WO2005073377A1 - Method of collecting dna from environmental sample - Google Patents

Method of collecting dna from environmental sample Download PDF

Info

Publication number
WO2005073377A1
WO2005073377A1 PCT/JP2004/011956 JP2004011956W WO2005073377A1 WO 2005073377 A1 WO2005073377 A1 WO 2005073377A1 JP 2004011956 W JP2004011956 W JP 2004011956W WO 2005073377 A1 WO2005073377 A1 WO 2005073377A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
extract
edta
soil
concentration
Prior art date
Application number
PCT/JP2004/011956
Other languages
French (fr)
Japanese (ja)
Inventor
Hiroki Rai
Shigeto Ohtsuka
Masaya Nishiyama
Keishi Senoo
Original Assignee
Toudai Tlo, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toudai Tlo, Ltd. filed Critical Toudai Tlo, Ltd.
Priority to JP2005517375A priority Critical patent/JP4665124B2/en
Publication of WO2005073377A1 publication Critical patent/WO2005073377A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • the present invention relates to a method for recovering DNA from an environmental sample.
  • genes that are commonly held by a wide range of organisms such as bacteria and fungi are applicable only to universal primers or target organisms (genus, species level). Analysis is performed by PCR amplification using primers that perform the above steps, and decoding the nucleotide sequence through DGGE, TGGE, or cloning.
  • the most important problem in this community structure analysis is "bias". The problem of PCR bias is not negligible, but before that, there is the question of whether DNA is extracted without bias from all microorganisms during the extraction from soil. In order to analyze the microbial community structure more accurately, it is first required that DNA be uniformly extracted from all microorganisms at the stage of extraction from soil.
  • DNA that is strongly adsorbed to soil particles such as a portion of actinomycetes, microorganisms that live in plant remains, or microorganisms that live inside aggregates cannot be extracted by this method.
  • direct extraction methods were successively developed by Ogram et al. (1987), Tsai & Olson (1991), Zhou et al. (1996), and others.
  • the bacteria are separated from the soil by treating the soil with an enzyme extract containing enzymes such as lysozyme and proteinase K and SDS, and denaturing the protein. Lysis in a solution where a matrix of soil exists.
  • the indirect extraction method may not accurately reflect the community structure of the soil, but once the DNA is extracted after separating microorganisms from the soil, not only the humic substances are less contaminated than the direct extraction method, However, it is possible to suppress the fragmentation of the DNA due to physical shearing caused by the collision of the soil particles with the DNA, and it is possible to obtain higher-molecular-weight DNA.
  • vectors that can introduce high-molecular-weight DNA with high cloning efficiency have been developed, and the indirect extraction method has been used more often in gene search research than in community structure analysis. (Bakken & Lindahl 1995, Saano et al. 1995, Berry et al. 2003, Gabor et al. 2003).
  • kits that prepare DNA from soil in a short time using a proprietary method such as the BiolOl Fast DNA spin kit (Qbio, USA) and the UltraClean Soil DNA kit (MoBio, USA), have also been commercialized. All of them use beads beater and can extract soil DNA in a short time.
  • Humic substances contaminated in DNA samples can inhibit the PCR reaction even in very small amounts (Tsai et al. L991). For this reason, Zhou et al. (1996) et al. Used a method in which the extracted soil DNA was electrophoresed once with an agarose gel to separate humic substances and DNA, and then only the DNA was recovered from the gel. (Zhou JZ, Bmns MA, Tiedje JM; Applied and environmental microbiology; 1996; 62: (2) p.316-322). An agarose-embedded preparation is another method that uses low-melting-point agarose gel (Moreim 1998).
  • a typical separation method based on the molecular weight is a gel filtration method. It uses a porous resin such as Sephadex or Sepharose to separate and recover relatively large DNA molecules from small humic substances by size fractionation (eg Jackson et al. 1997, Miller 2001).
  • a porous resin such as Sephadex or Sepharose
  • DNA is easily adsorbed on the glass surface due to the chaotropic effect in a chaotropic salt solution.
  • PVPP polyvinylpolypyrrolidone
  • CTAB cetyltrimethylammonium bromide
  • An object of the present invention is to provide a method for extracting high yield and high purity DNA from soil.
  • the present inventor first decided to use various substances such as soil-derived humic substances and clay as chemical conditions, which are suitable for lysing soil microbial cells.
  • soil-derived humic substances and clay as chemical conditions, which are suitable for lysing soil microbial cells.
  • surfactants that can sufficiently lyse microorganisms even under coexisting conditions.
  • physical conditions of cell destruction the conditions of crushing and heating by beads-beating were examined.
  • Tris-HCl buffer, EDTA solution, and phosphate buffer used in the extract, and the purification and precipitation of the obtained soil DNA were carried out by purification using CTAB, a positive surfactant, and polyethylene glycol. Investigating to obtain a high yield and high purity DNA by simpler operation from various soils with different properties, such as combining DNA precipitation operation with PEG under optimal conditions. Was performed.
  • the present invention is as follows. (1) A method for extracting DNA from an environmental sample, comprising treating the environmental sample in the presence of a DNA extract containing 5% or less of a surfactant.
  • surfactant is selected from the group consisting of SDS, CTAB, Triton X-100, and lauroyl sarcosine sodium.
  • the method comprising:
  • a method for extracting DNA from an environmental sample (a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 mM EDTA, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
  • the method comprising:
  • the above method comprising the step of subjecting an environmental sample to beads-beating treatment and / or heat treatment in the presence of a DNA extract containing 5% or less of a surfactant and a phosphate buffer of 250 to 2000 mM.
  • the method comprising:
  • Extract solution I after beads-beating treatment and / or heat treatment was mixed with 75 to: l200 mM EDTA, 250 to 3000 mM phosphate buffer, or the above EDTA and phosphate buffer. Extract II was prepared by mixing with
  • the method comprising:
  • Extract IV after beads-beating treatment is mixed with 400 to OmM EDTA, 750 to 2050 mM phosphate buffer, or a mixture of EDTA and phosphate buffer to prepare Extract IV.
  • the method comprising:
  • Remaining environmental sample is 5% or less of surfactant and 400mM or less of EDTA. And beads-beating treatment in the presence of DNA extract III containing phosphate buffer of up to 250 mM
  • the method comprising:
  • the method comprising:
  • the method comprising the steps of:
  • the method comprising:
  • a method for purifying DNA comprising purifying DNA derived from an environmental sample in the presence of a cationic surfactant and a salt.
  • a method for recovering DNA comprising precipitating in the presence of 2-propanol, ethanol or polyethylene glycol.
  • a DNA characterized in that the DNA extracted by the method according to any one of (1) to (9) above is precipitated in the presence of 2-propanol, ethanol or polyethylene dalicol. Collection method.
  • the method comprising: (46) The method according to (45), wherein the DNA extract further comprises' a phosphate buffer and / or EDTA.
  • the method comprising:
  • the method comprising:
  • the method comprising:
  • the method comprising:
  • the method comprising the steps of:
  • a method for recovering DNA from an environmental sample comprising:
  • Extract II EDTA, 250-3000 mM phosphate buffer or a mixture of EDTA and phosphate buffer described above to prepare Extract II,
  • the method comprising the steps of:
  • Extract IV after beads-beating treatment was mixed with 400-1000 mM EDTA, 750-2050 mM phosphate buffer, or a mixture of the EDTA and phosphate buffer to prepare Extract IV.
  • the method comprising the steps of:
  • Residual environmental samples should be prepared in the presence of DNA extract III containing 5% or less of detergent, 400 mM or less of EDTA, and 250 mM or less of phosphate buffer. beads-beatin processing
  • the method comprising the steps of:
  • the method comprising the steps of:
  • recovering the DNA comprises a step of mixing the supernatant after centrifugation with a cationic surfactant and a salt to purify the DNA.
  • recovering the DNA comprises a step of purifying the DNA by mixing the extract II with a cationic surfactant and a salt.
  • recovering the DNA comprises a step of purifying the DNA by mixing the extract IV with a cationic surfactant and a salt.
  • the step of recovering DNA includes a step of purifying DNA by mixing extract V after heat treatment and / or extract III after beads-beating with a cationic surfactant and a salt. the method of.
  • Recovering DNA includes purifying DNA by mixing Extract III after beads-beating and / or Extract V after beads-beating with cationic surfactant and salt (67 ) Described method.
  • step of recovering the DNA comprises a step of mixing the extract after the second heating with a cationic surfactant and a salt to purify the DNA.
  • recovering the DNA comprises a step of mixing the extract after the heat treatment, a cationic surfactant and a salt to purify the DNA.
  • recovering the DNA comprises a step of precipitating the DNA in the presence of 2-propanol, ethanol or polyethylene glycol.
  • kits according to (96), wherein the pH of the DNA extract can be adjusted to 7.0 or more (100) The kit according to (96), wherein the pH of the DNA extract can be adjusted to 7.0 or more.
  • a kit for purifying DNA from an environmental sample comprising a salt solution containing a pH buffer having a pKa on the acidic side, a cationic surfactant, or a mixture of the salt solution and a cationic surfactant.
  • pH buffer having a pKa on the acidic side is an acetate buffer, a phosphate buffer, a hydrochloric acid buffer, or a sulfate buffer.
  • the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate.
  • a kit for recovering DNA from an environmental sample comprising an alkaline buffer.
  • FIG. 1 is a diagram showing an example of quantification of soil DliA.
  • FIG. 2 is a diagram showing the amount of soil DNA extracted by various surfactants.
  • Figure 3 shows the effect of SDS concentration on DNA extraction from compost plotted soil in Yayoi field.
  • FIG. 4 shows the effect of SDS concentration on soil DNA extraction.
  • FIG. 5 is a diagram showing the effect of the pH of the extract on soil DNA. The numbers above each bar represent the pH of the extract after beads-beating.
  • Figure 6 shows the effect of EDTA concentration on DNA extraction from volcanic ash soil.
  • FIG. 7 is a graph showing the effect of EDTA concentration on DNA extraction.
  • FIG. 8A is a diagram showing the relationship between the EDTA concentration of the extract and the amount of extracted metal element.
  • FIG. 8B is a diagram showing the relationship between the EDTA concentration of the extract and the amount of extraction of the metal element.
  • FIG. 8C is a diagram showing the relationship between the EDTA concentration of the extract and the amount of extraction of the metal element.
  • FIG. 9A is a diagram showing the relationship between the EDTA concentration of the extract and the EDTA-metal element complex formation rate.
  • FIG. 9B is a diagram showing the relationship between the EDTA concentration of the extract and the EDTA-metal element complex formation rate.
  • FIG. 9C is a diagram showing the relationship between the EDTA concentration of the extract and the EDTA-metal element complex formation rate.
  • FIG. 10A shows the DNA yield when DNA is repeatedly extracted from soil.
  • FIG. 10B is a view showing DNA yield when DNA is repeatedly extracted from soil.
  • Figure 10C shows the DNA yield when DNA was repeatedly extracted from soil.
  • FIG. 11 is a diagram showing the calculation results of the amount of fresh soil DNA extracted from the re-added EDTA solution.
  • FIG. 12 is a diagram showing the amount of soil DNA considered to have been able to be extracted by newly added EDTA. '
  • FIG. 13A shows the amounts of metals (A1 and Fe) extracted from soil by newly added EDTA.
  • FIG. 13B is a diagram showing the amounts of metals (Ca and Mg) extracted from soil by newly added EDTA.
  • FIG. 14 is a diagram showing the results of extracting soil and DNA from the Yayoi field control plot soil to which a high-concentration EDTA solution was added after beads-beating.
  • FIG. 15A shows the effect of the EDTA concentration of the extract on the amount of soil DNA extracted.
  • FIG. 15B shows the effect of the P04 3 -concentration of the extract on the amount of soil DNA extracted.
  • Figure 16 ⁇ shows the effect of EDTA-phosphate concentration in the extract of volcanic ash soil on soil DNA extraction.
  • Figure 16B shows the effect of EDTA-phosphate concentration in the extract on non-volcanic ash soil on soil DNA extraction.
