JPH07322899A - Recovery and purification of dna originated from microorganism - Google Patents

Abstract

PURPOSE:To collect highly pure DNA originated from microorganisms in a high recovering percentage by simple operations comprising selectively and highly separating and removing free non-decomposed DNA from a microorganism suspension. CONSTITUTION:This method for recovering and purifying the DNA originated from microorganisms comprises disposing a well at a place near the center of a gel for electrophoresis vertically to the emigration direction, receiving a microorganism suspension in the well, applying an electric voltage to the gel for the emigration of the microorganisms, cutting out the get for the removal of the non-decomposed DNA at a position where non-decomposed DNA is emigrated, lysing the microorganisms in the suspension in the well with a lysing solution, applying an electric voltage to the gel in the direction that the minus and plus of the electrodes are reversed, subjecting the lysed products to an electrophoresis in the direction reverse to the emigration direction of the microorganism suspension, and subsequently cutting out the gel at a position where DNA originated from the microorganisms is emigrated.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is a suspension for studying various useful microorganisms that can be recovered from soil suspension, activated sludge of sewage / wastewater treatment tanks, and bottom sludge of rivers, lakes, seas, etc. Related to a technique for recovering and purifying DNA derived from the microorganism in high purity from, especially, undegraded DNA of unknown origin from the microorganism
The present invention relates to a method for efficiently separating / removing free undegraded DNA from a system containing a mixture of DNAs, and recovering and purifying DNA from a target microorganism with high purity and easily.

[0002]

2. Description of the Related Art In recent years, with the development of genetic engineering, the possibility of producing useful substances and decomposing harmful substances using microorganisms and enzymes, which is different from conventional chemical methods, has been actively investigated. As a result, research on microorganisms has become popular for the purpose of carrying out genetic engineering production while maintaining the advantages of existing chemical engineering production, for example, in special environments such as high temperature, low temperature, high alkali, high water pressure. Microbes that live and act are also being studied.

Under such circumstances, a very wide variety of microorganisms inhabit, and there is a possibility that there are also microorganisms having the above-mentioned properties, the activity of soil suspensions and sewage / wastewater treatment tanks. If the technique of isolating various microorganisms from sludge and the bottom mud of rivers, lakes, seas, etc. and studying their ability, or extracting DNA in which information on their functions is written is the basic research of genetic engineering, First, it has become very important for the further development of the applied technology field.

The following methods are the most commonly used methods for extracting DNA.

One is to dilute a suspension containing microorganisms and apply it to an agar medium containing components suitable for the purpose, and leave the medium for several days in an environment suitable for the purpose of culturing and proliferating. This is a culture and recovery method in which the microbial colonies are selected, further concentrated and cultivated to increase the concentration, and then precipitated by centrifugation or the like to recover the microorganisms and extract the DNA.

[0006] This method has an advantage that DNA of one kind of microorganism can be recovered in high purity, but has a problem that it cannot be used for microorganisms whose separation and culturing conditions are unknown. It is generally said that it is difficult to grow 99 to 99.9% of microorganisms in the soil on the agar medium and separate them. Therefore, most of the useful microorganisms in the sampled suspension are not recoverable. In addition, even if activated sludge in a wastewater treatment tank that is relatively easy to estimate culture conditions, the ratio of microorganisms changes due to a subtle difference between the actual conditions inside the tank and the conditions inside the laboratory. There is a high possibility that the priority species in the activated sludge cannot be recovered as the priority species in the double land.

[0007] The other is to remove contaminants from a suspension containing microorganisms by centrifugation or the like to separate and collect only the microorganisms.
This is a bacterial cell collection method for extracting NA.

This method has an advantage that DNAs of various microorganisms in the suspension can be recovered. However, depending on the suspension, the recovery rate exceeds 1 μg per 1 g of soil, or conversely decreases to about 10 ng. The recovery rate changes greatly in this way because it depends on how much the microorganisms adhere to the contaminants in the suspension and how easily they aggregate. Therefore, DNA cannot be recovered at a stable recovery rate.

Further, in order to obtain high-purity DNA, it is necessary to strongly separate microorganisms and contaminants, which inevitably reduces the recovery rate of microorganisms and DNA. Especially when the microorganisms adhere to the contaminants, the recovery rate becomes close to 0%. On the contrary, when trying to increase the recovery rate, contaminants are allowed to be mixed in, and the purity of the recovered DNA is lowered, and it is possible to perform work using DNA in the recovery such as cutting with digestive enzymes or amplification by PCR. There is a problem that it will disappear.

