, p495-503]. The carrier can be selected from glass, silica gel, anion exchange resin, hydroxyapatite and Celite such as Diatomaceous Earth. The shape of the carrier is not particularly limited, but preferably has a large surface area for adsorption.
The carrier can be, for example, a mesh filter, beads or powder. For example, the carrier can be, for example, a glass filter, glass beads or glass powder.

The solution for adsorbing DNA is a solution containing chaotropic ions. In addition, the lysis solution is composed of a cell wall decomposition solution (solution I), an alkali-ionized surfactant solution (solution II), and a neutralization solution (solution III),
Alternatively, it may be composed of a cell wall decomposing solution of the cells (solution I) and an alkali-ionized surfactant solution (solution II). However, in the latter case (when the lysis solution is solution I and solution
II), a solution for neutralization and DNA adsorption, which is a single solution containing a neutralizing agent and chaotropic ions, is used.

[0012] The cell wall decomposing solution (solution I) of cells has the function of converting cells into spheroplasts. For example, Tris-E
DTA-glucose-lysozyme aqueous solution (solution I). Further, the alkali-ionized surfactant solution (solution II) has a function of dissolving the cell membrane and the protein to lyse the bacteria and denaturing the DNA.
NaOH-SDS aqueous solution (solution II) can be mentioned. The neutralizing solution (solution III) has a function of neutralizing the solution made alkaline by the solution II, and examples thereof include an aqueous solution of potassium acetate. Lysis can be performed by sequentially adding these three or two types of solutions to the cells. The concentration and amount of each solution can be appropriately determined according to the type and amount of the cells.

By using a solution containing a neutralizing agent (for example, potassium acetate) and chaotropic ions as a solution for neutralization and DNA adsorption, D
There is an advantage that the adsorption of NA can be performed and the processing time can be reduced. When a solution containing a neutralizing agent and chaotropic ions is used as the solution for DNA adsorption, it is preferable to adjust the pH of the solution to a range of 6 to 12 from the viewpoint of preventing RNA from being mixed. .
However, since this pH range also depends on the ionic strength, it can be appropriately determined depending on the conditions. Incidentally, RNase may be added to the solution I in order to prevent contamination of RNA.

Further, a DNA adsorbing solution, neutralizing solution and D
Examples of the solution containing a chaotropic ion which is a solution for NA adsorption include an aqueous solution containing LiClO ₄ , KI, NaI, LiCl, NaCO ₂ H, guanidine hydrochloride and the like. The concentration and amount of the chaotropic solution can be appropriately determined according to the type and amount of the cells. The solution for DNA adsorption or the solution for neutralization and DNA adsorption is further added to a mixture with the cells to which the lysis solution has been added in the presence of a carrier.
By adding the solution for DNA adsorption, the DNA dissolved from the cells is adsorbed on the carrier. A feature of the present invention is that each of the added solutions is sequentially added without separating and removing the previously added solution, and there is no need to separate the solution each time the solution is added. In addition, it is preferable to add each solution by adding one solution, for example, leaving the solution to stand for about 1 second to 60 minutes, and then adding the next solution from the viewpoint of performing each operation reliably.

Next, the carrier on which the DNA is adsorbed is separated from the solution. Separation of the carrier and the solution can be performed by a method such as decantation, centrifugation, or filtration. The carrier separated from the solution can be washed and dried if necessary.
For washing, for example, a Tris-EDTA-NaCl / ethanol mixture,
Ethanol, a mixed solution of ethanol / glycerol and the like can be used. Next, the DNA adsorbed on the carrier
Is recovered by eluting with a DNA elution solution. As the DNA elution solution, for example, a Tris-EDTA buffer can be used.

In the second method of the present invention, a column in which a carrier is provided on a membrane filter having a solution holding ability and a solution permeating ability during suction is used. By using this column, the separation and collection operation can be performed more easily. Further, when a small amount and a plurality of samples are to be processed simultaneously in parallel, a bundle of a plurality of columns can be used. In addition, a membrane filter is provided in one opening of a plate having a plurality of through-holes (wells).
A column filled with a carrier can also be used as a column.

The membrane filter is not particularly limited as long as it has a solution holding ability and a solution permeating ability at the time of suction. A commercially available membrane filter can be used as it is. In addition, the same carrier as that described in the first method can be used. The size and shape of the column can be appropriately determined in consideration of the amount of the cells to be treated and the amount of the solution. Microbial cells are supplied into the column. The microbial cells to be supplied may be those separated from the culture solution by filtration or centrifugation separately, but the culture solution containing the microbial cells is directly supplied to the column, and the liquid is aspirated to filter the microbial cells. The microbial cells can also be supplied by capturing with.