  • FIG. 17 is a diagram showing the relationship between the phosphate ion concentration of the extraction solution and the yield of soil DNA in the two-step DNA extraction.
  • FIG. 18A is a diagram showing the yield of soil DNA extracted by a two-step operation using a high concentration of EDTA and a phosphate buffer.
  • FIG. 18B shows the yield of soil DNA extracted from Yayoi field control plot soil by the improved 2-step method using EDTA and phosphate buffer. Beauty P0 4 Oyo EDTA concentrations shown in Figure 3 - concentration is the concentration in the extraction solution at the time of beads-beating, beads-beating final concentration after addition of the solution prepared respectively after every 4 0 0 mMEDTA / 750mM PO 4 3 _.
  • Figure 19 is an electrophoresis photograph showing the results of DNA extraction by the improved 2 steps.
  • FIG. 2 OA shows the results of DNA recovery when each precipitant was used.
  • FIG. 20B is a diagram showing the effect of removing humus by the difference in the method of precipitating soil DNA.
  • FIG. 21 shows the effect of PEG concentration on soil DNA recovery.
  • FIG. 22A shows the amount of DNA recovered when simple purification was performed using CTAB.
  • FIG. 22B shows the effect of removing humic substances by CTAB treatment.
  • FIG. 22C is a diagram showing the effect of removing humic substances by CTAB treatment.
  • FIG. 23 shows a comparison of soil DNA yields under the four original conditions.
  • Figure 24 is an electrophoresis photograph showing the size of the extracted DNA under the original four conditions.
  • FIG. 25 is a diagram showing a comparison between the method of the present invention and a conventional method in soil DNA yield.
  • Fig. 26A is an electrophoresis photograph showing the results of a purity test using a PCR reaction of soil DNA extracted by the original one-step method.
  • the upper two panels have a type I concentration of 100ng / 50l, the two panels have a type I concentration of 50ng / 50l, and the other two panels have a type I concentration of 10ng / 50l.
  • Figure 26B is an electrophoresis photograph showing the results of a purity assay using PCR reaction of soil DNA extracted by the original two-step heating method.
  • the ⁇ type concentration in each panel is the same as in FIG. 26A.
  • FIG. 27A is an electrophoresis photograph showing the evaluation results of the extracted DNA.
  • Lanes 1-5 are the soils of the Yayoi field control plot
  • Lanes 6-10 are the Chiba agricultural trial forest soil
  • Lane 11-: L5 is the Ibaraki agricultural trial forest soil
  • Lane 16-20 is the migration of DNA extracted from Tanashi farm pasture soil
  • Lanes in each soil represent the original IStep method, 2Step method, 2Step heating method, LCB method, and PCR products of soil DNA extracted by Zhou et al. (1996) in order from the left.
  • FIG. 27B is an electrophoresis photograph showing the evaluation results of the extracted DNA.
  • Lane 1 8 to 8 are the Gunma animal pastures
  • lanes 9 to: L6 is the electrophoretic diagram of DNA extracted from Tochigi forest soil. The lanes in each soil are, in order from the left, the original IStep method, 2Step method, 2Step heating method, LCB method, the method of Zhou et al. (1996), the method of Cullen & Hirsch (1996), UltraClean Soil DNA kit, Bio 101 Fast DNA PCR product of soil DNA extracted by spin kit.
  • FIG. 27C is an electrophoresis photograph showing the evaluation results of the extracted DNA. Lanes 1 to 8 are the migration plots of DNA extracted from Tohoku University forest soil, and lanes 9 to 16 are the grass extractive soil from the permanent grass field. Lanes for each soil are similar to FIG. 27A.
  • FIG. 27D is an electrophoresis photograph showing the evaluation results of the extracted DNA.
  • Lanes 1 to 8 are the Saitama agricultural test field soil, and lanes 9 to 16 are swimming diagrams of DNA extracted from the Osaka agricultural test field soil. Lanes for each soil are similar to FIG. 27A.
  • FIG. 27E is an electrophoresis photograph showing the evaluation results of the extracted DNA.
  • Lanes 1 to 8 are the Hyogo Agricultural Experiment Station soils
  • Lanes 9 to 16 are the swimming diagrams of DNA extracted from the Nara Agricultural Experiment Station soil. Lanes for each soil are similar to FIG. 27A.
  • FIG. 28 shows the effect of lowering the pH due to the acidic buffering capacity of the salt solution on the purity of the DNA extract during the purification of DNA by CTAB.
  • FIG. 29 is a diagram showing the effect of lowering the pH due to the acidic buffering capacity of the salt solution during DNA purification by CTAB on the purity of the DNA solution after precipitation and recovery.
  • FIG. 30 shows the effect of lowering the pH due to the acidic buffering capacity of the salt solution on the amount of DNA recovered during the purification of DNA by CTAB (PEG solution was used for DNA recovery).
  • Figure 31 shows the effect of lowering the pH due to the acidic buffer capacity of the salt solution on the purity of the DNA extract during the purification by CTAB. (A mixed solution of CH 3 COONa and NaCl was used for the salt solution.) Case).
  • Figure 32 shows the effect of lowering the pH due to the acidic buffering capacity of the salt solution during purification by CTAB on the purity of the DNA recovered with the PEG solution (a mixed solution of CH3COONa and NaCl was added to the salt solution). If used).
  • Figure 33 shows the effect of reducing ⁇ due to the acidic buffering capacity of the salt solution on the amount of DNA recovered during purification by CTAB (using a mixture of CH3COONa and NaCl for the salt solution). Use PEG solution for DNA recovery).
  • FIG. 34 shows the effect of the presence or absence of the buffering capacity of the PEG solution on the purity of the recovered DNA.
  • FIG. 35 shows the effect of the presence or absence of the alkaline buffering capacity of the PEG solution on the amount of DNA recovered. .
  • Figure 36 shows the effect of the alkaline buffering capacity of the PEG solution used to recover DNA on the purity of the recovered DNA (EDTA 200 mM I Na2HP04 Soil DNA extract obtained with 375 mM extract) Targeting).
  • Figure 37 shows the effect of the salt solution composition during purification by CTAB and the buffering capacity of the PEG solution used for DNA recovery on the amount of DNA recovered (EDTA 200 mM I Na2HP04 375 mM extract).
  • Figure 38 shows the results of examining the extraction conditions for polymer soil DNA by heat extraction (for the Yayoi control plot soil).
  • Figure 39 is a diagram showing the results of examining the conditions for extracting polymer soil DNA by heat extraction.
  • FIG. 40 shows the effects of the composition of the extract and the physical treatment on the amount of DNA extracted from feces.
  • Figure 41 is an electrophoresis photograph of DNA extracted from feces.
  • FIG. 42 is a diagram showing the results of studies on a DNA extraction method from feces.
  • FIG. 43 shows the purity of DNA extracted from feces.
  • Figure 44 shows the effect of extract composition and physical treatment on the amount of DNA extracted from compost.
  • Figure 45 is an electrophoretic photograph of DNA extracted from compost and activated sludge.
  • Figure 46 is a diagram showing the results of studies on a method for extracting DNA from compost.
  • FIG. 47 is a diagram showing the purity of DNA extracted from compost.
  • FIG. 48 is a diagram showing the results of a study on a method for extracting DNA from activated sludge.
  • FIG. 49 shows the purity of DNA extracted from activated sludge.
  • Figure 50 is an electrophoretic photograph of DNA extracted from lake sediments.
  • Figure 51 shows the results of a study on DNA extraction from lake sediments.
  • Figure 52 shows the purity of DNA extracted from lake sediments.
  • FIG. 53 is a diagram showing a result of DGGE analysis of soil: DNA.
  • FIG. 54 is a diagram showing a DGGE analysis result of fecal DNA.
  • FIG. 55 shows DGGE analysis results of compost DNA and activated sludge DNA.
  • Figure 56 shows the results of DGGE analysis of lake bottom sediment DNA.
  • the present invention is a method for efficiently extracting, purifying, or recovering DNA from an environmental sample, and includes a step of extracting DNA and a step of removing contaminants other than DNA from a DNA extract.
  • the concentration conditions of EDTA, phosphate buffer, and a mixture thereof to obtain a high yield of DNA were examined even in a volcanic ash soil where DNA extraction would be difficult.
  • the optimal precipitation conditions and the optimal purification conditions that do not contain contaminants were examined, and found a method for efficiently recovering DNA from soil with high purity.
  • treatment with a DNA extract containing a surfactant is employed as a basic operation of DNA extraction.
  • Combining beads-beating or heat treatment at the time of extraction enables efficient extraction.
  • an environmental sample, microparticles, and a DNA extract containing a surfactant at a predetermined concentration are mixed, and the mixture is subjected to beads-beating treatment to extract DNA.
  • DNA is extracted by heat-treating a mixture of the above soil sample and a surfactant at a predetermined concentration.
  • the pH of the extract, EDTA or phosphate Concentration conditions of buffer solution and Huangwei solution, heating condition, concentration condition of cationic surfactant such as CTAB, kind and concentration of salt added during CTAB treatment, polyethylene glycol concentration condition, and polyethylene glycol By examining various conditions such as pH during the DNA precipitation operation by, it is possible to set appropriate extraction conditions, recovery conditions, and purification conditions according to the type and properties of the soil. .
  • the pH of the whole extract used to extract DNA from the sample is adjusted to ⁇ or more, and its optimum pH is 8.6 (details will be described later).
  • the basic extract composition to be mixed with a surfactant for each environmental sample includes an EDTA solution and a phosphate buffer.
  • DNA When DNA is extracted from soil, which is a representative example of environmental samples, the DNA is adsorbed by amorphous aluminum contained in the soil in the extraction of DNA from volcanic ash soil such as the Kanto-guchi layer. And the recovery rate is very poor.
  • This adsorption by soil can be solved by using a solution containing high concentrations of EDTA, phosphoric acid, or both.
  • EDTA, phosphoric acid, or both are also effective in extracting DNA from normal soil and other environmental samples containing microorganisms, such as compost and water-based sediments, activated sludge and feces. is there.
  • the concentrations of EDTA and phosphoric acid are 50 mM to 600 mM and 100 mM to 1500 mM, respectively.
  • EDTA is 50 mM to 600 mM
  • phosphoric acid is 100 mM to 750 mM.
  • the above concentration is an example, and the concentration can be appropriately changed depending on a difference in physical treatment after the addition of the extract during the extraction operation.
  • Quaternary ammonium salts such as CTAB can be used to purify DNA obtained from environmental samples.
  • the quaternary ammonium salt include cationic surfactants such as CTAB and DTAB.
  • the purification of DNA using CTAB is preferably performed in the presence of a salt.
  • the salt include sodium salts such as sodium chloride and sodium acetate, and potassium salts such as chloride chloride.
  • the present invention provides a purification method comprising adding a cationic surfactant and a salt solution to an extract. It is characterized in that the pH during production is lower than the pH during extraction.
  • addition in the present invention means not only adding one solution to the other solution, but also includes mixing one solution and the other solution.
  • the pH of the extraction solution containing DNA after the extraction operation is adjusted to 7.0 'or more, preferably 8.0 or more by the buffer solution contained in the extraction solution, but the pH is adjusted with a cationic surfactant such as CTAB.
  • the pH is lowered to less than 7.0 by mixing the extract with a salt solution that has a buffering capacity to lower the pH.
  • the salt solution preferably contains a pH buffer having an acidic pKa such as acetic acid.
  • the optimal salt solution in this case is a solution of 3.33M sodium acetate / 1.67M sodium chloride (pH 5.2).
  • the pH of the extract can be lowered to 6.0 or less.
  • the cationic surfactant and the salt solution can be added to the extract after mixing the two.
  • PEG polyethylene glycol
  • the solution containing PEG has a buffer capacity to raise the pH lowered during purification to alkaline again. Increasing the pH to the acidic side during purification and increasing the pH to the alkaline side during recovery enables more selective precipitation and recovery of DNA, and prevents co-precipitation of humic sugars and other contaminants. Can be.
