JPH0740029B2 - Method for treating sample containing DNA - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for automatically processing a sample containing DNA, more specifically, various bacterial cell components such as DNA, RNA, protein and polysaccharide. From the cell lysate containing
The present invention relates to a method for isolating and purifying DNA with high purity.

(Conventional Technology) With the development of biotechnology, the frequency of gene separation, purification, and gene sequencing is extremely high.
However, in spite of the progress of various technologies, the operation requires complicated manual work, and requires a long time and a lot of labor.

For example, in order to isolate and purify pure DNA from a sample containing DNA, RNA, polysaccharides and proteins, the following three-step process is performed. (1) Soluble DNA that disrupts the cell wall
To release. (2) The released DNA-protein complex is decomposed by protein denaturation or proteolysis.
(3) From a mixture of denatured or degraded protein and DNA,
Isolate and purify only DNA. However, such a processing operation requires a long time (for several days). Therefore, such work is a heavy burden for researchers in basic and applied development.

Especially for the development of applied technology of biotechnology,
A technique capable of completing these operations in a short time is desired. In particular, DNA sequence analysis, gene construction, constructed DN
For the purpose of checking whether the desired gene is correctly contained in the A sequence, it is necessary to isolate and purify the desired DNA for use in this in a short time.
For various studies, it is particularly desirable to rapidly separate and purify DNA from bacterial cells. The boiling method and the alkaline method have been adopted as a method for separating DNA from the cells relatively quickly, but they are both manual operations that require complicated steps and are difficult to automate. DN because it is manual
There are variations in the recovery rate and purity of A, and there are large individual differences due to technological differences. Further, the DNA obtained by such a method is contaminated with RNA, various proteins, polysaccharides and the like, and various experiments such as the sequence analysis of the above DNA cannot be carried out as it is. Since RNA interference is particularly large, this RNA is usually reduced to a low molecular weight by ribonuclease treatment and removed by polyethylene glycol precipitation. However, because ribonuclease is highly stable,
It is difficult to remove completely after use. Ribonucleases are highly active, and in some cases have the drawback of degrading the necessary RNA used in subsequent reactions.

A method using a hydroxyapatite column has been proposed as a method for isolating and purifying DNA from bacterial cells in one step without using ribonuclease. (FA Beland et al., JC
hromatography, 174 , 177-186 (1979)]. According to this method, first, the bacterial cells are lysed in advance, and then urea 8M
A buffer containing (8 mol /) phosphate buffer (0.24M phosphoric acid), sodium lauryl sulfate (SDS; 0.1%) and ethylenediaminetetraacetic acid (EDTA; 10 mM) is added. When this is passed through a hydroxyapatite column pretreated with the same buffer as above, only the DNA in the lysate is selectively adsorbed by hydroxyapatite (adsorbent). Under the above adsorption conditions, only double-stranded DNA [called double-stranded DNA (ds DNA)] is adsorbed, and single-stranded DNA
[Signal strand DNA (ss DNA)], RNA (1
It is not adsorbed (as long as the concentration of phosphoric acid is 0.15M or less). Since the urea concentration is as high as 8M, non-specific protein adsorption hardly occurs. To the hydroxyapatite column that adsorbed ds DNA,
Then, a 10 mM phosphate buffer solution is flowed to remove urea, SDS, etc. in the buffer solution used for the adsorption. Furthermore, the adsorbed DNA is melted by passing a 0.3 M phosphate buffer, and this is recovered.

In such a treatment method, the lysing operation as the pretreatment step is performed in a container completely different from the column. The lysate is supplied to the column with, for example, a pipette. However,
Supplying the lysate with a pipette is complicated and lacks accuracy. There is also a method in which a lysing container and a column are connected via a tube and a lysing solution is supplied by the power of a pump. With this method, the complexity of supplying the lysate is eliminated, but the lysate contaminates the tubes and pumps. Therefore, it is necessary to remove the contamination of the tube and pump for each measurement, and the operation in this respect becomes complicated.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and an object of the present invention is to provide a sample containing DNA through a pretreatment step with high purity in a short time. Another object is to provide a method for processing without requiring complicated operations.