Next, the lysis solution and DN were applied to the column.
The DNA for adsorption is added to the carrier by sequentially adding the solution for A adsorption, or the solution for bacteriolysis and the solution for neutralization and DNA adsorption are sequentially added to the column to adsorb the DNA on the carrier. The lysis solution, DNA adsorption solution, and neutralization and DNA adsorption solution used here are the same as those described in the first method. As described above, a feature of the present invention is that each solution added to the column is sequentially added as it is without separating and removing the previously added solution, and there is no need to separate the solution each time the solution is added.

After all of the solution has been added, the solution is removed from the column by suction through a membrane filter. As a result, the cell residue remains on the filter,
DNA is also adsorbed on the carrier and remains on the filter.
Next, the column including the carrier is optionally washed to remove impurities such as free RNA and protein, and then dried. From the viewpoint of increasing the purity of the DNA to be recovered, the above-mentioned washing is preferably performed. For washing, for example, a Tris-EDTA-NaCl / ethanol mixed solution, ethanol, an ethanol / glycerol mixed solution, or the like can be used. Next, a solution for DNA elution is supplied to the column, and the DNA is adsorbed on the carrier by suction to recover the DNA.
As the DNA elution solution, for example, a Tris-EDTA buffer can be used.

In both the first method and the second method of the present invention, a lysis solution and a DNA adsorption solution or neutralization and DN
It comprises three steps: a step of sequentially adding the solution for A adsorption, a step of separating the carrier from the solution, and a step of eluting the DNA from the carrier, and by these three steps, the DNA can be recovered from the microbial cells. Further, it is preferable to provide a step of washing impurities before the step of eluting the DNA from the viewpoint of increasing the purity of the recovered DNA. The DNA recovered by the method of the present invention is a double-stranded circular plasmid DNA, and includes cosmid DNA, Bacterial Artificial Chromos
ome (BAC), P1-derived Artificial Chromosome (PAC)
Is also included in the plasmid DNA.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments. Example 1 An E. coli SOLR strain having a plasmid pBluescript SK (+) into which a 5.6 kb cDNA derived from a mouse was inserted was used.
The cells were cultured overnight in an LB medium containing 100 μg / ml ampicillin. Of this, 0.6 ml of the culture solution was transferred to a well closed with a 96-well glass filter and a membrane, and the solution was filtered by suction to collect cells in the glass filter. 25 μl of solution I (50 mM glucose, 25 mM Tris-HCl buffer [pH 8.0], 10 mM EDTA, 10 mg / ml lysozyme) was added to each well containing the cells, and left for 5 minutes. Then, 50 μl of solution II
(0.2N sodium hydroxide, 1% sodium dodecyl sulfate) was added and left for 5 minutes. Then, 37.5 μl of solution III (3M potassium acetate [pH 4.8]) was added and left for 5 minutes. Plus 120
μl of a 7 M guanidine hydrochloride solution (adsorption solution) was added, and the mixture was filtered by suction.

Next, 300 μl of a washing buffer (100 mM Tris-HCl buffer [pH 8.0], 5 mM EDTA, 0.2 M sodium chloride,
(60% ethanol) and filtration by suction.
One time, once with 300 μl of 80% ethanol, 300 μl of 100
One run with% ethanol. Thereafter, suction was performed for 20 minutes to dry the plasmid DNA on the glass filter, and finally a TE buffer (10 mM Tris-HCl [pH 8.0]) warmed to 65% was used.
, 1 mM EDTA) was added, and the plasmid DNA was eluted by suction. By this operation, 4-6μ
g of plasmid DNA could be obtained. In addition, the purity was as high as about 2 at 260 nm to 280 nm, and could be used sufficiently for DNA sequencing by the dideoxy method.

Example 2 An E. coli SOLR strain having a plasmid pBluescript SK (+) into which a 5.6 kb cDNA derived from a mouse was inserted was used.
The cells were cultured overnight in an LB medium containing 100 μg / ml ampicillin. Of this, 0.6 ml of the culture solution was transferred to a well closed with a 96-well glass filter and a membrane, and the solution was filtered by suction to collect cells in the glass filter. 25 μl of solution I (50 mM glucose, 25 mM Tris-HCl buffer [pH 8.0], 10 mM EDTA, 10 mg / ml lysozyme) was added to each well containing the cells, and left for 5 minutes. Then, 50 μl of solution II
(0.2 N sodium hydroxide, 1% sodium dodecyl sulfate) and leave for 5 minutes, then add 160 μl of neutralization and adsorption solution (0.7 M potassium acetate [pH 4.8, 5.3 M guanidine hydrochloride solution]) Left for 5 minutes.