In contrast to these methods, a lysate such as cell membrane lysing enzyme / SDS / phenol is added to a suspension containing microorganisms to dissolve the microorganisms, and DNA is directly extracted to recover 100%. It is the direct lysis method that tried to get closer to.

According to this method, a wide variety of microorganisms in a suspension such as soil, activated sludge and bottom sludge are first dissolved, and DNA is suspended in the suspension.
Since it is recovered using the NA extraction method, there is no loss such as the loss of microorganisms when removing contaminants.
The amount of A recovered is, for example, 10 μg per 1 g of soil, which is advantageous in that it can be recovered at a higher yield than the bacterial cell recovery method.

However, unlike the liquid medium in which the microorganisms have been cultured, the above-mentioned microbial suspension has a free undegraded origin of unknown origin such as microorganisms that have died in the past in addition to the desired microorganisms and carcasses of plants and animals. Since DNA is mixed, there is a problem that when the direct lysis method is used, these free undegraded DNA and the DNA derived from the target microorganism obtained by the lysis treatment are mixed.

[0013] In this case, even if the DNA is recovered in a high yield, the library cannot be prepared.
There is a concern that problems such as amplification of DNA that is completely unrelated to microorganisms may occur during operations such as R amplification.
Since these free undegraded DNAs are attached to solid matter such as soil particles, washing the suspension to remove soluble matter has little effect.

Due to such drawbacks, the direct lysis method is used to estimate the amount of organisms in the soil from the amount of recovered DNA by utilizing the fact that the recovery rate is higher than that of recovering and utilizing DNA. It was only used.

Furthermore, when recovering nucleic acids such as DNA from the recovered microorganisms, the quality and purity of the recovered DNA are very important for the subsequent treatments such as enzymatic digestion, PCR and hybridization. However, organic substances such as humus adhere to the carrier for supporting microorganisms such as soil particles in a large amount like microorganisms, and if purification of microorganisms is insufficient,
These organic substances are recovered together with the microorganisms. For this reason,
In order to remove impurities such as humus from the recovered microbial DNA, purification using a gel filter such as cesium chloride equilibrium density gradient method or hydroxyapatite column chromatography is required (for these, "Molecular Clon"
ing, A LABORATORY MANUAL, SECOND EDITION ”, J. Samb
Rook et al., Cold Spring Harbor Laboratory Press, p.
1.40-48 and "Biochemical Experimental Method 11, Gel Filtration Method", Kensuke Shimura et al., Academic Publishing Center, p181-1
95). However, in these methods, the operation itself and pretreatment are complicated, and it is necessary to centrifuge for 24 to 48 hours in an ultracentrifuge for a long time. Use of etidium bromide which is a carcinogen, DNA recovery efficiency There was a problem such as low.

[0016]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned conventional circumstances, and an object thereof is to collect microorganisms by culturing, or to physically separate and collect only microorganisms,
By adopting electrophoretic treatment, it is possible to selectively separate and remove free undegraded DNA from a microbial suspension, and collect microbial-derived DNA efficiently and with high purity by a simple operation. is there.

Another object of the present invention is to more easily remove undegraded DNA and collect microorganism-derived DNA with high purity by performing the electrophoresis treatment in a gel medium having a specific structure. Is.

Still another object of the present invention is to obtain undecomposed D
It is to further simplify the treatment operation by removing NA and collecting the microorganism-derived DNA using a common electrophoretic medium.

[0019]

Means for Solving the Problems The present invention to achieve the above object is to separate a microbial suspension by subjecting the microbial suspension to electrophoresis to move free undegraded DNA present in the suspension. After removal, the microorganisms in the microorganism suspension are lysed, and electrophoresed again to recover the microorganism-derived DNA.
It is a method for collecting and purifying microorganism-derived DNA, which is characterized by purification. By applying electrophoretic treatment, undegraded DNA and microorganisms can be first separated by electrophoresis, then the microorganisms can be lysed and the DNA can be separated by electrophoresis. Further, it becomes possible to highly efficiently recover the microorganism-derived DNA from the suspension, and the recovered DNA is directly purified D
It can be used as NA. Here, by reversing the electrode during the electrophoresis of the suspension and the electrode during the electrophoresis of the bacteriolysis solution, it is possible to prevent the undegraded DNA from being mixed with the recovered DNA. It is possible to further increase the purity of the microorganism-derived DNA that can be produced.