  • a PEG solution having an alkaline buffering capacity for example, a PEG solution having a pH of 8.0 or more can be prepared and used by a Tris-HCl buffer system or the like.
  • the optimal condition is to mix a solution of 12% PEG / 1.5M Tris-HCl (pH 8.6) with the DNA extract and centrifuge. By mixing this solution, the pH of the solution when recovering DNA rises to 7.5 or more, preferably 8.0 or more.
  • the PEG solution and the Tris-HCl solution may be separately prepared and mixed so that the final pH is 7.5 or more.
  • the meanings of the abbreviations used in the present specification are as follows.
  • CTAB Cetyltrimethylammonium bromide bromide
  • EDTA ethylenediaminetetraacetic acid
  • PEG polyethylene glycol
  • the environmental sample to be subjected to DNA extraction is not particularly limited as long as it is a solid or liquid component existing in the environment.
  • soil, compost, water-based sediment, activated sludge, feces and the like can be mentioned, and these environmental samples can be appropriately selected according to the purpose of use.
  • the soil sample to be collected is not particularly limited, and any soil can be used.
  • any soil can be used.
  • volcanic ash soil is distributed over a relatively wide area, and volcanic ash soil is often the target soil for DNA extraction.
  • the target soil may be non-volcanic ash soil.
  • the volcanic ash soils include the Kanto-guchi layer, which is a deposit of Fuji volcanic ash mainly distributed in the Kanto region, as well as the alofen black-pok soil and the Tohoku region, which are based on volcanic eruption products and tephra fallen ash.
  • Non-arofenic soils such as those found in the southern part of the country are listed.
  • the non-volcanic ash soils include gray lowland soil, lowland paddy soil, and brown forest soil unaffected by fire ash in plains throughout Japan. , Red soil, yellow soil and the like.
  • Compost is an essential organic fertilizer for growing crops and maintaining soil productivity.
  • Compost is usually added to plant residues of field crops such as straw and straw, or wood-based materials such as leaves and wood chips, by adding inorganic fertilizers and livestock dung as a nitrogen source, depositing, and decomposing by microorganisms ( ⁇ A part of it is decomposed).
  • inorganic fertilizers and livestock dung as a nitrogen source
  • depositing, and decomposing by microorganisms ⁇ A part of it is decomposed
  • microorganisms After being added to the soil, these microorganisms are not only useful as fertilizer sources, but also important as useful microorganisms that promote crop growth. In recent years, compost-derived microorganisms are often used to treat persistent chemicals such as pesticides and PCBs.
  • Aqueous sediment refers to sediment, such as lakes, ponds, rivers and oceans, and sediment, such as soil, organic matter, and microorganisms.
  • plant and animal blanktons grown in water and their carcasses are deposited in large quantities, and there are many microorganisms that degrade them.
  • These dead bodies are broken down by microorganisms, and nutrients such as nitrogen contained therein are mineralized and released back into the water.
  • the microorganisms in the sediment play a role in circulating nutrients. It is considered that clarifying the number and type of such microorganisms is extremely important in clarifying the material cycle in water systems.
  • Activated sludge is one of the most common methods of treating wastewater.
  • Typical examples of sewage include general urban wastewater and livestock night soil.
  • the wastewater is purified by subjecting these wastewaters to aeration treatment with air, decomposing organic substances by microorganisms, and collecting the decomposition products and microbial cells grown by the decomposition.
  • the composition of the microorganisms grown in this treatment, that is, activated sludge differs depending on the quality of the wastewater to be treated and the treatment conditions, and the analysis of these microorganisms is extremely important in the purification treatment of wastewater.
  • Feces Feces such as humans, livestock, and insects, contain very large amounts of microorganisms.
  • Human feces contain an extremely large number of microorganisms, including Escherichia coli and lactic acid bacteria grown in the intestine. It has been clarified to date that useful microorganisms are included.
  • the analysis of microbes contained in these feces is extremely important because they include harmful microorganisms that cause food poisoning.
  • herbivore animals such as cattle, which are ruminants, partially degrade ingested plants using intestinal microorganisms, and it is extremely important to clarify these microbial communities by analyzing feces. .
  • Beads-beating is the process of adding soil and DNA extract to a screw cap tube and adding microparticles (glass beads, silica zirconium abs, aluminum beads, etc.) to physically destroy cells. It is a method, including the composition of beads, etc., which has been studied in detail by Rgmann et al. In this method, even gram-positive bacteria that have an extracellular polysaccharide membrane and are not easily affected by detergents such as SDS can be mechanically disrupted, so that DNA can be extracted in extremely high yield. In addition, since extraction is completed in a short time, a soil DNA sample with little humic substance contamination can be obtained.
  • a heat treatment can be performed during DNA extraction.
  • the heating conditions are 45 to 70 ° C. for 0.5 to 24 hours, preferably 6 (1 hour at TC).
  • Extraction of DNA from bacteria, fungi, etc. first requires the denaturation of cellular proteins with surfactants such as SDS and CTAB to destroy cell structures. It also has the ability to denature powerful proteins such as phenol and benzyl chloride. Organic solvents that also destroy the cell wall and cell membrane may be used.
  • SDS is a commonly used surfactant in molecular biology and is widely used for DNA extraction in microorganisms, animals and plants (Marniur 1961).
  • the method using CTAB was originally developed as a method for extracting DNA from plants (Murray & Thompson 1980) ', but it has also been partially used for extracting DNA from microorganisms (Velegraki et al. 1999). ).
  • CTAB also has the property of selectively binding and precipitating DNA at low salt concentrations (Murray & Thompson 1980).
  • CTAB is also known to be effective in removing polysaccharides from solutions (Sambrook 1989) and humic substances (Wilstrom et al. 1996, Zhou et al. 1996).
  • Guadizidine thiosinate is a powerful protein denaturant and is commonly used for RNA as well as DNA extraction (eg Chirgwin et al. 1979, Pitcher et al. 1989, Chomczynski & Sacchi 1987, Logemann et al. 1987, Ausubel et al. 2000).
  • sarkosyl is added as a surfactant.
  • the benzyl chloride method (Zhu et al. 1993) was developed as a simple and rapid method of using benzyl chloride to destroy cells.
  • Benzyl chloride reacts with polysaccharides, ie, cellulose, synthesized by bacteria, filamentous fungi, and plants as components of the cell wall, and OH groups in the micelle mouth to destroy cells.
  • DNA which is a water-soluble molecule, is extracted from the organic layer of benzyl chloride to the aqueous layer, and this method uses centrifugation to remove protein at the interface between the organic layer consisting of benzyl chloride and the aqueous layer.
  • Kits that use this method are also available (product names Isoplant, Nippon Gene, Japan).
  • Triton X100 is a nonionic surfactant that has a milder surface activity than ionic surfactants such as SDS and CTAB, and has a weak protein denaturing effect, thus maintaining its activity. It may be used for extraction of membrane proteins to be prepared as-is, or for maintaining or enhancing enzyme activity in enzyme reaction solutions such as PCR. Triton X100 does not inhibit the PCR reaction at concentrations up to 1%, and is used for simple DNA analysis in combination with colony PCR and enzyme reaction (Agei'sborg et al. 1997). In the present invention: The type of surfactant is not limited as long as DNA can be extracted. For example, SDS, TritonX-100, N-lauroylsarcosine sodium and the like are preferable, and SDS is more preferable.
  • the concentration of the surfactant used is 5.0% or less in the case of SDS, and is preferably from 0 ::! To 2.0%, more preferably from 0.5% to 2.0%, even more preferably from 0.5 to 1.0%.
  • the content is 5.0% or less, preferably 0.1% to 2.0%, more preferably 0.5% to 2.0%, and still more preferably 0.5% to L.0%.
  • a weak buffer having a pH of about 8.0 to 8.3 is usually used. This pH condition is considered to be applied to the extract because of the stability of DNA, and is the same pH as the TE buffer used to store the extracted DNA as a solution.
  • most soils in Japan are slightly acidic with a pH of about 5.5 to 6.5, and some volcanic ash soils and non-arofenic black pork soils have acidic soils with a pH of 4.5 to 5.5. Therefore, when extracting DNA from soil, not only the pH of the extract itself affects the amount of extraction, but also the pH of the extract is affected by the acidity of the soil, and the pH during extraction changes. Also need to be considered.
  • the pH of the whole extract is 7 or more, preferably 8.0 to 9.0, and more preferably around 8.6.
  • EDTA is used to prevent the DNA from being degraded by DNase released from cells, and its concentration is 1 to: L00 mM.
  • Tris-EDTA which is often used in the field of molecular biology, is also used.
  • EDTA in the extract is considered to be used mainly for the purpose of inactivating DNase, and its concentration is lOO mM, which is sufficient to achieve the intended purpose.
  • Volcanic ash soil a typical soil in Japan, is considered to be a soil to which DNA is easily adsorbed as described above.
  • Soil rich in amorphous aluminum, such as volcanic ash soil in Japan is scattered only in limited countries and regions, such as New Zealand, and is not distributed over a wide area of the world.
  • soils that are high in a-mouth fins were not considered, and therefore most conventional soil DNA extraction methods would have been developed without considering volcanic ash soils. Therefore, the setting of the EDTA concentration was also studied for soils other than volcanic ash soil.
  • EDTA which is a strong chelating agent for metal ions that form many polyvalent ions, is thought to form a complex with active aluminum, which causes DNA adsorption, in volcanic ash soil. It was suggested that concentrations of EDTA outside the specified range could be effective. In addition, it was considered that more DNA could be extracted by increasing the EDTA concentration even in soils other than volcanic ash soil if DNA adsorption to the soil occurred.
  • chelators with different complexing ability depending on the form of A1 and Fe in the soil, such as citrate-oxalic acid and pyrophosphate, are usually used. By using these chelators with different reactivities, the amorphous components of soil are selectively melted and quantitatively analyzed.
  • EDTA is a compound having an extremely excellent chelate stability constant for most metal ions among chelating agents. EDTA is used in DNA extraction and storage because DNase inactivates DNase by chelating the Mg ions required to maintain its activity.
  • the concentration of EDTA used as an extract in the present invention is, for example, 20 mM or more.
  • the EDTA concentration is 50 mM or more, preferably 100 mM to 600 mM, preferably 200 to 400 mM, and more preferably 300 mM to 400 mM.
  • the extract containing the above-mentioned concentration of EDTA is mixed with soil, and after beads-beating treatment, a high-concentration EDTA solution is further contained to bring the concentration to, for example, 600 to 1100 mM, preferably 600 to 800 mM.
  • Heat treatment can be used together in the elevated state.
  • the heating conditions are 45 ° C to 70 ° C for 0.5 to 24 hours, preferably 60 ° C for 1 hour. This allows higher yields of DNA to be extracted from soil.
  • the extract may also contain a buffer such as Tris-HCl.
  • concentration of Tris-HCl is, for example, 100 mM. Tris-HCl can be included in the DNA extract throughout the present invention.
  • the number of times of DNA extraction is not limited to one. After performing beads-beating on the DNA extract containing EDTA, centrifuge and collect the supernatant to obtain the DNA extract. The soil remains in the tube after collecting the supernatant. It is considered that this soil contains DNA that could not be extracted in one extraction operation and remained adsorbed on the soil.
  • the sampling process can be repeated multiple times.
  • the number of repetitions is, for example, 1 to 4 (the total number of times is 2 to 5).
  • Phosphate buffer is one of the representative buffers used in biological experiments.
  • many of the indirect extraction methods use phosphate buffer to separate microorganisms from soil (eg Torsvik et al. 1980). Buffers are often used (eg Ogram et al. 1987, Cullen & Hirsch 1998).
  • the phosphate concentration is set to 100 to 120 mM when a phosphate buffer is used for DNA extraction.
  • phosphate buffers cause more humic substances to elute than Tris-EDTA buffers, making their removal extremely difficult. It is said that Tris-EDTA buffer is better than phosphate buffer for DNA extraction.
  • the phosphate buffer that can be used in the present invention is not particularly limited, and examples thereof include a phosphate buffer such as a potassium phosphate buffer and a sodium phosphate buffer.