Next, after filtering the mixed solution from the well by suction, 300 μl of 80% ethanol three times with 300 μl
Was washed once with 80% ethanol-20% glycerol.
Thereafter, aspiration is performed for 20 minutes to dry the plasmid DNA on the glass filter. Finally, 25 to 50 μl of 65% warmed TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) is added and aspirated. To elute the plasmid DNA.

By this operation, 4 to 6 μg of plasmid D
NA was obtained. In addition, the purity was as high as about 2 at 260 nm to 280 nm, and could be used sufficiently for DNA sequencing by the dideoxy method. Further, since a mixed solution of potassium acetate and guanidine hydrochloride was used as the solution for adsorption, the treatment time could be shortened by about 15 minutes as compared with Example 1. Also, by using 80% ethanol-20% glycerol for washing,
Compared with Example 1 using 100% ethanol, there was an advantage that the TE buffer used for elution permeated the glass filter better, and the amount of plasmid DNA recovered was slightly improved.

Example 3 Instead of a glass filter, Diatomaceous was used as a carrier.
Earth (Bio RAD Co & Ltd.), glass powder (Liken),
Porous high surface area glass (Bio101) or anion exchange resin
The same operation as in Example 1 was repeated except that (Qiagen) was used, to obtain 4 to 6 μg of plasmid DNA for each carrier. The yield was similar to that of Example 1 because 4-6 μg of plasmid DNA was the maximum yield. Carrier mg
The yield of plasmid DNA per carrier is proportional to the surface area of the carrier, and the recovery efficiency of plasmid DNA per 10 mg of carrier is shown below. Diatomaceous Earth (Bio RAD Co & Ltd.) 15-20 μg Glass powder (Liken) 5 μg Porous high surface area glass (Bio101) 10-20 μg Anion exchange resin (Qiagen) 5 μg

Claims (14)

[Claims] 1. A method for lysing microbial cells, adsorbing free DNA on a carrier, and recovering the DNA adsorbed on the carrier, comprising lysing the microbial cells in the presence of the carrier, and lysing the cells. Adsorbing the obtained DNA on the carrier, separating the solution used for lysis and adsorption from the carrier, and eluting and recovering the DNA adsorbed on the carrier with a DNA elution solution. A method for recovering DNA, characterized in that: 2. A method for lysing microbial cells, adsorbing free DNA on a carrier, and recovering the DNA adsorbed on the carrier, wherein the DNA is collected on a membrane filter having a solution holding ability and a solution permeating ability at the time of suction. Supplying microbial cells to a column provided with a carrier, then lysing the microbial cells in the column, and adsorbing the DNA obtained by lysis to the carrier, for lysis and adsorption in the previous step Removing the solution used in step (a) from the column by suction, supplying a solution for DNA elution to the column, and collecting the DNA adsorbed on the carrier by suction. 3. The method according to claim 2, wherein the microbial cells are supplied by supplying a culture solution containing the microbial cells, and then sucking the liquid to capture the microbial cells with a membrane filter. 4. The method according to claim 1, wherein the lysis solution and the DNA adsorption solution are sequentially added to the microbial cells to adsorb the DNA to the carrier. 5. The lysis solution is a cell wall decomposing solution of a cell (solution I) or an alkali-ionized surfactant solution (solution II).
The method according to claim 4, wherein the solution for DNA adsorption comprises a chaotropic ion-containing solution. 6. The method according to claim 5, wherein the solution I is an aqueous solution of Tris-EDTA-glucose-lysozyme, the solution II is an aqueous solution of NaOH-SDS, and the solution III is an aqueous solution of potassium acetate. 7. The method according to claim 1, wherein the lysing solution and the neutralizing and DNA adsorbing solution are sequentially added to the microbial cells to adsorb the DNA to the carrier. 8. A lysis solution comprising a cell wall decomposing solution of a cell (solution I) and an alkali-ionized surfactant solution (solution
8. The method according to claim 7, wherein the solution for neutralization and DNA adsorption comprises a single solution containing a neutralizing agent and chaotropic ions. 9. The solution I is an aqueous solution of Tris-EDTA-glucose-lysozyme, the solution II is an aqueous solution of NaOH-SDS, and the solution for neutralization and DNA adsorption is a solution containing potassium acetate chaotropic ions. 9. The method according to 8. 10. The method according to claim 5, wherein solution I contains RNase. 11. The method according to claim 7, wherein the neutralization and DNA adsorption solution is adjusted to a pH in the range of 6-12. 12. The method according to claim 1, wherein the carrier before elution is washed with a DNA elution solution and dried. 13. The method according to claim 1, wherein the carrier is selected from the group consisting of glass, silica gel, anion exchange resin, hydroxyapatite and celite. 14. The method according to claim 13, wherein the carrier is a mesh filter, beads or powder.