(Means for Solving the Problems) The present invention was carried out in a DN which was conventionally carried out in a container different from the column.
The pretreatment process of the sample containing A (especially the cell lysis procedure),
By performing in the reservoir part (lysis container) integrally formed with the adsorbent layer made of hydroxyapatite of the column, complicated operations such as supply of the lysis solution with a pipette etc. are not necessary; Since it is integrated, it was completed based on the inventor's knowledge that contamination of tubes and pumps that conventionally connected a lysis container and a column can be avoided.

In this way, if the reservoir part integrated with the column is used as a lysis container, the adsorbent layer of the column and the reservoir part only pass through the filter, and the bacterial cells supplied to the reservoir part immediately absorb the adsorbent. It is thought that it will flow into the layer. However, in reality, surprisingly, the bacterial cells cannot easily permeate the filter, and almost never flow into the adsorbent layer. From these facts, the present inventor has concluded that the lysis of cells can be performed even in the reservoir portion of the column.

The method for treating a sample containing DNA of the present invention has an adsorbent layer made of hydroxyapatite and a reservoir portion,
A method for treating a sample containing DNA using a column in which the adsorbent layer and the reservoir portion are integrally connected via a filter, wherein the reservoir portion is filled with a sample containing DNA and a pretreatment liquid. And then pretreating the sample, supplying the pretreated sample to the adsorbent layer to adsorb the components in the sample to the adsorbent, and after washing the adsorbent layer, A step of eluting the component, whereby the above object is achieved.

The column 1 used in the method for treating a sample containing DNA of the present invention has, for example, as shown in FIG. 1, an adsorbent layer 10 and a reservoir portion 11 formed on the adsorbent layer 10. . The sample is lysed in the reservoir portion 11, and the components in the sample are adsorbed in the adsorbent layer 10. A filter 12 is mounted between the adsorbent layer 10 and the reservoir portion 11. Filter 13 on the inner bottom of column 1
Is mounted, and the adsorbent layer 10 is provided on the filter 13. At the bottom of the column 1, an eluate outlet 14 protruding downward and a recovery tube 15 connected to the outlet 14 are arranged. A column stopper 16 for hermetically sealing the inside of the column 1 is attached to the upper end of the column 1, and a tube 17 and a valve 18 for opening the inside of the column 1 are used.
Is installed. A tube 19 for supplying a treatment liquid (washing liquid, eluent, regenerating liquid, etc.) is connected to the column 1.

In this column 1, for example, as shown in FIG. 2, the adsorbent layer 10 and the reservoir portion 11 have a tube 20 of an appropriate length.
May be connected via. In another example of the column 1, for example, as shown in FIG. 3, a structure in which an adsorbent layer 10 and a reservoir portion 11 are separate bodies, but are almost integrally connected via a connecting portion 21. There is.

In the method of the present invention, the sample pretreatment step includes a microbial cell lysis step, a protein lysis step, an extraction step, and the like.
The sample to be treated by this method is a sample containing DNA, and particularly a sample containing cells such as bacterial cells is suitable. The method of the present invention is suitable for the extraction of DNA from a sample containing DNA, particularly the extraction of plasmid DNA (10 ⁶ dalton order) to higher molecular weight DNA (10 ⁸ dalton order) from E. coli cells. However, this method can also be used for cells other than E. coli cells, such as eukaryotic cells.

The adsorbent used in the present invention is hydroxyapatite, and when the DNA is eluted by treating the sample, dsDN
It is preferably used because only A can be selectively adsorbed. The pore size of the filter is in the range of 5 to 100 μm, preferably 20 to 70 μm. In particular, it is suitable if it is 40 μm or less.
If it is less than 5 μm, the resistance of the filter increases, so that the pretreated sample does not sufficiently penetrate into the adsorbent layer. Above 100 μm, the sample and pretreatment liquid cannot be filled in the reservoir portion of the column and immediately flow out to the adsorbent layer, so that the sample is not pretreated.