  • the concentration of the phosphate buffer used in the DNA extract is 250-2000 mM. In the case of volcanic ash soil, for example, it is 250 to 2000 mM, and in the case of non-volcanic ash soil, it is, for example, 250 to 1000 mM. (4-6) Combined use of high concentration EDTA and phosphate buffer
  • the concentration of EDTA is 100 to 800 mM, for example, 200 to 800 mM
  • the concentration of the phosphate buffer (for example, potassium phosphate buffer) is 250 to 2000 mM, for example, 250 to 1250 mM. be able to.
  • concentrations of EDTA and phosphate buffer can be kept lower than when each is used alone.
  • the combination to be used for volcanic ash soil is preferably 100 to 600 mM EDTA and the phosphate buffer is 250 to 1500 mM (for example, 250 to 1250 mM), and the combination to be used for non-volcanic ash soil is Preferably, the EDTA is 100-400 ⁇ and the phosphate buffer is 250-:! 250mM.
  • a combination of 400 mM EDTA and 750 mM phosphate buffer is most preferred. Since the combination of the above 400 mM EDTA and 750 mM phosphate buffer can provide almost the maximum yield of DNA for almost all soils, this combination can be said to be a so-called “universal buffer composition”.
  • the above-mentioned universal buffer composition may cause low molecular weight of soil DNA (20 to 7 kbp) depending on the soil.
  • soil DNA 20 to 7 kbp
  • the purpose can be sufficiently achieved even with a somewhat low molecular weight DNA.
  • extraction conditions EDTA and phosphate buffer Solution concentration
  • a soil sample can be mixed with an extract containing SDS or TritonX-100, EDTA and a phosphate buffer, and then heat-treated.
  • concentration of SDS or ritonX-100 is 5% or less
  • concentration of EDTA is 100-800 mM, for example
  • concentration of the phosphate buffer (for example, potassium phosphate buffer) can be used in the range of 250 to 2000 mM, for example, 250 to 1250 mM.
  • the heating condition is 45 to 70 ° C for 0.5 to 24 hours, preferably 60 to 1 hour. (4-7) Two-step method
  • extract I 5% or less of SDS (preferably 1% SDS) or a mixture of 5% or less of TritonX-100 and Tris'HCl buffer (hereinafter referred to as "extract I") is used.
  • Extract II After bead-beating the soil sample in the presence of DNA Extract I above, perform a simple high-speed centrifugation for a few seconds to collect the soil solution at the bottom of the tube, and then contain a high concentration of EDTA and phosphate buffer Prepare the extract (referred to as “Extract II”) and mix well.
  • the composition of Extract II is the same as that of Extract I above, for example,
  • the concentration of EDTA is preferably 400 to 800 mM, and more preferably the concentration of phosphate buffer is 750 to 1500 mM. Most preferably, the concentration of EDTA is 400 mM and the concentration of the phosphate buffer is 750 mM.
  • DNA is extracted by centrifuging the extract II, and DNA is recovered from the supernatant.
  • This improved method is called the “two-step improved method”.
  • the extract used in the first step should contain 5% or less of SDS (preferably 1% SDS) or 5% or less of Triton X-100, Tris-HCl buffer, 400 mM or less of EDTA, and 250 mM or less of phosphorus. It contains an acid buffer (referred to as "Extract III").
  • EDTA is preferably 300 mM
  • phosphate buffer is preferably 100 mM.
  • extract IV OOmM (preferably 400 to 600 mM) EDTA, 750 to 2050 mM phosphate buffer, or a mixture of the EDTA and the phosphate buffer.
  • the concentration of EDTA is 400 raM and the concentration of phosphate buffer is 750 mM.
  • extract the extract II after preparing the extract II in the two-step method when performing the above two-step method and the two-step improvement method, extract the extract II after preparing the extract II in the two-step method, and extract the extract IV after preparing the extract IV in the two-step method. It is also possible to extract soil DNA by heating the soil sample in the presence of liquid (eg, 60 ° C for 1 hour) to increase the recovery of soil DNA from the soil. Further, in the present invention, before performing the two-step method and the two-step improvement method, an extract containing 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer (hereinafter referred to as “extract V”) is used before performing the two-step method and the two-step improvement method.
  • extract V an extract containing 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer
  • the soil sample is heated (eg, at 60 ° C for 1 hour) or beads-beating, centrifuged after the extraction, and the supernatant is collected (referred to as “supernatant I”).
  • Add solution I or extract solution III perform beads-beating treatment, centrifuge the treated solution to obtain a supernatant (referred to as supernatant II), and collect soil DNA from supernatant I and supernatant II. it can.
  • a solution containing 100 to 800 mM EDTA and 250 to 2000 mM phosphate buffer was used.
  • the soil sample is heated (eg, 60 hours for 1 hour) or beads-beating in the presence of SDS, and SDS or Extract III is added to the treated solution, heat-treated, centrifuged, and centrifuged. It is also possible to recover soil DNA.
  • DNA can be extracted by combining heat treatment and beads-beating in the absence of SDS first (see (i) to (i) below).
  • a soil sample is heat-treated in the presence of a DNA extract containing 100-400 mM EDTA and 250-1500 mM phosphate buffer (referred to as “extract V”).
  • the heat treatment conditions are, as described above, 0.5 to 24 hours at 45 ° C to 70 ° C, preferably 1 hour at 60 ° C.
  • the extract V is preferably 400 mM in EDTA and 750 mM in phosphate buffer.
  • the extract V after the heat treatment is centrifuged to collect the supernatant.
  • a soil sample remains in the tube, and it is necessary to remove the DNA containing 5% or less of SDS, Tris-HCl buffer, 400 mM or less of EDTA, and 250 mM or less of phosphate buffer.
  • extract III is centrifuged to collect the supernatant, and DNA is recovered from the two collected supernatants.
  • the soil sample is beads-beated in the presence of DNA extract V containing 100-400 mM EDTA and 250-1500 mM phosphate buffer.
  • the extract V after beads-beating treatment is centrifuged to collect the supernatant.
  • the remaining soil sample is subjected to beads-beating treatment in the presence of a DNA extract IV containing 5% or less of SDS, Tris-HCl buffer, 400 mM or less of EDTA, and 250 mM or less of a phosphate buffer.
  • extract IV is centrifuged to collect the supernatant, and DNA is recovered from the two collected supernatants.
  • the first heat treatment of the soil sample is performed in the presence of a DNA extract containing 200-800 mM EDTA and 250-2000 mM phosphate buffer. Mix this with less than 5% SDS A second heat treatment of the soil sample. After centrifuging the extract after the second heat treatment, DNA is recovered from the supernatant.
  • the extract is preferably 400 mM EDTA and 750 mM phosphate buffer.
  • V Phosphate has a high DNA extraction effect. However, when used in volcanic ash soil, it is necessary to use extremely high concentrations, so care must be taken to reduce the molecular weight of DNA.
  • PCR cloning
  • sequencing cloning
  • hybridization a technique that uses PCR to generate cloning
  • gene expression tests include PCR, cloning, sequencing, hybridization, and gene expression tests.
  • the PCR reaction is an important and indispensable elemental technology for many gene analyses, and is also an essential operation for analyzing the microbial community structure based on genetic information.
  • the PCR product can be obtained using the extracted DNA as type III, and its success or failure can be used as an index to determine the purity of soil DNA. Yes (Tsai & Olson 1992, Watson & Blackwell 2000)
  • DNA extracts from soil include debris such as cell membranes and cell walls of microorganisms and plants, proteins denatured by surfactants, soil organic matter such as humic substances accumulated in soil itself, and heavy metals. It is included, and it is desirable to remove these contaminants as much as possible for subsequent analysis.
  • humic substances are known to strongly inhibit the PCR reaction even in trace amounts of nanograms' (Tsai & Olson 1992, Boon et al. 2002, Watson & Blackwell 2000). Therefore, it is important to remove this humic substance from the DNA solution as much as possible.
  • the DNA excision operation after agarose gel electrophoresis is to separate the macromolecule DNA and the lower molecular humic substance and recover only the DNA (Zhou et al. 1996, Kurien et al. 2001, Kurien & Scofield 2002, Chandler et al. 1997), but the cutting operation from agarose gel is extremely complicated.
  • the extract after beads-beating or heat treatment described above is mixed with CTAB and salt to purify DNA, or the extract after beads-beating or heat treatment is centrifuged. And mixing the supernatant after centrifugation with CTAB and salt to purify DNA.
  • Purification of DNA means removing contaminants other than DNA contained in the DNA extract.
  • the purification method of the present invention not only targets the extraction solution or the extracted DNA in the above-mentioned extraction step, but also purifies the DNA (or a solution containing DNA) extracted by a method other than the above-mentioned extraction method. Can also be purified.
  • the CTAB and any salt solution are mixed with an extract of soil DNA (made weakly acidic with a buffer having a buffering capacity on the acidic side), and then 45 ⁇ -70 (for example, (60 ° C) to remove protein by incubate and black mouth form.
  • the concentration of CTAB is 1-3%, preferably 2-3%.
  • DNA is more easily extracted from soil under alkaline conditions.
  • the extract contains highly buffered substances such as Tris-HCl, EDTA, and phosphate buffer, and the extract is kept alkaline. You. Therefore, it is necessary to neutralize these buffer capacities and make the pH weakly acidic. Therefore, it is preferable to use a salt having a buffer capacity on the weakly acidic side.
  • Salt means that a monovalent cation of 1.0 M or more can be added to the extract and the pH is 5.0 A substance that can be adjusted to a weak acidity of ⁇ 6.5.
  • Such salts include, for example, sodium chloride (NaCl), sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate.
  • concentrations of NaCl, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate and ammonium phosphate are 0.7 to 2.1M.
  • NaCl, sodium acetate, potassium acetate and ammonium acetate 1.0M or more is preferable, and for sodium phosphate, potassium phosphate and ammonium phosphate, 0.7M or more is preferable. All salts are 1.0 to 1.4M. Is more preferred.
  • the pH of sodium acetate, acetate acetic acid, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate is preferably 5.0 to 6.0.
  • Na + and monovalent cations, K +, ⁇ 4 + refers to such.
  • the salts are not limited to the above substances, and all salts that can add 1.0% or more of monovalent thione to the extract and can adjust the pH to 5.0 to 6.5 are included in the present invention.
  • humic substances can be removed from a soil DNA solution to a level that does not inhibit PCR by adding CTAB to the extract. Then, it is possible to search for a method suitable for sedimentation of the soil D ⁇ ⁇ and determine the optimal purification conditions in combination with the soil D ⁇ ⁇ extraction method developed above.
  • the concentration of CTAB is 2-3% and the concentration of salt is 1.4M.
  • Methods for separating and recovering DNA from a solution include a method of precipitating DNA, a method of adsorbing on a silica surface by a chaotropic effect, and a method of separating from a solution using magnetic beads or the like.
  • the most commonly used method is DNA precipitation, which utilizes the precipitation of DNA in the presence of a certain concentration of ethanol or 2-propanol. Since ethanol precipitates DNA at a concentration of about 70%, it is necessary to add 2-2.5 times the amount of ethanol to the extract, and when extracting on a mini-scale using a microphone-mouth tube, test ethanol precipitation. Low extractable volume Poor efficiency.
  • 2-propanol has a stronger effect of precipitating DNA than ethanol, and can be used for miniscale DNA extraction because it can precipitate DNA in 6/10 equivalent of the extract.
  • the DNA is recovered by weakening the acid side with an acetic acid or phosphate buffer having a buffering capacity on the acidic side, and then precipitating the soil DNA.
  • the precipitation includes both (i) the step of precipitating the sample after the extraction and purification steps, and (ii) the step of precipitating the sample after the purification step.
  • the substance used for the precipitation includes 2-propanol, ethanol or polyethylene dalicol, but polyethylene glycol (PEG) is preferred.
  • the concentration of PEG is 10-15%, preferably 12% (PEG concentration in solution: 5-7.5%).
  • polyethylene glycol 8000 (PEG) is also used.