The present invention also relates to the above-mentioned microorganism-derived DNA.
In the recovery / purification method, electrophoresis of a microbial suspension and / or electrophoresis of a lysate-treated solution are carried out by using a gel provided with a well as a medium and containing a sample in the well, and the target DNA migrated in the gel. Is a method for collecting and purifying DNA derived from a microorganism, which is collected by cutting out a gel corresponding to the moving position. By providing a well in the gel medium for electrophoresis, a predetermined electrophoresis can be performed simply by storing a sample in the well and applying a voltage, so that the separation operation by electrophoresis can be further simplified. Here, the electrophoresis of the microbial suspension and the electrophoresis of the lysate are performed using a common gel medium, and a well is provided near the center of the gel in a direction perpendicular to the migration direction to perform electrophoresis of undegraded DNA. Microbial origin D
In the electrophoresis of NA, the positive and negative of the electrodes are reversed and a voltage is applied to migrate the undegraded DNA to one side of the gel.
On the other hand, if the microbial-derived DNA after lysis is electrophoresed, the undegraded DNA can be easily removed and the microbial-derived DNA can be collected and purified simply by cutting out the necessary parts of the gel (the gel at the positions where the DNA has moved). Since it can be performed extremely easily, the procedure for recovering and purifying the microorganism-derived DNA is further simplified. In addition, if a gel having a well containing a DNA size marker in parallel with the well containing a reagent is used as the gel, the excision position after electrophoresis can be specified by the DNA size marker. It becomes possible to selectively collect and purify only a DNA of a specific length.

[0021]

[Function] Since DNA is negatively charged, free undegraded DNA is moved to the positive pole by applying a voltage to a microbial suspension containing free undegraded DNA of unknown origin and microorganisms. The microbial sample for the purpose of recovering and purifying the microbial DNA can be easily prepared. It was beyond expectation that effective separation and removal would be possible with such a simple method. In addition, a suspension containing free undegraded DNA is put in the well portion of the gel for electrophoresis, the undegraded DNA is electrophoretically removed by applying a voltage, and then a lysate such as cell membrane degrading enzyme or phenol is added. The microorganisms in the suspension are lysed by placing them in a well so that the DNA in the microorganisms is suspended in the liquid, and after re-electrophoresis, the gel portion containing the required DNA is cut out to obtain D
NA can be separated and purified very easily from undegraded DNA and soil particles. Furthermore, if the size of the DNA is limited when the gel is cut out, only the DNA of the desired length such as only the genome or only the plasmid of the specific size is selectively recovered among the desired microbial DNA, and the unnecessary length is removed. You can work at the same time.

The present invention has been filed for Japanese Patent Application No. 6-38.
Method for pretreatment of microbial suspension described in No. 91 and D
This is a method for further improving the method for recovering NA, which is simpler in operation than the method and can remove soil fine particles.
It has a feature that the operation of selectively recovering only the DNA of the desired length can be simultaneously performed in the DNA recovery step. Further, in the same method, a rack for electrophoresis equipped with a filter, a semipermeable membrane, etc. must be prepared for each operation, whereas in the embodiment using an agarose gel or the like, it suffices to solidify the gels. So it's easy to prepare for work.

Free undegraded DNA of unknown origin
When lysing microorganisms directly from a suspension in which microorganisms are mixed with impurities such as soil and soil particles and recovering DNA directly, lysing treatment immediately causes undegraded DNA derived from microorganisms and undegraded. There is an inconvenient problem that DNA cannot be separated due to mixing, and a problem that it is very difficult to separate soil microparticles from microorganisms or DNA derived from microorganisms. There is no concern that free undegraded DNA of unknown origin will be mixed into the DNA, and it is also possible to prevent mixing of DNA other than the target length.
By gel electrophoresis of P., it is possible to separate and remove fine particles of soil, contaminants such as salts and organic substances in the suspension.
It is extremely effective for CR amplification and preparation of DNA library.

The present invention will be described in detail below.