As the sample pretreatment solution, for example, a solution containing detergent, salt, lysing enzyme, alkaline agent, chelating agent, chaotropic agent, urea or the like can be used. Chelating agents and lytic enzymes are used to weaken the cell wall during cell lysis. Chelating agents include EDTA and 8-hydroxyquinoline. Examples of the lytic enzyme include lysozyme and proteinase K. Detergents are used to lyse cell walls weakened by lytic enzymes or chelating agents. Examples of detergents include sodium dodecylbenzene sulfonate (SDS), Triton X-100, Nonidet, lauroyl sarcosine. The concentration of sodium dodecyl sulfate can be changed in the range of 0.1 to 2%. However, a range of 0.5-1% is preferred. Urea is usually used at a concentration of 2-8M. 8M urea corresponds almost to the saturated concentration. When the sample to be treated is E. coli, the preferred combination of pretreatment solutions is 0.2M NaOH, 1% SDS, 50mM Tris-HCl (pH8.0), 10m
M EDTA, 1 mg / ml lysozyme. If the sample is a eukaryotic cell, 1M NaCl, 1% SDS, 50mM Tris-HCl (pH8.0), 10mM
EDTA, 1 mg / ml proteinase K.

As the eluent used in the elution step, a solvent having a strong interaction with at least one of the components (anion and cation) constituting the adsorbent hydroxyapatite, preferably both, can be used. Carbonic acid; and organic acids such as formic acid, acetic acid and propionic acid are preferred as the anion component of the salt used. As the cation component,
Ammonia; alkylamines such as diethylamine and triethylamine; alkanolamines such as ethanolamine and propanolamine; and aromatic amines such as pyridine and aminobenzene are preferable. A triethylamine carbonate buffer solution is particularly suitable as an eluent containing a salt composed of such components. Since the triethylamine carbonate buffer solution is volatile, it is possible to recover only DNA with high purity by distilling off the solvent of the eluate containing DNA or by freeze-drying.

The concentration of the eluent varies depending on the type of the eluent used. For example, when using a triethylamine carbonate buffer solution, the concentration is 10 mM or more, preferably 50 mM to 2 M, more preferably
It is in the range of 100 mM to 1M. Thus, even at a low concentration of 50 mM or less, it can be used when eluting DNA.

Examples of the cleaning agent used for cleaning the adsorbent layer include a chelating agent and a chaotropic agent. These chelating agents,
Examples of chaotropic agents include the reagents used in the pretreatment liquid.

The method for treating a sample containing DNA of the present invention is performed, for example, as follows when the treatment of the sample is the isolation / purification of DNA from the bacterial cells: The bacterial cell to be treated is placed in the reservoir portion of the column. Add lysing enzyme (lysozyme, proteinase K, etc.), chelating agent (EDTA, etc.), detergent (sodium dodecyl sulfate, etc.). The column is agitated or shaken to lyse the cells. The cell walls of the cells are weakened by chelating agents and lytic enzymes, and dissolved by detergent. The obtained lysate is brought into contact with hydroxyapatite which is an adsorbent. The column packed with hydroxyapatite, for example 0.15 ~
By passing 0.25M phosphate buffer as a solvent,
The ds DNA in the lysate is selectively adsorbed. RN in lysate
A, proteins and polysaccharides are not adsorbed. Next, the lysate is brought into contact with an adsorbent that adsorbs and carries DNA. For example, when triethylamine carbonate buffer is brought into contact with hydroxyapatite that adsorbs and supports DNA, carbonate ions combine with calcium of hydroxyapatite to form sparingly soluble calcium carbonate, while triethylamine forms a phosphate moiety and salt of DNA. As a result, DNA is desorbed from hydroxyapatite. For example, almost the entire amount of adsorbed DNA can be recovered by passing a triethylamine carbonate buffer solution in an amount twice the bed volume of the hydroxyapatite column. The purified DNA can be obtained by subjecting the eluate (extract) thus recovered to a usual ethanol precipitation treatment. Since triethylamine carbonate in the eluate is compatible with ethanol, it does not mix with DNA and reduce its purity. By distilling off the solvent under reduced pressure or freeze-drying instead of the ethanol precipitation treatment, the triethylamine carbonate is volatilized and removed (desalting is achieved), and high-purity DNA is obtained.