  • PEG is commonly used to remove primers from PCR products before the sequence reaction (Kusukawa et al. L990).
  • the PEG solution has high selectivity for the substance to be precipitated, and does not precipitate RNA that is structurally very similar to DNA. Even with the same DNA, short-chain primers did not precipitate, indicating their selectivity in size (Pai thanker & Prasad 1991, Lis 1980, Sambrook et al 1989).
  • PEG is often used in research to extract DNA from samples that are easily contaminated with soil or compost. (Ogram et al.) 1987, Porteous et al 1997, Howeler et al 2003, LaMontagne et al 2002).
  • the present invention can preferably employ one of the following two means.
  • soil DNA can be obtained even if each step is used independently without performing all of them, or extracted by a method other than the above extraction method. Purification of soil DNA is possible. 6. DNA extraction, purification and / or recovery kit
  • the present invention provides a kit for extracting, purifying, or recovering the above soil DNA.
  • the kit for extracting DNA from environmental samples contains 5% or less of a surfactant or a combination of the surfactant and beads for beads-beating. That is, the DNA extraction kit of the present invention has a basic composition of a surfactant (SDS, CTAB, Triton X-100 or E. laurel sarcosine sodium), and the beads used for beads-beating. , EDTA, phosphate buffer, alkaline buffer (Tris buffer such as Tris-HCl), pH adjuster, etc.
  • the DNA extraction kit of the present invention can adjust the pH to 7.0 or more with an alkaline buffer. '
  • the concentration of EDTA can be arbitrarily selected from the range of 50 mM to: L200 mM.
  • concentration of the phosphate buffer can be arbitrarily selected from the range of 50 mM to 3000 mM.
  • the DNA extract should be prepared so that the concentration can be set appropriately according to the soil used.
  • concentration can be divided stepwise.
  • the SDS may be adjusted in several steps from 0.1% to 2.0%, and one type of high-concentration SDS is prepared and diluted with a diluent so that the experimenter can adjust the concentration as desired. You can also make it available.
  • EDTA or phosphate buffer be sure to include those whose concentration is adjusted stepwise such as 50 mM, 100 mM, 200 mM, 300 mM, or 400 mM, or prepare high concentration (for example, 1 M). It can be prepared so that it can be diluted to any concentration with a diluent.
  • a combination of 400 mM EDTA and 750 mM phosphate buffer can be included in the kit as a universal buffer composition.
  • the present invention relates to a method for purifying DNA from an environmental sample, comprising a salt solution containing a pH buffer having an acidic pKa, a cationic surfactant, or a mixture of the salt solution and a cationic surfactant.
  • a kit for purifying DNA from an environmental sample, comprising a salt solution containing a pH buffer having an acidic pKa, a cationic surfactant, or a mixture of the salt solution and a cationic surfactant.
  • the pH buffer having a pKa on the acidic side include an acetate buffer, a phosphate buffer, a hydrochloric acid buffer and a sulfate buffer.
  • This kit can contain a cationic surfactant (such as CTAB) for DNA purification and the above salt. It is possible to adjust the pH at the time of purification to less than 7.0 by using the pH buffer having the pKa on the acidic side mixed with a salt.
  • the present invention provides a kit for recovering DNA from an environmental sample, which contains an alkaline buffer (for example, Tris buffer).
  • the kit can also include 2-propanol, ethanol or PEG for use in precipitation.
  • the pH of the DNA solution at the time of recovery can be adjusted to 7.0 or more.
  • the present invention provides a DNA extraction and purification, a DNA extraction and recovery, a DNA purification and recovery, A kit set for DNA extraction, purification and recovery is also provided.
  • obtaining DNA such a combination of two or more of DNA extraction, purification and recovery is referred to as obtaining DNA. Therefore, DNA acquisition (extraction, purification, recovery, or a combination thereof) can be performed by using a combination of each kit. Wear.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • concentration range in the present invention is described in Examples, the concentration range is an example and is not limited at all without departing from the object of the present invention.
  • Table 1 Physicochemical properties of test soils Acid oxalate extraction Pyrophosphate extraction alofen pH pH (KCI) Total nitrogen total ash tea (mg / g soil) (mg / g soil) (mg / g soil) (mg / g soil) (mg / g soil)
  • a 7001 aqueous layer was collected, 1/10 volume of 3 M sodium acetate was added, 6/10 volume of 2-propanol was added, and the mixture was centrifuged at 20000 X g to precipitate DNA. After centrifugation, the DNA precipitate was washed with 70% ethanol, dried, dissolved in 100 Hi TE buffer, and used as extracted DNA. Because the extracted soil DNA solution was not purified, substantial amounts of humic substances were mixed. As it was, it was not possible to measure the amount of DNA by absorbance. Therefore, in order to quantify the DNA without the influence of humic substances, the extracted DNA was electrophoresed on a 1% agarose gel, and the humic substances that traveled a long distance due to small molecules were sufficiently separated from the DNA.
  • Table 2 shows the buffer composition and physical conditions of each surfactant. Table 2 Overview of buffer solution S and surfactants
  • Beads composition Use 2 ml tube with screw cap.
  • the beads composition was prepared by referring to the knowledge of Burgmann et aL (2001). All experiments were performed in triplicate.
  • Figure 2 shows the results for the amount of soil DNA extracted. Irrespective of the heat treatment and beads-beating treatment, the extraction efficiency of soil DNA was better when SDS and Triton X100 were used, and there was no DNA degrading that could be attributed to the surfactant. The combination of beads-beating treatment and SDS resulted in a higher extraction volume than the other combinations. It was possible to extract DNA close to g.
  • CTAB and guanidin thiosinate failed to extract sufficient amounts of DNA from soil samples, but the extract was almost colorless and very effective in preventing humic substances from being extracted from soil. It became clear.
  • the beads-beating treatment was able to perform extraction in a very short time, and the yield of DNA was higher than that of the heat treatment. Also, it was presumed that the heat treatment extracted more humic substances than the beads-beatin treatment, and it was difficult to remove this humic substance.
  • the size of the DNA estimated from the electrophoresis gel, which was obtained by beads-beating, was slightly reduced to a molecular weight of 20 kbp to 7 kbp, mainly about 20 kbp. On the other hand, the DNA obtained by the heat treatment had a size of 23 kbp or more, and almost no low molecular weight was observed.
  • Example 2 based on the results of Example 1, the optimal concentration of SDS used in the extract of soil DNA was examined.
  • the level of SDS concentration was set from 0% to 2%, The optimal SDS concentration conditions for the extraction of DNA were examined.
  • the experimental conditions other than the SDS concentration were the same as in Example 1 except that the extract was 100 mM Tris-HCl lOOmM EDTA (pH 8.6). All experiments were performed in triplicate.
  • the composition of the extract was' 100 mM Tris-HCl 300 mM EDTA (pH 8.6), and the same conditions as in Example 1 were used except that the SDS concentration was changed from 0% to 2%. Then, the optimal SDS concentration for soil DNA extraction was examined. Details regarding the EDTA concentration will be described later. In the experiment, six types of soils shown in Table 4 were used as test soils.
  • Table 4 Physicochemical properties of test soils Arophen (mg / g soil) Al Yayoi field compost continuous use soil Arofen black pork soil 35.73 Tochigi agricultural test forest soil Arofen black pok soil 23.63 Tohoku University forest soil Non-arofen black po H ⁇ 20.82 Saitama agricultural test field soil Gray lowland soil 1.22 Osaka agricultural test field soil Gray lowland soil 0.59 Hyogo agricultural test field soil Gray lowland soil 0.56
  • Yayoi Farm Compost Continuous Use Area and Tochigi Agricultural Test Forest Soil Tohoku University Forest Soil is volcanic ash soil, Osaka Agricultural Test Field Soil and Hyogo Agricultural Test Field soil, Saitama Agricultural Test Field soil is non-volcanic ash soil (alluvial soil) It is. The experiment was performed in triplicate.
  • the extract used was a Tris-EDTA buffer extract. 1.2 ml of an extract consisting of 100 mM Tris-HCl / 100 mM EDTA / 1% SDS with pH set to 9.0, 8.0, 7.0 and 6.0 was added to 0.5 g of soil, and beads-beating treatment was performed. The DNA was extracted by DNA extraction, and after the protein was removed, the DNA was recovered and quantified.
  • the amount of soil DNA extracted and the concentration of Al, Fe, Ca, and Mg dissolved in the crude DNA extract (supernatant centrifuged after beads-beating) were analyzed. These metal elements were predominant in soil and were considered to be the elements that could cause DNA adsorption by soil.
  • the amount of soil DNA extracted was quantified in the same manner as in Example 1, and the metal elements were appropriately diluted and quantified by ICP emission analysis (Seiko SPS-1200). The experiment was performed in triplicate.
  • Figure 6 shows an example of the effect of EDTA concentration on soil-extracted DNA.
  • Figure 6 shows an example of a photograph of DNA stained after agarose gel electrophoresis. The result is shown.
  • Figures 8A to 8C show the results of quantification of metal elements in the crude extracted DNA solution. Metal elements that are considered to be chelated and extracted by EDTA are considered to be EDTA-metal element complexes. The ratio of EDTA-metal element complex formation relative to the total amount of EDTA, that is, the ratio of actual EDTA used, is calculated as the complex formation rate of each of the four elements. EDTA metal element complex formation combining the four elements The rate was determined. This is shown in FIGS.
  • the amount of DNA extracted in all three types of soil increased as the EDTA concentration in the extract increased.
  • the EDTA concentration was 100 mM or higher, and more high-molecular DNA was obtained by using 300 mM or higher.
  • phosphate groups which are presumed to be the main cause of DNA adsorption to soil, are present in each of the DNA constituent units, nucleotides, and are part of the phosphate groups of long DNA molecules. It is thought that DNA is not released into the solution due to the adsorption of the soil particles.
  • DNA is also extracted from the Yayoi field compost continuous use soil with relatively low concentration of EDTA.
  • Most of the metal elements extracted from the soil of the Yayoi Field Compost Zone are Ca, and A1 is low.
  • the Yayoi field is a volcanic ash soil
  • Ca is accumulated in the soil due to long-term continuous use of compost, indicating that the metal element composition contained in the soil was greatly affected.
  • A1 was significantly affected by the EDTA concentration, with a high complexation rate until the EDTA concentration reached 100 mM, and A1 was eluted from the soil corresponding to the EDTA concentration. 'Also, even at concentrations of 100 mM or more, the amount of A1 eluted gradually increased.
  • the EDTA concentration was 50 mM
  • the soil DNA began to be extracted at an EDTA concentration of 100 ⁇ . Above that, the DNA extraction amount increased in proportion to the EDTA concentration. ing.
  • non-volcanic ash soil soil DNA was extracted even when the EDTA concentration was less than 50 mM. In all three types of soil, the yield was maximum at EDTA concentrations of about 100 to 200 mM, and at higher concentrations, the DNA yield was conversely reduced. Looking at the amount of metal ions extracted from non-volcanic ash soil, most of it is Ca. In these soils, the amount of Ca that is considered to be extracted by EDTA cannot be fully extracted.Even in the case of low-concentration EDTA of 50 mM or less, that is, even when Ca is not sufficiently removed from the soil. Sufficient DNA yields were obtained (see Figures 7 and 8A), indicating that Ca contained in the soil did not adsorb enough to affect DNA extraction.
  • Saitama Agricultural Trial contains a small amount of volcanic ash soil, and the amount of A1 eluted is larger than that of Osaka Agricultural Experimental Soil and Hyogo Agricultural Experimental Soil. There was almost no difference in the elution amount of A1 even when the EDTA concentration was increased.
  • EDTA at a low concentration of about 10 to 20 mM chelates a metal element at a rate of 100%, and at a high concentration of 300 to 400 mM, about 20% of the EDTA chelate a metal element. Therefore, even if high-concentration EDTA is not used from the initial stage of extraction, it is possible to extract soil DNA by repeating extraction with low-concentration EDTA solution or increasing the ratio of extract to soil. Was thought.