The microbial suspension which is the object of the present invention can be used for various purposes from soil suspensions, activated sludge of sewage / wastewater treatment tanks, suspensions of river / lake / sea bottom mud, etc. Various microbial suspensions are collected and prepared, and can be targeted without any particular limitation. For example, a microbial suspension or the like for the purpose of collecting a microbial sample prepared for extracting DNA in which information regarding the functions of various microorganisms is written. When the target is a solid substance such as soil, distilled water or an aqueous solution of an electrolyte described below may be added to form a suspension. Such a microbial suspension is usually a system in which undegraded DNA of unknown origin and microorganisms are mixed,
Such undegraded DNA hinders the study of microbial DNA. Here, free undegraded DNA is
This is a general term for DNA other than the target microbial DNA, and includes, for example, microorganisms that have died in the past, carcasses of plants and animals, etc. regardless of their origin, unidentified or identified.
Here, the microorganisms include naturally occurring substances such as bacteria, actinomycetes, yeasts, molds, mushrooms, microalgae, protozoa, and the like, and those obtained by application of biotechnology.

The concentration of solid matter in the microbial suspension subjected to electrophoresis should be at most 50%, preferably about 20% or less for efficient electrophoresis, and the pH of the suspension is Is preferably 3 to 9, and preferably about 4 to 8, because electrophoresis can be rapidly carried out, and microorganisms are not lysed or inactivated. It is also possible to remove large contaminants from the microbial suspension in advance by centrifuging at several thousand rpm for 10 to 30 seconds, and finely separate the soil particles with a blender, mixer, homogenizer, etc. If pulverized, the recovery rate of microorganisms will be further improved.

An electrolyte must be present for the microbial suspension to be electrophoresed. In the case of electrophoresis using a gel, an electrolyte may be included in the preparation of the gel, but in the case of using a filter or the like, DN may be added to the suspension.
An aqueous solution of an electrolyte that conducts electricity such as a buffer solution used in A or protein electrophoresis is added, or an appropriate electrolyte is added to prepare an electrolyte solution. When electrophoresis is performed for a long time, a buffer solution may be used, and examples thereof include neutral to weakly acidic ones such as a phosphate buffer solution, an acetate buffer solution, and a citrate buffer solution, and preferably TA
A buffer solution for DNA electrophoresis such as E, TPE and TBE. Further, in the case of adding an electrolyte, a substance that lyses or inactivates microorganisms by extremely lowering or raising pH is not suitable. As an electrolyte,
Examples thereof include phosphoric acid, acetic acid, citric acid and the like, and combinations thereof with sodium, potassium and the like, and preferably, a mixture of Tris-EDTA and acetic acid, phosphoric acid, boric acid or the like.

Next, this suspension is placed in an electrophoretic bath for electrophoresis. Here, as a means for separating the microorganisms and the undegraded DNA by electrophoresis, a gel having wells, which will be described later, is preferable.
3891, ie, 20-50%
Electrophoresis is performed in an electrolyte solution whose viscosity is adjusted with an aqueous sucrose solution to separate undegraded DNA due to the difference in migration speed, or a filter is provided between electrodes to selectively separate undegraded DNA. Is also possible. As a method of separating into two fractions using a filter, for example, a filter is installed on the positive electrode side of the electrophoresis tank to prevent the microorganisms from migrating out simultaneously with the DNA.
Fraction A should be permeated and separated. As such a filter, any filter can be used without limitation as long as it has a function of permeating undegraded DNA without permeating microorganisms, but the material of the filter maintains its shape even when immersed in water, and is impregnated with an aqueous electrolyte solution. And it must be a good conductor of electricity. Examples of materials that can be used include nitrocellulose, polyvinylidene difluoride, and other membrane filters having uniform pore diameters, cellulose, glass,
A fiber filter such as silica can be used.
It is advisable to select a filter having a pore size smaller than the size of the microorganisms to be recovered from among those actually marketed.
For example, in order to separate bacteria and DNA, it is preferable that the pore diameter is 1 μm or less, preferably about 0.2 μm.

The method for separating and removing undegraded DNA by electrophoresis is preferably a method using a gel provided with wells for containing a suspension, and compared with an embodiment using a filter, a semipermeable membrane, etc., an agarose gel, etc. In the embodiment using, it is only necessary to solidify the gels, so it is not necessary to prepare a special electrophoresis rack for each work, and the preparation for work is easy.