In the method of treating a sample containing DNA of the present invention, a column is used that can perform a pretreatment step and a lysis step in succession.
By combining this column with a container containing pretreatment liquid, eluent, cleaning liquid, etc., and supplying these treatment liquids from this container by a supply means (tube, pump, etc.),
Processing of the sample can be automated. Furthermore, if a fraction collector is placed downstream of the column, recovery of the eluate will be automated. The supply means and fraction collector are
Usually controlled by a computer.

(Examples) The present invention will be described below with reference to Examples.

Example 1 (A) Culture of bacterial cells Escherichia coli HB101 strain containing plasmid pBR322 was cultured overnight in 5 ml of LB medium. 1.5 ml of this culture broth was centrifuged at 5,000 rpm for 5 minutes to precipitate bacterial cells.

(B) Lysis of bacterial cells To the bacterial cells obtained in (A), 20 mM Tris-HCl buffer (pH
8.0) -10mM ethylenediaminetetraacetic acid (2 x TE) 250μ
To suspend and add this to the reservoir part 11 of column 1.
I put it in. Attach the column stopper 16 to the column 1 and attach the tube.
17, 19 and valve 18 were connected. The tube 19 was connected to a supply means (having a pump) and a container (not shown).
Further, a valve 18 was provided to this reservoir portion 11 to make it an open system, and then 50 μm of 1 mg / ml lysozyme dissolved in 50 mM Tris-HCl buffer was added from a tube 19. valve
18 was closed, the reservoir portion 11 was used as a closed system, and the cells were shaken at room temperature for 10 minutes to lyse them.

Next, after opening the valve 18 to open the system, 20 μm of 0.5 M EDTA was added from the tube 19, and the column was gently shaken and stirred at room temperature for 10 minutes. Further, 10% of 2% Triton X100 was added to the tube 19 and left at room temperature for 45 minutes. During this time, the column was gently stirred and shaken.

To this solution, for the purpose of calculation of the recovery, in vitro ³ H (in vitro) labeled plasmid DNA ⁽³ H-pBR322D
NA) was added at about 0.1 μCi.

(C) Purification of DNA: (C) -1 Column operation (adsorption) To the aqueous solution containing the lysed cells (lysate) obtained in (B), 9M urea-0.27M phosphate buffer (pH 7.0) ) -1% SDS containing buffer was added from a tube 19 by opening the valve 18 and opening the system with 8 volumes. The mixed solution was charged into a column filled with 0.5 ml of hydroxyapatite (adsorbent) at a flow rate of 5 ml / hour by closing the valve 18 to make the column 1 a closed system to adsorb the bacterial cell component.

(C) -2 Column operation (washing 1) About 20 ml of 8M urea-0.24M phosphate buffer solution (pH 7.
0) was eluted at a flow rate of 20 ml / hour for about 1 hour and passed until the absorbance at 260 nm (OD ₂₆₀ ) became almost zero.

(C) -3 Column operation (Washing 2) Next, 10 mM Tris / hydrochloric acid-1 mM ethylenediaminetetraacetic acid (p
H7.5) (TE) buffer solution of about 10 ml at a flow rate of 20 ml / hour
The solution was passed for a minute to wash away the urea and phosphate buffer solutions, which are the components of the cleaning solution of (C) -2.

(C) -4 Column operation (elution) 0.1M triethylamine carbonate buffer solution (pH 8.
0) at a flow rate of 5 ml / hour. When an ultraviolet absorption monitor (UV monitor) was connected to the outlet of the column and the absorbance at 260 mn was monitored, the results shown by the curve in FIG. 4 were obtained. From FIG. 4, it can be seen that most of the plasmid DNA was eluted in the range of 0.2 to 1.2 ml after the start of the eluent flow. The radioactivity of each fraction of the eluate from the column was measured by a liquid scintillation counter to examine the recovery rate of ³ H-labeled plasmid contained in each fraction. The results are shown in a bar graph in FIG. From Figure 4,
It can be seen that approximately 95% or more of the plasmid pBR322-DNA was recovered.