  • a point to keep in mind when using EDTA is that when EDTA exceeds 400 mM, the molecular weight of DNA was reduced. In a solution with a pH of about 8.3, about three sodium ions exist as one counter ion per EDTA molecule.
  • the 400 mM EDTA solution is a high concentration salt solution corresponding to a 1.2 M NaCl solution in ion intensity. Therefore, it was thought that DNA was electrically neutralized due to the sodium ions contained in such a large amount, and this effect might have caused the beads to be easily sheared during beads-beating treatment.
  • EDTA is a compound with an excellent chelate stability constant for most metal ions (Catalog 22nd Edition, Dojin Chemical Laboratory Co., Ltd.). This means that, even at very low concentrations, most EDTA will complex with the target metal ion.
  • the amount of extract was 1.0 ml for 0.5 g of soil. By repeating extraction and increasing the ratio of extract to soil, even if the concentration of EDTA solution is low, amorphous A1 in the target soil may be removed.
  • the EDTA concentration was set at several levels, and the total DNA obtained when DNA was repeatedly extracted several times from the same soil was determined. The yield was examined.
  • the test soil used was three types of soil: Yayoi field compost continuous use soil, Tochigi agricultural test forest soil, and Tohoku University forest soil.
  • the basic extract composition is the same as in the previous section, using Tris-HCl 100 mM and SDS 1%, and using EDTA concentrations of 0 mM, 50 mM, 100 mM, 200 mM, 300 mM and 5 mM extract.
  • 0.5 g of soil was dispensed into a tube with a screw cap containing beads lg, and 1.0 ml of the extract was added, and beads-beating treatment was performed at a strength of 5 m / sec for 30 seconds.
  • the extract was re-added at each stage of the extraction, and that it was newly obtained.
  • the amount of soil DNA and the amount of leached metal elements are described by the following formulas according to the above operation.
  • the extraction amount of DNA or metal element at the nth time is Xn (value converted to the yield of DNA or metal element per lg of soil, the unit is g / g soil).
  • New amount obtained from the second extraction X2-X1 X 1/2
  • New amount obtained by the third extraction X3-X2X 1/2-X1 X 1/4
  • New amount obtained from the fourth extraction X4-X3 X 1/2-X2 X 1/4-XI X 1/8
  • Figure 10A shows the yield of each step of the soil DNA (converted to the yield per lg of soil) and the total yield of soil DNA extracted five times from the soil obtained by repeated extraction using EDTA extracts at different concentrations.
  • Figure 12 shows the amount of soil DNA that was considered to have been extracted by the added EDTA during repeated extraction (values according to the above calculations; calculation examples are shown in Figure 11).
  • Figures 13A and 13B show the amounts of metal elements considered to have been extracted by the chelation of the added EDTA.
  • the amount of soil DNA obtained by extracting five times was the highest in all three types of soils containing the extract containing 300 mM EDTA (Figs. 10A to 10C).
  • the recovered solution (the part contained in the gap between the soil and beads after centrifugation) was recovered little by little and the amount of extraction increased, and the DNA adsorbed on the soil was separated and recovered. It didn't seem to be what I was doing.
  • the absolute amount of EDTA used for soil extraction is the same, for example, when the absolute amount of EDTA is 300 mol per 0.5 g of soil, that is, when the first extraction amount using 300 mM EDTA extract and 200 mM EDTA
  • approximately the same amount of soil DNA was obtained in the Yayoi field compost continuous use soil and the Tohoku University forest soil.
  • the absolute * of EDTA was 200 nmol or 100 nmol.
  • this calculation did not apply to the Tochigi Agricultural Forest Soil, and there was a large difference in the amount of extracted DNA even if the absolute amount of EDTA was the same. That is, even with the same amount of EDTA, the amount of DNA extracted at the first high concentration was larger than that of the first extraction, and even with repeated extraction, it did not reach the amount of DNA extracted at a high concentration at one time.
  • the Yayoi field compost continuous use soil and Tohoku University forest soil have a relatively weak effect of amorphous Ai that adsorbs DNA, and a certain amount of soil DNA is obtained with a relatively low concentration of about 50 mM EDTA solution.
  • amorphous A1 is strong in Tochigi agricultural test forest soil, and EDTA of 200 mM or more is required for DNA extraction, and it is difficult to dissociate and recover DNA once adsorbed on the soil. It was thought there was.
  • A1 that caused adsorption was hardly extracted from the soil of the Yayoi compost plot.
  • Ca was accumulated in the soil in large amounts due to long-term use of compost, and the amount of A1 in the soil was smaller than that of the control area where no compost was used, and the soil was strongly affected by Ca.
  • Another characteristic of this soil is that it is rich in amorphous Si, and most of the amorphous A1 forms aluminosilicate (or arophen with a high silicon ratio) with this Si. I thought it was possible.
  • A1 was extracted in large amounts from Tohoku University forest soil.
  • This soil is a special soil among the non-arofenous black pork soils of volcanic ash soil, which is rich in pyrophosphate-extracted A1 (that is, in the form of humus-A1 complex) and low in arofen content.
  • Can be A1 in the humus-A1 complex accounts for about half of the total amount of amorphous A1 in soil, and many A1s are complexed with humic substances.
  • Tochigi forest soil is also rich in pyrophosphate extract A1, but also rich in arofen.
  • repeated extraction in this soil did not increase the DNA yield, suggesting that the newly added EDTA from the second time onward may have little effect on the elimination of DNA adsorption.
  • A1 was newly removed by EDTA from the soil added after the second time.
  • A1 that causes adsorption soil where DNA is gradually extracted as A1 that causes adsorption is removed by repeated extraction
  • Tochigi Agricultural Forest soil soil where DNA extraction does not increase even if ⁇ is removed
  • the forest soil of Tohoku University is a non-arofenous black pork soil, in which most of the amorphous A1 is a humus-A1 complex, and the content of arophene is low as compared with the degree of apofenic black pork. Therefore, in A1 that adsorbs soil DNA, in addition to A1 in the humus-A1 complex, A1 in the arophen state exists, and EDTA mainly eliminates the adsorption of DNA by humus. - ⁇ It was presumed to be A1 of the complex.
  • Example 6 DNA extraction from soil from which DNA is not extracted even with high-concentration EDTA From the results so far, the biggest cause of the difficulty in extracting DNA from volcanic ash soil by the conventional extraction method is the adsorption of DNA by soil. Was strongly suggested. Therefore, in order to further examine the optimal concentration of EDTA that eliminates DNA adsorption, soil that is considered to have strong DNA adsorption is targeted, and after beads-beating, high-concentration EDTA is added again, and heat treatment with high-concentration EDTA is performed. The method and conditions for recovering DNA adsorbed on soil by using a combination of these were investigated.
  • the soil in the control section of the Yayoi field was used for the test. The following three points were considered.
  • the EDTA concentration of the first extract extract at the time of beads-beating
  • the EDTA concentration of the DNA recovery solution the EDTA solution to be re-added
  • the treatment after adding the recovery solution stirring or heating.
  • Table 7 shows the combinations of the treatment contents and the extracts and recovered liquids.
  • EDTA forms a 1: 1 complex with A1. Assuming that 1 ml of a 1000 mM EDTA solution reacts 100%, it is possible to clean lmniol, that is, 27 mg of A1. On the other hand, amorphous A1 is approximately 100 mg in 1 g (dry soil) of a volcanic ash soil with much amorphous components.
  • 0.5 g of soil contains up to about 40 mg of amorphous A1, and if 1 ml of 1 M EDTA solution is used as the recovery solution, almost all This is because EDTA that can dissolve amorphous A1 is used.
  • humic substances are stabilized by forming a complex with amorphous A1 in soil by the same principle as the adsorption of DNA to soil. Since EDTA gradually removes amorphous A1 that has retained humus and other soil organic matter during the heat treatment, the aggregates and humic substances retained inside the soil particles are gradually eluted into the extract. To DNA samples There is a problem that humus is often mixed.
  • Phosphoric acid was added to the extract at various concentrations, and the amount of soil DNA extracted was measured.
  • alofenic materials were used as the volcanic ash soil: the Yayoi field control plot soil, the Tanashi farm uncultivated land soil, and the Tochigi agricultural test forest soil, for which DNA extraction was difficult even when highly concentrated EDTA was used as the test soil.
  • Black alluvial soil and three types of alluvial soil were used as non-volcanic ash soil: Saitama Agricultural Experiment Station Soil, Grassland Experimental Station Permanent Grassland Soil, and Hyogo Agricultural Experiment Station Soil (Table 8).
  • the conditions for beads-beating were set in the same manner as in Example 1.
  • the extract composition 100 mM Tris-HCl I 1% SDS (pH8.3) as the base, to which ⁇ 2 ⁇ 4 the ( ⁇ 8.3) 0, 50, 100, 250, 500, 750, 1000, 1250, 1500, Extraction buffer solutions were prepared to which the concentration was 1750 and 2000 mM, and 1.2 ml each was added to 0.5 g of soil to extract soil DNA.
  • the permanent grassland soil at the grassland test site showed a tendency to extract the volcanic ash soil type (Fig. 15A).
  • the grassland test site is located in the Nasu volcanic belt, and most of the soil in this area is originally arofenic black pork soil.
  • the perennial grassland which is the collection site, loses its volcanic ash layer due to river erosion, and the original stratum that was covered by volcanic ash appears on the ground surface and is the base material of the current topsoil. Therefore, it shows the properties of non-volcanic ash soil while located in the volcanic zone.
  • EDTA Comparing EDTA and phosphoric acid at the same molar concentration, except for Tochigi Agricultural Forest and Grassland Experimental Grassland Grassland Soil, EDTA was more effective in extracting DNA from the soil, but it had higher phosphate solubility and was more soluble in water.
  • the solubility of EDTA is 1.5 M (pH 8.3) at the maximum, whereas the solubility of potassium phosphate is as high as 3 M. Therefore, it can be used at a much higher concentration than EDTA.
  • phosphate ions were used at high concentrations, it was possible to extract more than twice the maximum yield of DNA obtained using EDTA from soil in the control plot of Yayoi field and uncultivated soil in Tanashi farm.
  • the phosphate buffer is excellent in DNA yield.
  • the following items need to be considered.
  • Example 7 Six types of soils shown in Example 7 were used.
  • EDTA and phosphoric acid were used in a combination of lOO mM Tris-HCl 1 1% SDS and the combinations shown in Table 9 Nos. 1 to 16.
  • the extraction conditions and DNA quantification conditions are the same as in Example 6.
  • Figures 16A-B show the results of soil DNA extraction. Compared to using EDTA and phosphate alone, a high DNA extraction effect was obtained even at a lower concentration in the mixed solution, and a complementary effect of EDTA and phosphate on soil DNA extraction was observed. In addition, in non-volcanic ash soil, a decrease in DNA yield was previously observed with an increase in EDTA concentration.However, when EDTA and phosphate buffer were combined, the decrease in yield was high even at high EDTA concentrations. Was kept very low. ⁇
  • EDTA-phosphate buffer The advantage of the combined use of EDTA-phosphate buffer is that, in part, if one wants to obtain the same DNA yield, the concentration of each can be kept lower than the concentration when extracting alone. Second, the concentration at which the maximum yield of DNA is obtained also varies slightly depending on the soil, but combining 400 mM EDTA with 750 mM potassium phosphate will yield the maximum yield of DNA from most soils. (Figs. 16A-B). This means that the composition of the universal DNA extract was determined regardless of the type of soil. At this concentration, it was not necessary to perform a dilution operation to reduce the salt concentration during subsequent DNA purification and precipitation operations.Therefore, the above composition should be applied to the soil DNA extraction buffer in the future. did.
  • This universal buffer composition provides the maximum yield from almost all of the soil tested.
  • the soil shown in Table 10 was used as the test soil.
  • the extract with a concentration of 400 mM EDTA and 750 mM phosphate gave the highest DNA recovery, as was the case with the extract containing high concentrations of EDTA and phosphate buffer from the beginning.