The electrophoretic tanks used for the electrophoresis of the microbial suspension and the electrophoresis of the lysate treated solution according to the present invention can be constructed according to any of the above-mentioned methods. Separation with a filter can be carried out in the electrophoresis tank for electrophoresis of the suspension, and gel separation can be carried out in the electrophoresis tank for electrophoresis of the lysate-treated solution. However, it is preferable to carry out the electrophoresis of the microbial suspension and the electrophoresis of the lysate treatment solution in a common migration tank, and in order to do so, the wells should be placed near the center of the gel perpendicular to the migration direction. By using the provided gel and reversing the electrodes for electrophoresis in the case of the microbial suspension and in the case of the lysate treatment solution, the target DNA can be separated and recovered very easily and efficiently. Next, a gel provided with wells will be described.

Any gel may be used as long as it does not pass gels (such as microorganisms and soil particles) having a particle size exceeding 0.1 μm (does not migrate), and passes gels (migrates) if it is smaller than DNA. And the like, and preferably agarose gel or acrylamide gel used for electrophoresis of DNA or protein in the field of molecular biology, and those without a DNA degrading enzyme are preferred. In the case of agarose gel, when recovering plasmid DNA of several kbp or less, a concentration of about 0.8 to 1.5% is desirable, but Genome D having a size of several tens kbp.
In the case of NA, if the concentration is high, it becomes difficult to migrate, and if it is too low, the physical strength of the gel itself decreases, so
About 3 to 0.6% is desirable. Further, it is desirable that agarose or acrylamide be dissolved in an electrolytic solution to form a gel. Examples of the electrolytic solution include phosphate buffer solution, acetate buffer solution, citrate buffer solution and the like having a pH of 4 to 8 and the like, preferably DAE such as TAE, TPE and TBE.
A buffer solution for NA electrophoresis and the like.

The length (thickness) of the well in the electrophoretic direction may be appropriately determined according to the amount of the suspension to be electrophoresed. However, if only the DNA of a specific length is desired to be recovered, too much thickness lowers the resolution. Therefore, it is difficult to collect it, so about 2 to 10 mm is preferable. In addition, in electrophoresis of the suspension and electrophoresis of the lysate, it is better to run both of them in the opposite directions in order to avoid remixing of the free DNA and the DNA derived from the microorganism. It is advisable to provide a well at a position where one side can be used for migrating undegraded DNA and the other can be used for migrating microbial-derived DNA. Simply, it is provided near the center of the gel migration direction. Is good. The width of the well (length in the vertical direction from the migration direction) is about 10 to 50 mm, the depth is about 5 to 15 mm,
The capacity is usually about 0.1 to 5 cc.

The gel in which the suspension was injected into the wells was set in an electrophoresis tank for DNA electrophoresis, and then a DC voltage of 10 to 1000 V, usually 20 to 200 V, was applied to the gel for 10 to 30 V.
After allowing to stand for a minute, undegraded free DNA is migrated to a gel and removed. At this time, those having a size larger than the mesh of the gel (approximately 10 to 100 nm) such as microorganisms and fine particles of soil remain in the well.

After the electrophoresis, the gel on the side on which the free DNA has migrated is cut and removed, and the free DNA once separated is again mixed into the lysis solution during the subsequent electrophoresis of the lysis solution. Prevent from doing. If microorganisms are attached to the wall on either side of the well in the well, it is better to reverse the positive and negative poles and apply a voltage for about 1 minute to sufficiently detach the attached microorganisms and resuspend them in the DNA. The collection efficiency of is increased. Next, the lysate is added to the well and the inside of the well is stirred with a pipette or the like. Examples of the lysate at this time include cell membrane degrading enzymes such as lysozyme, phenol, and SDS.
After this, it is left to stand for 10 minutes to 2 hours to sufficiently lyse the microorganisms. When the lysate is a cell membrane degrading enzyme, the gel may be stored in a thermostatic chamber at the optimum temperature for the enzyme. For example, 37 ° C. is suitable for lysozyme.

When the cell membrane is sufficiently decomposed and the DNA in the microorganism becomes suspended in the liquid, the gel is again added to 50 to 10
A DC voltage of 0 V is applied to allow the microbial DNA to migrate in the gel. At this time, the positive pole and the negative pole are opposite to those when the free DNA is removed by electrophoresis. The migration time at this time varies depending on the size of the DNA to be recovered. A few k
A plasmid of about pb may take about 30 to 60 minutes.
Further, in the case of a genome having a length of several tens of kpb or more, lowering the voltage to about 10 to 50 V and performing electrophoresis for 1 to 2 hours can minimize contamination without the target DNA without lowering the yield. it can. At this time, substances having a size larger than the mesh of the gel such as microbial residues and soil fine particles remain in the well, and organic substances such as salts, ions, and humus have a faster migration speed than DNA or the migration direction is opposite. So it can be separated.