(C) -5 Isolation of DNA and Evaluation of Purity 0.1 ml of 3M sodium acetate was added to about 1 ml of the obtained eluate to perform ethanol precipitation. 4 μg of plasmid DNA (pBR322) was recovered. When 1/5 amount of this plasmid DNA was subjected to agarose gel electrophoresis, the migration pattern shown in FIG. 5 was obtained (FIG. 5 shows that plasmid DNA (pBR
322) is a marker). RNA was not detected at all, and the host DNA was comparable or less (5% or less) compared to the conventional method using ribonuclease (comparative example, migration pattern 5).
Met. In Fig. 5, oc DNA is nicked pBR322 plasmid DNA, and cc DNA.
Indicates pBR322 plasmid DNA without nick.

Furthermore, when the amount of protein in the recovered solution was quantified, the protein content was below the detection limit of 2 ng, and it was estimated that the DNA was sufficiently pure. It was found that this DNA was completely cleaved by the restriction enzyme Hinf I and was satisfactory in terms of purity.

Example 2 Except that the alkaline method was used as the method for lysing the cells,
The cells were lysed by the same method as in Example 1.

(A) Culturing and lysis of bacterial cells 1.5 ml of the centrifuged bacterial cells obtained by the same operation as in Example 1 (A), was added to 20 mM Tris-HCl buffer (pH 8.0) -10 mM.
100 μl of ethylenediaminetetraacetic acid (2 × TE) was added and suspended, and this was put in the reservoir portion 11 of the column 1.
The column stopper 16 was attached to the column 1, and the tubes 17 and 19 and the valve 18 were connected. The tube 19 was connected to a supply means (having a pump) and a container (not shown). Furthermore, 0.2 M of 50 mM Tris-HCl buffer was added to this reservoir part 11.
Sodium hydroxide-0.1% sodium dodecyl sulfate 200
μ was added. The valve 18 was closed, the reservoir portion 11 was used as a closed system, and the cells were shaken at room temperature for 10 minutes to lyse them.

Then, in this strong alkaline aqueous solution, potassium acetate 150μ
Was added to neutralize. About 0.1 μCi of plasmid DNA ( ³ H-pBR322 DNA) labeled with ³ H in vitro was added to this solution for the purpose of calculating the recovery rate.

(B) Purification of DNA A column operation was performed in the same manner as in Example 1 using the solution containing ³ H-pBR322 DNA obtained in the item (A) of this Example. Most of the plasmid DNA was eluted in the range of 0.2 to 1.2 ml after starting the eluent flow. From the radioactivity of the ³ H-labeled plasmid, the recovery rate of DNA was found to be approximately 95% or higher. These operations were all controlled by a computer and completed in about 2 hours.

About 1 ml of the collected eluate was added with 0.1 ml of 3M sodium acetate and ethanol precipitation was performed to recover 3 μg of plasmid DNA (pBR322). When this plasmid DNA was subjected to agarose electrophoresis, the migration pattern shown in FIG. 5 was obtained. No RNA was observed, and the host DNA was considerably lower than in the conventional method (comparative example; migration pattern 4), and it was difficult to detect it by ethidium bromide staining.

Furthermore, when the amount of protein in the recovered solution was quantified, the protein content was below the detection limit of 2 ng, and it was estimated that the DNA was sufficiently pure. It was found that this DNA was completely cleaved by the restriction enzyme Hinf I and was satisfactory in terms of purity.

Comparative Example Examples 1 and 2 are all examples of a method for treating a sample (DNA purification) by an automated device using a minicolumn method based on the principle of liquid chromatography. On the other hand, a conventional manual DNA purification example using the alkaline method is shown below.

