JPS63263093A - Purification of dna - Google Patents

Abstract

PURPOSE:To isolate DNA from a bacteriolysis solution in high purity, by subjecting a cell to bacteriolysis treatment to give a bacteriolysis solution, bringing the bacteriolysis solution into contact with an adsorbent to adsorb DNA in the solution on the adsorbent and bringing an eluting solution into contact with the DNA to elute the DNA. CONSTITUTION:A cell is subjected to bacteriolysis treatment to give a bacteriolysis solution, which is brought into contact with an adsorbent (preferably hydroxyapatite), DNA in the bacteriolysis solution is adsorbed on the adsorbent and an eluting solution is brought into contact with the adsorbent having adsorbed the DNA to elute the DNA. The eluting solution is preferably a solution of salt. Preferably the anionic solution the salt is ammonia and/or an amine and the cationic component of the salt is a volatile acid. Especially the eluting solution is preferably triethylamine carbonate buffer solution.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for purifying DNA, more specifically, from a bacterial cell lysate containing various bacterial cell components such as DNA, RNA, proteins, and polysaccharides. DN using adsorbent
This invention relates to a method for isolating A with high purity.

(Prior Art) With the development of biotechnology, the frequency of gene isolation, purification, and gene nucleotide sequencing is extremely high.

However, despite advances in various technologies, the operation requires complicated manual labor, requiring a long time and a great deal of effort. Therefore, such work places a heavy burden on researchers in both basic and applied development. For the development of biotechnology, especially applied technology, a technology that can complete these tasks in a short time is desired. In particular, for purposes such as DNA sequence analysis, gene construction, and checking whether a desired gene is correctly included in the constructed DNA sequence, it is possible to quickly obtain the desired DNA for use in this process. Let go.

It is necessary to purify it. For various research.

In particular, it is desired to rapidly separate and purify DNA from bacterial cells. Boiling methods and alkaline methods are used as methods to relatively quickly isolate DNA from bacterial cells, but both are manual processes that require complex processes and are difficult to automate. There are variations in the recovery rate and purity of DNA, and there are large disparities between individuals due to differences in technology. moreover.

The DNA obtained by such a method is contaminated with RNA, various proteins, polyvyi species, etc., and various experiments such as the above-mentioned DNA sequence analysis cannot be carried out correctly if it is as it is. Since the interference caused by RNA is particularly large, this RNA is usually reduced in molecular weight by ribonuclease treatment and removed by polyethylene glycol precipitation. However, due to the high stability of ribonucleases, it is difficult to completely remove them after use. Due to the high activity of ribonucleases, the disadvantage is that in some cases the necessary RNA used in subsequent reactions is degraded.

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.

(F, A, Ba1and et al., J, Chrollato
graphy, 174.177-186 (197
9) ). According to this method, first, the bacterial cells are lysed in advance, and then urea 8M (8 mol/1) phosphate buffer (
phosphoric acid 0.24M), lauryl sulfate (0.24M), sodium lauryl sulfate (0.24M),
SDS; 0.1%) and ethylenediaminetetraacetic acid (
Add a buffer containing EDTA (10 mM). this.

When the solution is passed through a hydroxyapatite column that has been previously treated with the same buffer solution as above, only the DNA in the lysate is selectively adsorbed to the hydroxyapatite (adsorbent). Under the above adsorption conditions.

Double helix DNA (to double strand DN)
s DNA) is adsorbed, and single-stranded DNA
C jingle strand DNA (ss DNA))
, RNA (single-stranded), etc. are not adsorbed (if the phosphoric acid concentration is 0.15M or less, they are adsorbed to some extent)
. Since the urea concentration is as high as 8M, non-specific protein adsorption hardly occurs. Next, a loa+M phosphate buffer was passed through the hydroxyapatite column that had adsorbed the ds DNA to remove urea and SO3 in the buffer used for the adsorption.
etc. to be removed. Further, the adsorbed DNA is eluted by passing 0.3 units of phosphate buffer through it and collected.

However, since the recovered DNA is obtained as a solution in the above-mentioned 0.3M phosphate buffer, it cannot be used as it is. This is because phosphoric acid has low solubility in alcohols such as ethanol, so when attempting to recover DNA by ethanol precipitation in the first step, a large amount of phosphoric acid precipitates simultaneously with DNA.