In order to grasp the position of the DNA electrophoresed in the gel, it is advisable to use a DNA size marker and run the sample at the same time as the sample. The size marker well and the sample well are arranged in parallel (perpendicular to the running direction). It is good to install it in. If the size marker is appropriately selected, the migration position of the DNA fragment having the desired size can be grasped, so that the desired DNA fragment can be easily recovered.

After the electrophoresis, the DNA is stained with ethidium bromide, etc., and the necessary portion of the gel is cut out with a cutter or the like, and then the gel is removed from the gel with a DNA dialysis tube or a centrifuge tube with a DNA recovery filter (Takara Shuzo Co., Ltd.). DN
The liquid containing A is separated, and the known techniques such as ethanol precipitation, isopropanol precipitation and PEG precipitation are applied to DN.
A can be collected and purified. Since the obtained DNA has high purity, PCR amplification, hybridization, etc., and library preparation can be performed with high reliability.

[0038]

EXAMPLES The present invention will be described below with reference to examples.

Example 1 Escherichia coli was placed in the LB liquid medium of Table 1 and shake-cultured overnight in a temperature-controlled room at 37 ° C. to grow 3 g of the bacterial solution.
4.5 ml of distilled water was added to the soil of (wet weight), and after stirring, the mixture was allowed to stand for 30 seconds and immediately mixed with 2 ml of the liquid prepared by removing sand that had settled, and further prepared separately beforehand at about 2 kbp. About 10 ng of DNA fragment of
A soil suspension containing NA fragments was prepared. 2 ml of this suspension was placed in a 0.4% agarose gel which was gelled using the buffer for electrophoresis shown in Table 2 (well of about 2.5 ml, length 5 mm, width). 50 mm,
It was injected to a depth of 12 mm). In addition, a small well next to the soil suspension well (volume: about 20 μl, length: 5 mm,
In a width of 0.5 mm and a depth of 10 mm, as a DNA size marker, an aqueous solution in which about 2 kbp of the same DNA as that mixed in the soil suspension was dissolved was put. The gel was set in an electrophoresis tank for DNA electrophoresis filled with an electrophoresis buffer (FIG. 2), and a DC voltage of 100 V (about 500 mA) was applied for about 30 minutes.

After the migration, the gel was cut and removed at a position 5 mm from the well on the plus pole side. As a precaution, when this gel was stained with ethidium bromide and observed, a band was observed at the same position as the size marker, and the band was approximately 2 kbp.
It was confirmed that about 10 ng of the DNA fragment of No. 1 was electrophoresed in the gel.

Next, add 1 to the remaining space of the gel well.
200 μl of 0% SDS solution was added and stirred with a pipette, and at the same time, microorganisms adhering to the wall surface on the plus side were scraped off.

After leaving it for 30 minutes to lyse the microorganisms in the treatment solution, the DNA size marker (λ /
Hind III digest, manufactured by Toyobo Co., Ltd. This one is 23,
Bands such as 9.4 and 6.5 kbp appear. ), Set the positive and negative poles in reverse, and then again for about 1 hour 1
A DC voltage of 00V was applied. After the electrophoresis, the gel was removed from the electrophoresis tank, stained with ethidium bromide and observed. As a result, about 20 kbp or more of genome DNA was found to have migrated in the gel. In addition, brown solid matter, which is considered to be fine particles of soil, stuck to the wall surface of the gel, but did not enter the gel.

Next, the portion of the gel containing the genomic DNA was cut out and divided into appropriate sizes, and a centrifuge tube with a filter for recovering DNA (trade name SUPREC-0
1 Takara Shuzo Co., Ltd.), store in a refrigerator at -20 ° C for 1 hour, freeze, then melt at room temperature and set in a centrifuge 1
It was centrifuged at 5000 rpm for 10 minutes. Then, 200 μl of TE solution was added and the mixture was centrifuged again at 15000 rpm for 10 minutes. Ethanol was added to the recovered solution to precipitate the DNA, which was recovered and dissolved in 20 μl of distilled water to obtain a DNA solution.