  • a mixed solution of 400 mM EDTA and 750 mM phosphoric acid prevents DNA released from the lysed microorganisms from adsorbing on the soil, The effect of recovering the adsorbed DNA was also considered to be the highest in the range of the combinations examined.
  • the use of a buffer solution with an upper limit concentration that does not cause the lowering of DNA in the extract during beads-beating was examined. That is, the final concentration of the DNA extract was fixed with 400 mM EDTA-750 mM phosphoric acid, beads-beating was removed by flash centrifugation with 8001 extract at the concentration shown in Table 12, and the concentration shown in Table 12 was removed. 4001 of the recovered solution was added and stirred to separate the DNA from the soil, and an improved method for recovery was studied.
  • the extract with the highest yield while suppressing DNA depolymerization that is, a buffer containing 300 mM EDTA and 100 mM phosphoric acid, was used as the extract during beads-beating treatment.
  • Use (1st step) then add a high concentration of EDTA-phosphate buffer, stir and recover the DNA adsorbed on the soil (2nd step).
  • the recovery rate of DNA and the amount of humic substances precipitated together with DNA were measured for the method of DNA precipitation using three types of 2-propanol, ethanol and PEG.
  • humic substances have a visible light absorption of 400 nm or more.
  • the humus analysis method (Yamamoto 1997) defines the absorbance at wavelengths of 400 nm and 600 nm as the amount of humus in the soil. Therefore, in order to estimate the amount of humic substances mixed into the soil DNA solution, these two wavelengths were used. The absorbance was measured. UV / VIS Spectrophotometer V-550 (JASCO) was used for the measurement.
  • the recovered amount of soil DNA is shown in FIG. 20A, and the absorbance of the soil DNA solution at 400 nm and 600 nm is shown in FIG. 20B.
  • PEG has the advantages of high DNA recovery rate, low humic substance contamination, and the ability to remove RNA. Therefore, this method was adopted as a method for precipitating soil DNA.
  • this method was adopted as a method for precipitating soil DNA.
  • the PCR reaction was performed on the 16S I 'RNA gene using the soil DNA obtained by this method as type I, almost no amplification was observed, and further contamination of the humic substances was observed. Needed to be removed
  • Soil DNA was extracted from the Yayoi field control plot soil and the Tochigi agricultural test forest soil by beads-beating treatment using 400 mM EDTA / 750 mM phosphoric acid / 1% SDS. After protein removal, soil DNA was precipitated and recovered using equal amounts of 10, 11, 12, 13, 14, and 15% PEG solutions, and the relationship between the recovered amount and the PEG concentration was examined. The PEG concentrations in the solution during DNA precipitation were 5, 5.5, 6, 6.5, 7, and 7.5%, respectively.
  • CTAB was unable to extract soil DNA, but the solution after extraction with CTAB was very transparent. It was shown that the removal of humic substances was extremely effective. Zhou et al. (1996), Porteous et al. (1997), and Wilstrom et al. (1996) report that CTAB is effective in removing humic substances. Therefore, we attempted to prepare PCR DNA soil DNA by combining the simple purification of soil DNA with CTAB and the precipitation with PEG as described in the previous section.
  • Example 10 As the test soil, three types of soil were used as in Example 10, namely, Tochigi Agricultural Forest Soil, Tohoku University Forest Soil, and Saitama Agricultural Test Field Soil.
  • 100 mM Tris-HCl I 300 mM EDTA I 1% SDS extract was used for extraction of soil DNA.
  • 100 mM Tris-HCl I 300 mM EDTA I 1% SDS extract was used for extraction of soil DNA.
  • a bead-beateing treatment was performed and centrifuged to obtain a crude extract.
  • the crude extract 3001 was used in four stages of final CTAB concentrations of 0%, 1%, 2%, and 3%, and a NaCl concentration of 0.7%.
  • M, 1.4 M, and 2.1 M were added in one of three combinations.
  • the extract was mixed well, an equal amount of black-mouthed form was added, and the protein was removed.
  • the aqueous layer was recovered and the 20% 6/10 equivalents of PEG were added, and soil DNA was recovered and dissolved in TE buffer.
  • the humic substances combined with CTAB are collected and removed in the denatured layer during protein removal by black-mouthed form.
  • the amount of soil DNA recovered was quantified, and the absorbance at 400 nm was measured for humic substances in the same manner as in 2-4-2.
  • Figure 22A shows the amount of soil DNA recovered.
  • Figures 22B to 22C show the absorbance at 400 mn of the soil DNA after the purification operation.
  • CTAB is a cationic surfactant. It binds quickly with hydrophilic groups to SDS, which is an anionic surfactant, and forms micelles or salts with hydrophobic groups facing outward and precipitates.
  • CTAB when CTAB is used at 1%, most of it is consumed in the reaction with SDS remaining in the solution, and the amount of CTAB used to remove humic substances is reduced, resulting in higher absorbance. It was considered.
  • Use of more than 2% of CTAB means that after the remaining SDS has been removed by CTAB, there is a sufficient amount of surplus CTAB, and humic substances are removed to remove humic substances, resulting in low absorbance. It was considered.
  • Zhou et al. (1996) have already considered adding CTAB to the extract, but use 2% SDS for 1% CTAB. It is presumed that CTDS has lost its function due to SDS, and that humus has not been sufficiently removed.
  • Example 13 Comparison of soil DNA yield between the method of the present invention and the existing method DNA was extracted from various soils using the method of the present invention and the existing method, and the yield of soil DNA was compared.
  • Previous methods include the method of Zhou et al. (1996) and two methods of Cullen & Hirsch (1998), as well as Bio101 Fast DNA spin kit (Qbio, USA) and UltraClean TM Soil DNA kit (MoBio, USA) Soil DNA was extracted by the method using two kinds of kits. Soil DNA extraction was performed according to each protocol.However, Cullen & Hirsch (1998) and UltraClean Soil DNA kit (MoBio USA) required special equipment for crushing with beads. For both, extraction was performed by substituting Fast Prep FP101 (Qbio, USA) with a treatment at an intensity of 5 m / sec for 30 sec.
  • the suspension of the soil and the extract is centrifuged to precipitate the soil (or a mixture of soil and beads if beads-beating is used), and the extract is collected. At this time, unextracted extract remains in the soil and in the gaps between beads. 'The amount of this extract recovered differs depending on the method of purification. When calculating the extraction efficiency from soil, it is assumed that the supernatant contained by centrifugation and the solution contained in the space between the soil and beads contain the same concentration of DNA. The amount was calculated by converting to the amount corresponding to the total amount of the added extract.
  • Amount of DNA extracted per gram of soil Amount of precipitated and recovered DNA X 1250/1000 X 1200/750 X 2
  • DNA was extracted from 12 kinds of soils by the above four kinds of original methods, which are examples of the method developed in the present invention.
  • FIG. Figure 24 shows the size of DNA extracted by the four methods.
  • Figure 25 shows the amount of soil DNA extracted by the original IStep method, the 2-step heating method, and the typical methods already used.
  • Soil DNA obtained by the original I Step method had a molecular weight of 20 to 7 kbp
  • soil DNA obtained by the original 2 Step method, the original 2 Step heating method, and the original LCB method had a molecular weight of 20 kbp. It was kept at a high molecular weight of kbp or more, and underpolymerization was suppressed.
  • the original IStep method and the 2Step heating method of the present invention were able to extract more soil DNA than the existing methods.
  • soil DNA can be obtained only from the arophenic black pork soil and the grassland of the grassland test site by the method of Zhou et al. (1996), and even in that case, the yield of DNA is low. It was low.
  • Non-volcanic ash soils and Tohoku University forest soils were also obtained by conventional methods except for the UltraClean TM Soil DNA kit.
  • the amount of extraction by Bio 101 FastSpinKit was large, and soil DNA was also extracted by the method of Cullen & Hirscli (1998).
  • the amount extracted by the method of Zliou et al. (1996) was small even in non-volcanic ash soil, and this difference in yield was considered to be caused by the presence or absence of heat treatment and bead-beating treatment.
  • polymer soil DNA of 23 kbp or more was obtained.
  • the original method of the present invention can extract soil DNA from various soils with high yield, and does not use the original method from soil rich in arophenic form A1. And found that sufficient or no soil DNA was obtained.
  • the soil DNA content is completely different, even if the final DNA solution volume is constant. Therefore, based on the quantified soil DNA concentration, the purity of the soil DNA was tested by testing the success or failure of the PCR reaction by using the soil DNA in three stages of 100 ng, 50 ng, and 10 ng.
  • the soil DNA studied was obtained by the original one-step method and the original two-step heating method.
  • the crude extract after bead-beating is colored brown or black due to the incorporation of humic substances, and it is considered that the amount of humic substances mixed is greater than in the other two methods. This is because it was considered suitable for examining the efficiency of humic substances removal by purification.
  • PCR was performed in triplicate, and the reaction conditions and primers used were as follows.
  • the composition of the reaction solution is as follows.
  • the reaction conditions were as follows: a reaction was first performed at 94 ° C for 2.5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, and elongation at 722 minutes as one cycle. The reaction was carried out at 72 for 10 minutes.
  • 26A to 26B show the results of the soil DNA purity test by PCR. Except for the two types of soils with high accumulation and contamination of humic substances, that is, except for Tochigi Agricultural Forest and Tohoku University forest soils, the soil DNA obtained by the original IStep method was used in a 50 l PCR reaction solution. Using 50 ng: PCR reaction was successful. Similar results were obtained with the soil DNA obtained by the original 2Step heating method. In Tochigi agricultural test forest soil and Tohoku University forest soil obtained by the original 1 Step 'method, PCR was successful when 10 ng soil DNA was changed to a cylin type.
  • composition of soil DNA obtained by various methods of the present invention was compared and examined by PCR-DGGE method.
  • the V3 region of the 16S rRNA gene was amplified by PCR and analyzed by DGGE.
  • the conditions of the PCR reaction and the primer and DGGE conditions used are shown below.
  • the reaction conditions are as follows: First, the reaction is performed at 94 ° C for 2.5 minutes, and then the denaturation at 94 ° C for 30 seconds, annealing for 30 seconds at 55 ° C, and the elongation reaction at 72 ° C for 1 minute are performed for 24 or 30 cycles. The reaction was finally performed at 72 ° C for 10 minutes.
  • the composition of the reaction solution is as follows.
  • the soil DNA solution obtained by the four original methods and the method of Zhou et al. (1996) is approximately 5-50 ng / l. Using this 10 to 20 g as type III, 24 cycles of PCR reaction were performed. In the other existing methods, sufficient amounts of soil DNA could not be obtained from the volcanic ash soil. However, in order to adjust the conditions, 30 cycles of PCR were performed using 11-types for each type. Soil DNA obtained from non-volcanic ash soil by the previous method was subjected to 24 cycles of PCR using the same type of lll.
  • FIGS. 27A to 27E show the results of DGGE analysis of soil DNA obtained by each method.
  • the amplification products obtained by these PCR reactions were analyzed by DGGE, and the composition of the biological origin of the soil DNA was examined.As a result, rare DGGE bands that appeared to have been contaminated from sources other than the soil during the extraction process were found to be rare. However, all extraction methods yielded similar DGGE profiles. That is, at least for the bacteria having 16S rRNA targeted in this experiment, it was shown that the ratio of the DNA extraction ratio between the bacterial group from which DNA was extracted and each bacterial group was the same in any of the extraction methods.
  • Humus Acid is chemically defined as a substance that is extracted from the soil with pulchali, loses its charge under acidic conditions of pH 2, and precipitates.
  • a DNA extract from soil was obtained using the following three types of extract with respect to 10 g of Tohoku University forest soil.
  • FIG. 29 shows the results obtained by appropriately diluting the DNA solution and measuring the absorbance at 400 nm. Results also for measuring the concentration of DNA in the finally obtained DNA solution was quantified for DNA extraction exudates by a recovery of soil DNA in 1% SDS I 200 mM EDTA / 375 mM Na 2 HP0 4 (pH 8.6) Is shown in FIG.