This is because it is difficult to remove the precipitated phosphoric acid. Therefore, it is necessary to remove phosphoric acid by a method such as i3 folding. As described above, even if DNA can be isolated from a bacterial cell eluate by adsorption in a one-step process, it requires a complicated operation of dialysis, which has the disadvantage of a poor DNA recovery rate.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and its purpose is to extract DNA from a bacterial cell lysate in a short time, with high purity, and without any hassle. The objective is to provide a method for isolation without requiring extensive manipulation. Another object of the present invention is to selectively adsorb the DNA in the lysate using an appropriate adsorbent, and further to adsorb the DNA in the lysate without requiring complicated post-treatment operations using an appropriate eluent. can be easily recovered. The purpose of the present invention is to provide a DNA isolation method (purification method).

(Means for Solving the Problems) The inventors first investigated the adsorption/desorption mechanism of hydroxyapatite listed in the prior art section. When hydroxyapatite (its main component is calcium phosphate) is used as an adsorbent, phosphoric acid and calcium, which are slightly dissociated upon contact with a solvent, are involved, and the substance to be isolated is adsorbed and absorbed through an ion exchange reaction. It was thought to be removable. However, there are many phenomena that cannot be explained by such simple ion exchange reactions.

For example, DEA, commonly known as an ion exchanger
When an isolation target substance is adsorbed on E-cellulose (diethylaminoethyl cellulose) and an eluent with a high salt concentration is passed through the adsorbed substance, the affinity between the adsorbed isolation target substance and DRAB decreases, and the adsorbed substance is eluted.

However, the DNA (
The substance to be isolated does not elute with saline solvents even at high concentrations. Therefore, it is thought that the adsorption of DNA onto hydroxyapatite is not just a simple ion exchange reaction.

Considering the above, the inventors used an adsorbent that selectively adsorbs ds DNA and has a strong interaction with the components (for example, calcium phosphate in the above-mentioned hydroxyapatite) that constitute the adsorbent. investigated eluents that can easily elute adsorbed DNA. For example, in the above-mentioned hydroxyapatite, we focused on the phosphoric acid component and the calcium component of calcium phosphate, which is its main component, and identified the ion 1 that has a strong interaction with at least one of them.
In particular, we attempted to desorb and recover DNA adsorbed on hydroxyapatite using a salt solution (a combination of anion and cation) containing ions that form poorly soluble salts. Anionic components that can form salts that have a strong interaction with calcium, which is the cationic component of hydroxyapatite, include phosphoric acid (also an anionic component of hydroxyapatite), which can form poorly soluble salts, and sulfuric acid (sulfuric acid, which is a poorly soluble salt). There are inorganic acids such as calcium (forming gypsum)), carbonic acid (forming the poorly soluble salt calcium carbonate (slaked lime, plaster)); and organic acids such as formic acid, acetic acid, and propionic acid, all of which are effective for DNA desorption and Elution was possible.

On the other hand, in the case of hydrochlorides, nitrates, etc. that can form easily soluble salts, even if high concentration solutions (14 or higher) are used, DNA
was not eluted. Effective cation components that can form salts that have a strong interaction with phosphoric acid, which is the anion component of hydroxyapatite, include calcium (also a cation component of hydroxyapatite), which can form poorly soluble salts, ammonia, and amines. It turned out to be.

In this way, DNA purification can be effectively achieved by selecting a specific adsorbent that can selectively adsorb DNA and an eluent that has some strong interaction with the adsorbent and can desorb and elute the adsorbed DNA. It turned out that it was done. Therefore, the DNA purification method of the present invention includes the steps of: contacting a lysate obtained by lysing bacterial cells with an adsorbent, and adsorbing the DNA in the lysate to the adsorbent;
The method includes the step of contacting an adsorbent that adsorbs and supports A with an eluent that has a strong interaction with the adsorbent to elute the DNA.

Thereby, the above objective is achieved.

Examples of the adsorbent used in the present invention include two examples.

As mentioned in the prior art section, hydroxyapatite, which can selectively adsorb only to ds DN, is preferably used. As the eluent, for example, as mentioned above, a solvent that has a strong interaction with at least one, preferably both, of the components (anions and cations) constituting the adsorbent, especially a salt solution that can form a sparingly soluble salt. can be used.

Among such salt solutions, it is a salt solution consisting of a combination of an anion and a cation with a small dissociation constant, and D
Those having high affinity with nucleic acids such as NA are preferred. Furthermore, a salt is selected that is soluble in water or alcoholic solvents and does not precipitate out by ethanol precipitation. For example, when hydroxyapatite is used as an adsorbent, carbonic acid; and organic acids such as formic acid, acetic acid, and propionic acid are suitable as the anion component of the salt used. Suitable cationic components include ammonia; alkylamines such as diethylamine and triethylamine; alkanolamines such as ethanolamine and propanolamine; and aromatic amines such as pyridine and aniline. A triethylamine carbonate buffer is particularly suitable as an eluent containing a salt made of such components. Since the triethylamine carbonate buffer is volatile, the DNA can be removed by distilling off the solvent of the eluate containing DNA or freeze-drying it.
This makes it possible to recover only high purity.