When 10 μl of this DNA solution was electrophoresed on an agarose gel, Genome DN of several tens kbp was obtained.
A was detected. When the remaining 10 μl was added with typical restriction enzymes BamHI and EcoRI in the composition shown in Table 3 and heated at 37 ° C. for 2 hours and then electrophoresed, DNA was digested and shortened, It was confirmed that the length was about 10 kpb. From this, it was confirmed that the DNA lysed and extracted from the microorganism purified and recovered by this method had such a purity that it could be digested with a restriction enzyme without any special DNA purification treatment.

[0045]

[Table 1]

[0046]

[Table 2]

[0047]

[Table 3] Example 2 Except for lysing lysozyme for 2 hours in an atmosphere of 37 ° C. instead of the SDS solution, the same experiment as in Example 1 was performed to recover DNA.

As a result, a genom DNA purified in the same manner as in Example 1 was obtained.

Example 3 Insert of about 1 kbp and pUC19 (Takara Shuzo Co., Ltd.)
E. coli into which plasmid A prepared by conjugating
And 10 ng of plasmid B having the same length as the insert of plasmid A but different inserts were
2 ml of the soil suspension containing the same state was prepared, and the DNA was purified and recovered in the same manner as in Example 1.

The recovered DNA solution is diluted 100 times,
Primer A (DN manufactured by ABI, Inc.) that reacts only with the insert of plasmid A to synthesize a DNA fragment of about 1 kbp
A) (synthesized by A synthesizer) or Primer B (synthesized by ABI DNA synthesizer) that reacts only with the insert of plasmid B to synthesize a DNA fragment of about 1 kbp.
Etc. were added in the composition shown in Table 4, and then PCR amplification was performed under the temperature conditions shown in Table 5. After amplification, the reaction solution was electrophoresed on an agarose gel. As a result, in the case where Primer A was added, the DNA fragment was amplified and the recovered DNA was directly converted into PC.
It was confirmed that the purity was sufficient for R and that the plasmid A was selectively present in the recovered DNA. In addition, in the case where Primer B was added, the DNA fragment was not amplified, and the plasmid B was not present in the recovered DNA, which confirmed that the extracellular DNA was removed.

[0051]

[Table 4]

[0052]

[Table 5] Comparative Example 1 2 ml of a soil suspension containing the same plasmid A-introduced E. coli as in Example 3 and 10 ng of the plasmid B in the state of DNA was prepared and treated with 10% SDS solution as in Example 1. After the cell membrane degradation treatment, as in Example 1, the cells were placed in the wells of the agarose gel shown in FIG. 1 and electrophoresed to about 1 kbp.
Was cut out to recover the DNA.

The recovered DNA solution was treated with 1 as in Example 3.
When diluted with 00 times and added with primer A or primer B, PCR amplification was carried out, and it was confirmed that the DNA fragment was amplified in both primers, and both plasmids A and B were mixed and not separated. It was

Comparative Example 2 E. coli having about 1 kbp of plasmid A and 10 ng of plasmid B having the same length as plasmid A but different from that of plasmid A.
2 ml of a soil suspension containing DNA in the form of DNA was prepared and subjected to cell wall decomposition treatment with a 10% SDS solution in the same manner as in Example 1, and then DNA was recovered without electrophoresis. At this time, the recovered DNA had a brown color like the soil suspension.

The recovered DNA solution was treated with 1 as in Example 3.
When diluted with 00 times and added primer A or primer B, etc. and PCR amplification was performed, it was confirmed that the DNA fragment was not amplified in either primer and the recovered DNA could not be PCR amplified due to contaminants. Was given.

[0056]

As described above, according to the present invention,
DNA can be recovered with high recovery efficiency, and there is no concern that unresolved undegraded DNA of unknown origin is mixed in the recovered DNA, and PCR amplification or DNA
It is possible to create a library, and the collected DNA is free from contamination by contaminants such as soil particles and humus.
Digestion with restriction enzymes or P without NA purification
There are advantageous effects that the conventional method does not have, such as CR amplification.