(A) Cell culture and lysis The procedure of Example 2 (A) was repeated to obtain a supernatant, to which ³ H-pBR322 DNA was added. An equal amount of phenol-chloroform (1: 1) was added to the solution for extraction, the protein was removed, the supernatant was separated, and an equal amount of isopropanol was added to the solution for alcohol precipitation. The obtained precipitate was centrifuged at 15,000 rpm for 10 minutes, the precipitate was washed with 70% ethanol and then vacuum dried to collect a DNA fraction. This DNA fraction was dissolved in 50 µ TE buffer. Then 1m into this solution
2 μg of ribonuclease (g / ml) was added and left at 37 ° C. for 1 hour to lower the molecular weight of RNA. Add 0.2 volume of 5M NaCl solution and 0.33 volume of 30% polyethylene glycol 6000.
An aqueous solution was added, and the mixture was cooled at -10 ° C for 1 hour. The resulting precipitate (plasmid DNA) was collected by centrifugation at 15,000 rpm for 10 minutes. Since RNA with reduced molecular weight does not precipitate,
To be removed. The obtained DNA was dissolved in 50 µ TE, and 5 µ 3M NaCl aqueous solution was added thereto. To this, 100% of 99% ethanol was added and cooled at -78 ° C for 10 minutes. This one
DNA was collected by centrifugation at 5,000 rpm for 10 minutes. By this operation, polyethylene glycol was removed. Got
The DNA was washed again with 70% ethanol and then vacuum dried. D
The yield of NA was 3 μg. Electrophoretic patterns of DNA with and without ribonuclease treatment are shown in FIGS. 4 and 5. In this comparative example, a large amount of RNA was contaminated when the ribonuclease treatment was not performed, while an extra 3 hours was required when the treatment for RNA removal was performed.

(Effect of the Invention) According to the method of the present invention, the pretreatment step of the sample containing DNA (for example, the cell lysis step) is thus formed integrally with the adsorbent layer of hydroxyapatite of the column. Since it is performed in the reservoir portion, the sample can be processed in a short time with high purity and high yield and without requiring complicated operations. Such a method is used for DN from bacterial cells.
It can be widely used in each field of genetic engineering such as isolation and purification of A.

[Brief description of drawings]

1, 2 and 3 are sectional views showing an embodiment of an apparatus used for the method for treating a sample of the present invention; FIG. 4 is a method for treating a sample of the present invention in Example 1. By
FIG. 5 is a graph showing the results of monitoring the ultraviolet absorption of the eluate when the DNA was purified and the results of monitoring the ³ H radioactivity added as an index of DNA; and FIG. 2 is an electrophoretic pattern of purified DNA obtained by the method. 1 …… Column, 10 …… Adsorbent layer, 11 …… Reservoir part, 1
2,13 …… Filter, 14 …… Outlet, 15 …… Recovery tube, 16 …… Column stopper, 17, 19, 20 …… Tube, 18 …… Valve, 21 …… Connection part.

Claims (7)

[Claims] 1. A method for treating a sample containing DNA using a column having an adsorbent layer made of hydroxyapatite and a reservoir part, wherein the adsorbent layer and the reservoir part are integrally connected via a filter. And a step of pretreating the sample with a sample containing DNA and a pretreatment liquid in the reservoir portion, supplying the pretreated sample to the adsorbent layer, and And a step of adsorbing the adsorbent on the adsorbent, and washing the adsorbent layer and then eluting the component. 2. The method for treating a sample containing DNA according to claim 1, wherein the pore size of the filter is in the range of 5 to 100 μm. 3. The method for treating a sample containing DNA according to claim 1, wherein the pretreatment step is at least one of a bacteriolysis step, a protein lysis step and an extraction step. 4. A sample containing the DNA according to claim 1, wherein the pretreatment liquid contains at least one of detergent, salt, lysing enzyme, alkaline agent, chelating agent, chaotropic agent and urea. Processing method. 5. The method for treating a sample containing DNA according to claim 4, wherein the detergent is sodium dodecylbenzenesulfonate. 6. The method for treating a sample containing DNA according to claim 4, wherein the chelating agent is EDTA. 7. The method for treating a sample containing DNA according to claim 1, wherein the detergent used for the washing is a chelating agent and / or a chaotropic agent.