The concentration of the eluent varies depending on the type of eluent used, but for example, when triethylamine carbonate buffer is used, it is in the range of 10 mM or more, preferably 50 mM to 2, and more preferably 100 mM to IM.

In this way, even at low concentrations below 50mM, DNA
It is possible to elute

To purify DNA according to the method of the present invention, first, a lysate prepared by a conventional method is brought into contact with an adsorbent 2 such as hydroxyapatite. A column packed with hydroxyapatite is preferably used.

For example, by passing a 0.05 to 0.25 M phosphate buffer as a solvent, ds DNA in the lysate is selectively adsorbed. RN^, protein, poly# in the lysate! etc. are not adsorbed. Next, the eluent is brought into contact with an adsorbent that adsorbs and supports DNA. For example, DNA
When hydroxyapatite 1-, which adsorbs and supports DNA, is brought into contact with a triethylamine carbonate buffer, carbonate ions combine with calcium in hydroxyapatite to produce poorly soluble calcium carbonate, and DNA is desorbed from hydroxyapatite. For example, by passing a triethylamine carbonate buffer in an amount twice the bed volume of the hydroxyapatite column.

Almost the entire amount of adsorbed DNA can be recovered. Purified DNA can be obtained by subjecting the eluate (extract) thus recovered to normal ethanol precipitation treatment. Since triethylamine carbonate in the eluate is compatible with ethanol, it does not mix with ])NΔ and reduce its purity. Triethylamine carbonate can also be volatilized and removed (desalting can be achieved) by distilling off the solvent under reduced pressure or by freeze-drying instead of ethanol precipitation, and highly pure DNA can be obtained.

(Example) The invention will be explained below with reference to examples.

Large stream ■ Soil (A) Cultivation of bacterial cells and lytic niblasmid pBR322
Escherichia coli II B 101 strain containing the following was cultured overnight in 5-LB medium. 1.5mf of this culture solution at 5.00O
Centrifugation was performed at rpm+ for 5 minutes to precipitate the bacterial cells.

The obtained bacterial cells were added with 20mM) lithium-hydrochloric acid buffer (pH 8,
0)-10mM ethylenediaminetetraacetic acid (2X TE)
Add 250 μl and suspend, and add 10 μl of 10 μl/-lysozyme dissolved in 50 mM Tris-HCl buffer.
! Addition.

The cells were lysed at 0°C for 10 minutes. Then 0.5M HD
20 μl of TA was added, and the mixture was kept at 0° C. for 10 minutes. Furthermore, 10 μl of 2% Triton X100 was added, and the mixture was left at 0° C. for 45 minutes. In addition to this, plasmid 0 labeled in vitro with ffH was added for the purpose of calculating the recovery rate.
About 0.1 μCi of NACH-pBR322 DNA) was added.

(B) Purification to DN: (II)-1 Force ram operation (adsorption) To the aqueous solution (lysate) containing lysed bacterial cells obtained in section (A), add 9M urea-0.27M phosphate buffer ( pH7,0)
, 8 volumes of a buffer containing -1% SOS were added. This mixture was charged to a column packed with 0.51111 hydroxyabatai I (adsorbent) at a flow rate of 5 to 7 hours.

Bacterial body components were adsorbed.

(B) - Double force ram operation (washing 1) Add 8M urea-0.24M phosphate buffer (pH 7.0) to the above column at a flow rate of 20-7 hours for about 1 hour, absorbance of eluate at 260 nm. The solution was passed until (ODtbo) became O.

(B) - Triple force ram operation (washing 2) Next, 1011M Tris-HCl-IIIM ethylenediaminetetraacetic acid (pH 7,5) (TE) buffer solution approx.
0- for 30 minutes at a flow rate of 20 ml/hour,
(B) Urea and phosphate buffer, which are components of the cleaning solution in Section 2, were washed away.

(B) - Four-force ram operation (elution) 0.1 M I-ethylamine carbonate buffer W (pH 8,0) was passed through this column at a flow rate of 5 to 7 hours. When an ultraviolet absorption monitor (UV monitor) was connected to the outlet of the column and the absorbance at 260 nm was monitored, the results shown by the curve in FIG. 1 were obtained. From FIG. 1, it can be seen that most of the plasmid DNA was eluted within 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 using a liquid scintillation counter, and the 11
The recovery rate of the 1-labeled plasmid was investigated. The result is the first
Shown in the figure as a bar graph.