That is, by applying the electrophoretic treatment, the undegraded DNA and the microorganism can be separated by electrophoresis first, and then the microorganism can be lysed and the DNA can be separated by electrophoresis. Microorganism-derived DNA can be recovered from a suspension with high purity and high efficiency, and the recovered DNA can be directly used as a purified DNA. In addition, by providing a well in the gel medium for electrophoresis, a predetermined electrophoresis can be performed simply by storing a sample in the well and applying a voltage, so that the separation operation by electrophoresis can be further simplified. In particular, the electrophoresis of the microbial suspension and the electrophoresis of the lysate-treated solution are performed using a common gel medium, and a well is provided near the center of the gel in a direction perpendicular to the migration direction so that the undecomposed D
Electrophoresis of NA and electrophoresis of DNA derived from microorganisms are applied to the electrodes with the positive and negative electrodes reversed, and undegraded D
If NA is electrophoresed on one side of the gel and DNA derived from the microorganism after lysis is electrophoresed on the other side, the unresolved DNA can be easily removed simply by cutting out the necessary part of the gel (the gel at the position where the DNA has moved). Since the microorganism-derived DNA can be recovered extremely easily, the operation for recovering the microorganism-derived DNA is further simplified. In addition, if a gel having a well containing a DNA size marker in parallel with the well containing a reagent is used as the gel, the excision position after electrophoresis can be specified by the DNA size marker. It becomes possible to selectively collect only the DNA of a specific length.

[Brief description of drawings]

FIG. 1 is a schematic perspective view of an agarose gel for purification and recovery of DNA that can be used in the present invention.

FIG. 2 is a schematic front cross-sectional view showing a state in which a gel is set in an electrophoresis tank that can be used in the present invention.

[Explanation of symbols]

1 Agarose gel (0.4%) 2 Well for soil suspension injection (volume about 3 ml) 3 Well for DNA size marker (volume about 20 μ
m) 4 Electrophoresis buffer (see Table 1 for tissue) 5 Platinum wire electrode 6 Agarose gel for purification and recovery of DNA

Claims (8)

[Claims] 1. A microorganism in a microbial suspension obtained by subjecting a microbial suspension to electrophoresis to move free undegraded DNA present in the suspension to separate and remove it from the microbial suspension. A method for recovering and purifying a microorganism-derived DNA, which comprises subjecting to a bacteriolytic treatment and re-electrophoresing to recover and purify the microorganism-derived DNA. 2. The method according to claim 1, wherein, when the lysate-treated solution is electrophoresed, the positive and negative electrodes of the electrodes are made opposite to those in the first electrophoresis to reverse the migration direction of the DNA. A method for collecting and purifying DNA derived from a microorganism, which prevents the contamination and purification of degraded DNA. 3. The electrophoresis of a microbial suspension and / or the electrophoresis of a lysate-treated solution according to claim 1 or 2,
A method for collecting and purifying a microorganism-derived DNA, which is performed by using a gel provided with a well as a medium to store a sample in the well, and collecting the target DNA migrated in the gel by cutting out a gel corresponding to the migration position. 4. The method according to claim 3, wherein the electrophoresis of the microbial suspension and the electrophoresis of the lysate-treated solution are performed using a common gel medium,
Wells are provided in the vicinity of the center of the gel in a direction perpendicular to the migration direction. First, a microbial suspension is placed in the wells and a voltage is applied to migrate the gel to cut out the gel at the position where undegraded DNA has migrated. Then, the undegraded DNA is removed, and then the microorganism in the suspension in the well is lysed with a lysing solution, and a voltage is applied by reversing the plus and minus of the electrode, and the direction of migration of the microbial suspension is reversed. A method for recovering and purifying a microorganism-derived DNA, which comprises subjecting a gel at a position where the microorganism-derived DNA has migrated by unidirectional electrophoresis to cutting out the gel to collect the microorganism-derived DNA. 5. The gel according to claim 3 or 4, wherein the gel is provided in parallel with the well containing the reagent and in parallel with the well.
It is equipped with a well that accommodates size markers,
A microorganism-derived D in which electrophoresis is performed using the gel and the excision position after electrophoresis is specified by the DNA size marker
NA recovery and purification method. 6. The method according to any one of claims 3 to 5,
A method for collecting and purifying a microorganism-derived DNA whose gel is selected from agarose gel, acrylamide gel, and gelatin gel. 7. The method according to any one of claims 1 to 6,
Lysate is phenol solution, cell membrane degrading enzyme solution or S
A method for collecting and purifying microbial DNA, which is DS. 8. The method according to claim 1, wherein
Microorganism-derived DN whose microbial suspension is soil suspension, activated sludge in sewage / wastewater treatment tanks, or river / lake / sea bottom mud
Method of collecting and purifying A.