From Figure 1, more than 95% of the plasmids ρBl? 32
It can be seen that 2-DNA has been recovered.

(B) Isolation to -5ON and evaluation of purity: The eluate obtained was approx. The total amount of sodium acetate in n1 is 0.11n! In addition, ethanol precipitation was performed. Plasmid DNA (pB
4 μg of R322) was recovered. This plasmid DNA
When 115 amounts of the sample were subjected to agarose gel electrophoresis,
The migration pattern shown in Fig. 2 2 was obtained (Fig. 2 1 shows
plasmid DNA (pBR322) marker)
.

Conventional method in which no RNA is detected and host DNA is also detected using ribonuclease (Comparative Example 2. Migration pattern 6)
It was at the same level or lower (5% or lower) compared to . In Figure 2, O(1, DNA is nicked pB11322 plasmid ON8, and cc
ON8 is a pB without a nick [? 322 plasmid DNA is shown.

Furthermore, when the protein content of the two recovered solutions was quantified, the protein content was less than the detection limit of 20 g.
The DNA was presumed to be sufficiently pure. This DN
A is restriction enzyme t! It was found that it was completely cleaved by fnfl, and the purity was sufficiently satisfactory.

sword salmon roux An alkaline method was used to lyse the bacterial cells.

(8) Cultivation and lysis of bacterial cells: Examples! G
T [! Suspend in 100μl of L buffer, add 200μl of 0.2M sodium hydroxide-0.1% sodium dodecyl sulfate! The cells were then subjected to fjl-1 at 0°C for 10 minutes to completely lyse the bacterial cells. Next, add 3 to this strong alkaline solution.
150 μl of M potassium acetate was added to neutralize.

15. Centrifugation was performed for 10 minutes at OOOrpm.

The precipitate fraction contains cell shells (cell debris) and host DNA.
A.

It contains host proteins, which must be removed. The obtained supernatant fraction contains, in addition to the target DNA, some host DNA, proteins, and most of RNA as impurities. This supernatant was incubated at 11 in vitro (in v
itro) labeled plasmid DNA C'H-pB
R322 DNA) equivalent to about 0.1 μCi was added. This is to calculate the recovery rate.

(B) Purification of DNA: Column operation was performed in the same manner as in Example 1 using the supernatant containing the 111-p[1R322 DNA obtained in section (8) of this example. Most of the plasmid DNA was eluted in a range of 0.2 to 1.2 d after the start of passing the eluent. The DNA recovery rate was found to be over 95% based on the radioactivity of the 3H-labeled plasmid.

Add 0.1 ml of 3M sodium acetate to about 1 liter of the collected eluate.
Add 1R1 and perform ethanol precipitation to obtain plasmid DNA.
(pBR322) was recovered. When this plasmid DNA was subjected to agarose electrophoresis, the electrophoresis pattern shown in FIG. 2 was obtained. No RNA was observed at all, and host DNA was considerably lower than in the conventional method (Comparative Example 2; migration pattern 6) and could not be detected by ethidium bromide staining.

Furthermore, when the protein content of the recovered solution was quantified, the protein content was below the detection limit of 2 ng.
The DNA was estimated to be sufficiently pure. This D
It was found that NA was completely cleaved by the restriction enzyme tlinfl, and the purity was sufficiently satisfactory.

In this method, most of the impurities were removed by centrifugation at the stage of bacterial lysis, so the column operation was much easier than in Example 1, and the recovery rate was also high.

(8) Volume of bacterial cells and lysis: After lysis using the same alkaline method as in Example 2, the lysate was directly proceeded to the next step without centrifugation.

(B) Purification of DNA: The lysate obtained in Section (A) of this Example was directly used in Example 1. The column was applied in the same manner as in section (B). Compared to the case of Example 2, the column was slightly clogged and the liquid flow was difficult, but the plasmid ONΔ
Approximately 3 μg of was recovered.

When this plasmid DNA was subjected to agarose electrophoresis, an electrophoresis pattern as shown in FIG. 2 was obtained. RNA
was not detected at all, and the host DNA was also less than the same level (less than 5%) compared to the conventional method (Comparative Example 2; Migration Pattern 6).
Met. When the protein content of the recovered solution was quantified, the protein content was less than the detection limit of 2 μg, and it was estimated that the solution was sufficiently pure. Furthermore, it was found that this DNA was completely cleaved with the restriction enzyme nf+ and was sufficiently satisfactory in terms of purity.

In this way, by charging the column with the lysate itself, centrifugation and the subsequent operation of collecting the supernatant can be omitted. This is considered to be a desirable method for labor saving.

Triethylamine carbonate buffer (pt18.
0) instead of 0.3M sodium phosphate buffer (
The procedure was carried out in the same manner as in Example 1, except that pH 7.0) was used.

If the column eluate is left as it is, a large amount of phosphate will be precipitated by ethanol precipitation. Therefore, dialysis was performed. After dialysis, plasmid DNA was recovered by ethanol precipitation, and the amount was less than 2 μg. This amount was extremely low compared to that in Example 1, and it required about one day for dialysis, which proved to be not an effective method. The electrophoretic pattern of the DNA obtained is shown in FIG. 24.

Examples 1 to 3 and Comparative Example 1 are all examples of DNA purification using an automated device using a mini-column method based on the principle of liquid chromatography. On the other hand, an example of conventional manual purification of DNA using an alkaline method is shown below.

(8) Cultivation and lysis of bacterial cells: Perform the same procedure as in Example 2 (A) to obtain a supernatant, and add 31t-pRR32 to the supernatant.
2DNA was added. An equal amount of isopropanol was added to this to perform alcohol precipitation. The obtained precipitate was heated to 15.0
After centrifugation at 00 rpm for 10 minutes, the precipitate was washed with 70% ethanol and vacuum dried to collect the DNA fraction. This DNA fraction was dissolved in 50 μl of TE buffer. Next, add 2μ2 of 1μ/ml of bonuclease to this solution.
The mixture was then left at 37°C for 1 hour to degrade the RNA.

To this, 0.2 times the volume of 5M NaCl aqueous solution and 0.3
Three times the amount of 30% polyethylene glycol 6000 aqueous solution was added, and the mixture was cooled at -10°C for 1 hour. The resulting sediment ′R(
plasmid DNAΔ) at 15,000 rpm.
Centrifugation was performed for 1 minute, and 8 samples were recovered. Since the reduced RNA does not precipitate, it is removed. 50 μl of the obtained DNA
of TH, and 5 μl of 3M NaCl aqueous solution was added thereto. Add 100 μl of 99% ethanol to this and -
Cooled at 78°C for 10 minutes. This is 15. OOOrp
The DNA was collected by centrifugation for 10 minutes. This operation removed polyethylene glycol. The obtained DNA was washed again with 70% ethanol and then vacuum dried.

The yield of DNA was 3 μg. The DNA electrophoresis pattern is shown in FIG. 2.6. The electrophoresis pattern of the crude DNA without the ribonuclease treatment is also shown in FIG. 2. In this comparative example, I? Approximately 3 hours were required for the treatment to remove NA.

(Effect of the invention) According to the method of the present invention, DN can be obtained from the bacterial cell lysate as described above.
A in a short time with high purity and high yield.

In addition, it can be purified into single gt without the need for complicated operations. Such methods can be widely used in various fields of genetic engineering such as DNA sequencing and gene construction.

Figure 1 shows the results of monitoring the ultraviolet absorption of the eluate when DNA was purified using a hydroxyapatite column according to the method of the present invention, and as an indicator of DNA. A graph showing the results of monitoring added '11 radioactivity; and FIG. 2 is an electrophoresis pattern of purified DNA obtained by the method of the present invention and other methods.

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Claims (1)

[Claims] 1. A step of contacting a lysate obtained by lysing bacterial cells with an adsorbent and adsorbing DNA in the lysate to the adsorbent, and adsorbing and supporting the DNA. A method for purifying DNA, comprising the step of contacting an adsorbent with an eluent that has a strong interaction with the adsorbent to elute the DNA. 2. The purification method according to claim 1, wherein the adsorbent is hydroxyapatite. 3. The purification method according to claim 1 or 2, wherein the eluent is a solution of a salt that has a strong interaction with the adsorbent. 4. The purification method according to claim 3, wherein the anion component of the salt is ammonia and/or amines. 5. The purification method according to claim 4, wherein the amines are selected from the group consisting of alkylamines, alkanolamines, and aromatic amines. 6. The purification method according to claim 3, wherein the cationic component of the salt is a volatile acid. 7. The purification method according to claim 6, wherein the volatile acid is carbonic acid and/or an organic acid. 8. The purification method according to claim 3, 4, 5, 6, or 7, wherein the eluent is a volatile buffer. 9. The purification method according to claim 8, wherein the eluent is a triethylamine carbonate buffer. 10. The concentration of the triethylamine carbonate buffer is 10 m
The purification method according to claim 9, wherein the purification method is M to 2M.