  • the experimental procedure was the same as (1) in Example 16, and a mixed solution of NaCl and CH 3 COONa was used as the salt to be added during the purification of CTAB. 250 l of a 10% CTAB solution and 250 1 of the following salt mixture were added to 750 of the DNA extract.
  • composition of the salt after addition was 0.5 M / 0.5 M, 0.33 M / .67 M, 0.25 MI 0.75 M, 0.67 MI 0.33 M, 0.75 M / 0.25 M, 1 M / 0.25 M, 1 M in CH 3 COONa I NaCl, respectively.
  • FIG. 31 shows the results obtained by adding these solutions, adding an equal amount of black form, collecting the aqueous phase after vortex and centrifugation, and after appropriate dilution, measuring the absorbance at 400 nm.
  • FIG. 32 shows the absorbance at 400 nm of the DNA solution finally recovered with a 12% PEG solution and dissolved in TE Buffer.
  • Figure 33 shows the amount of soil DNA recovered.
  • Example 18 Use of PEG solution with buffer capacity for DNA precipitation recovery of DNA DNA from soil is thought to be mostly humic acid. As described above, it has the property of precipitating under acidic conditions and ionizing again under alkaline conditions and dissolving in an aqueous solution.
  • the removal rate of humic substances could be increased by adding a salt solution having an acidic buffering capacity during purification by CTAB.
  • humic substances that could not be completely removed have precipitated together with the DNA, and this must be removed. Therefore, the pH that was reduced during purification by CTAB was increased again during the precipitation and recovery of DNA by PEG, so that the humic substances were ionized again and adjusted to conditions under which humic substances were less likely to precipitate, and only DNA was selected. It was examined whether it was possible to precipitate sedimentarily.
  • a solution with an alkaline buffering capacity is required. Since the PEG solution is an aqueous solution, an attempt was made to dissolve the PEG and a reagent having an alkaline buffering capacity, and to prepare and use the solution as a single solution.
  • Tris trishydroxyaminoaminomethane
  • CAPS CAPS
  • CAPSO CAPSO
  • CHES CHES
  • TAPS Tris-HCl
  • Bicine Tris-HCl
  • Soil DNA was extracted and purified by CTAB in the same manner as in Example 16.
  • CH 3 COONa having an acidic buffer capacity and a mixed solution of NaCl and CH 3 COONa were used as a salt solution under the same conditions as in Example 17.
  • FIG. 34 shows the results obtained by appropriately diluting the fermented soil DNA solution and measuring the absorbance at 400 ⁇ .
  • Figure 35 shows the effect of using these PEG solutions with alkaline buffering capacity on soil DNA recovery.
  • the highest purity DNA could be obtained by combining it with the operation of lowering the pH by using a salt solution having acidic buffer capacity during CTAB treatment.
  • the PEG solution having the alkaline buffering ability showed higher purity than the PEG solution having no alkaline buffering ability.
  • 12% PEG / 3 M Tris-HCl (pH 8.6) was used for the soil DNA extract purified by CTAB with 0.67 M CH 3 COONa / 0.33 M NaCl, which was used as the salt condition for purification by CTAB.
  • 12% PEG I 2 M Tris-HCl (pH 8.6)
  • 12% PEG 1 1.5 M Tris-HCl pH 8.6).
  • High-molecular-weight DNA requires less DNA damage and is needed for cloning and studies that target longer nucleotide sequences. No physical way to get high molecular weight DNA It is desirable to treat the soil under mild conditions such as heat treatment instead of the BeadsBeating method to avoid cutting.
  • Figure 38 shows the amount of DNA extracted using extracts of various compositions using soil from the Yayoi field control plot.
  • Fig. 39 shows the results of the extracted conditions for the DNA extraction amounts extracted from the Yayoi field control plot soil, the Tanashi pasture soil, the Tochigi agricultural test forest soil, the Saitama agricultural test field soil, and the Hyogo agricultural test field soil.
  • Environmental samples containing microorganisms other than soil include feces of humans and livestock, compost, activated sludge, and water-based sediments such as lake bottoms. '
  • DNA extraction was performed on stool samples sampled for three days from two adult males.
  • the composition of the extract was examined using sample R-1.
  • the extract used had the following composition and both BeadsBeating and extraction by heating were examined.
  • Extraction by a heat treatment method was performed by incubating at 65 ° C. for 1 hour, and then centrifuged at 12000 ⁇ g at 25 ° C. for 5 minutes to obtain a supernatant.
  • the supernatant 750 l to 10% CTAB solution 250 1 l Oyobi 3.33MCH 3 COONa / 1.67MNaCl solution was added 250 l, was added an equal amount of black hole Holm After vortex, vortex after 12000Xg 25 ° C 20 Centrifugation was performed for minutes. Collect the aqueous phase and add an equal volume of 12% PEG / 1.5 M Tris-HCl (pH 8.6)!
  • Figure 40 shows the amount of DNA extracted by each method.
  • FIG. 41 shows the results of electrophoresis of the obtained fecal DNA.
  • Figure 42 shows the results for the yield of fecal DNA obtained by the BeadsBeating method, the heat extraction method, and the conventional method
  • Figure 43 shows the results for the purity.
  • the extraction amount with 1% SDS 1100 mM Tris-HCl / 50 mM EDTA (pH 8.6) was the largest in both the BeadsBeating method and the heat extraction method. From Fig. 42, it became clear that the amount of DNA extracted by the newly developed BeadsBeating method and the heat extraction method was lower than that of the conventional method in terms of yield. However, in the BeadsBeating method and the heat extraction method developed this time, the purification operation is performed as a series of operations, and some DNA is lost during the operation.
  • RNA is not precipitated because the DNA is recovered using the PEG solution, whereas in the conventional method, RNA is also precipitated because 2-propanol is used for DNA precipitation. Therefore, apparently, this RNA affected the results of DNA quantification, resulting in a difference in yield.
  • Figure 43 shows that the purity of fecal DNA extracted by the BeadsBeating method and the heat extraction method developed this time is much higher than that of the conventional method.
  • DNA was extracted from compost and activated sludge samples.
  • Extraction by the heat treatment method was performed by incubating at 65 ° C for 1 hour, and then centrifuged at 12000Xg at 25 ° C for 5 minutes to obtain a supernatant.
  • the supernatant 750 l of 10% CTAB solution 250 / X 1 and 3.33 M CH 3 COONa 11.67 M NaCl solution 250 1 was added to, was added an equal amount of black hole Holm After vortex, vortex after 12000Xg 25 ° C 20 Centrifugation was performed for minutes. Collect the aqueous phase, add an equal volume of 12% PEG I 1.5 M Tris-HCl (pH 8.6), vortex, centrifuge at 20000 for 20 minutes, collect the DNA precipitate and collect with 70% ethanol. After washing and drying, it was dissolved in TE buffer (pH 8.0) to obtain a compost DNA solution. .
  • FIG. 44 shows the amount of DNA extracted by each method.
  • the extraction operation was performed by adding an equal amount of 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6) as described above.) After adding vortex, the mixture was centrifuged at 12000 X g at 25 ° C for 20 minutes. Collect the aqueous phase, add 0.6 volumes of 2-propanol, vortex, centrifuge at 20,000 X g at 4 ° C for 20 minutes, collect the DNA precipitate, wash and dry with 70% ethanol, and add TE buffer ( pH 8.0) to give a compost DNA solution and an activated sludge DNA solution.
  • TE buffer pH 8.0
  • Figure 46 shows the results for the yield of compost DNA obtained by the BeadsBeating method, the heat extraction method, and the conventional method
  • Figure 47 shows the results for the purity
  • Fig. 48 shows the results for the weight of the activated sludge sample
  • Fig. 49 shows the results for the purity.
  • the newly developed DNA purification and sedimentation method can extract DNA from compost samples and activated sludge samples with high yield, and can selectively extract only DNA.
  • DNA was extracted from a lake bottom sediment sample.
  • BeadsBeating treatment was performed at 4 m / sec for 30 seconds, and then a supernatant was obtained by centrifugation at 12000 ⁇ g25t for 5 minutes.
  • Extraction by the heat treatment method was performed by incubating at 65 ° C for 1 hour, and then centrifuged at 12000Xg at 25 ° C for 5 minutes to obtain a supernatant. Add 250 l of 10% CTAB solution and 250 l of 3.33 M CH 3 COONa / 1.67 M NaCI solution to 750 U of this supernatant, add vortex and equal volume of clonal form, and after vortex 12000 Xg 25 ° C 20 Centrifugation was performed for minutes.
  • Figure 50 shows the results of electrophoresis of the obtained lake sediment DNA.
  • an extraction operation was performed by adding an equal amount of 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6) to 500 l of the sample as described above and heating at 65 ° C. for 1 hour. To this supernatant, an equal amount of black-mouthed form was added. After vortexing, centrifugation was performed at 12000 X g at 25 ° C for 20 minutes. Collect the aqueous phase, add 0.6 volumes of 2-propanol, vortex, centrifuge at 20000 X g at 4 ° C for 20 minutes, collect the DNA precipitate, wash and dry with 70% ethanol, and add TE buffer (pH 8.0) to give a lake bottom sediment DNA solution.
  • Figure 51 shows the results for the yield of lake sediment DNA obtained by the BeadsBeating method, the heat extraction method, and the conventional method
  • Figure 52 shows the results for the purity.
  • the newly developed DNA extraction method can extract DNA from lake sediment at higher yields than the conventional method, and the purification and sedimentation method selectively extracts only DNA from the DNA extract obtained from lake sediment samples. It was shown to be very effective for removal.
  • the DNA extraction and purification method developed this time can extract DNA from environmental samples other than soil in high yields, and the obtained DNA has a much higher purity than the conventional method. And suitable for DNA extraction from all environmental samples was thought to be.
  • DGGE analysis was performed on DNA samples extracted from soil, feces, compost, activated sludge, and lake bottom sediments using the BeadsBeating method and heat extraction method using the final extraction and purification method developed this time.
  • V3 region of the 16S rRNA gene was amplified by PCR using the DNA solution 11 as type III, and this was analyzed by DGGE.
  • the conditions of the PCR reaction and the primers used and the conditions of DGGE are shown below.
  • reaction conditions were as follows: reaction at 94 ° C for 2.5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and elongation at 72 for 1 minute. The reaction was finally performed at 72 ° C for 10 minutes.
  • the composition of the reaction solution is as follows.
  • Voltage 100V Fig. 53 shows the results of DGGE analysis of soil DNA for 10 hours.
  • Figure 54 shows the results of DGGE analysis of fecal DNA.
  • Figure 55 shows the results of DGGE analysis of compost DNA and activated sludge DNA.
  • Figure 56 shows the results of DGGE analysis of lake sediment DNA.
  • Tomoto Arao Masago Okano, Tetsuo Kanamori 1998 Soil Phospholipids and Microbial Biomass ⁇ Community Structure Soil and Microorganisms 51; 49-58
  • Tomoto Arao Masago Okano, Tetsuo Kanamori 1998 Analysis of phospholipid fatty acid composition in light-colored black pork soil and its relationship to microbial biomass Japanese Journal of Soil Fertilizer Science 69; (1) 38-46 Tomoto Arao, Masago Okano, Tetsuo Kanamori 1998 Analysis of phospholipid fatty acid composition in various soils Japanese Journal of Soil Fertilizer Science 69; (1) 47-52

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A method of extracting DNA from an environmental sample characterized by comprising treating the environmental sample by beads-beating in the presence of a DNA extract containing not more than 5% of a surfactant and/or heating the same.

Description

' 明 細 書  ' Specification
環境サンプルからの DNAの回収方法 Methods for recovering DNA from environmental samples
技術分野 Technical field
本発明は、 環境サンプルから DNAを回収する方法に関する。  The present invention relates to a method for recovering DNA from an environmental sample.
背景技術 Background art
土壌等の環境中には様々な微生物が生息しており、 膨大なる多様性を秘めてい る。 しかし現在その 99%以上が培養できない、 もしくは極めて難培養な微生物で あるとされており、 培養法に頼つた土壌微生物の群集構造解析には限界があり、 また培養法に頼る限りにおいて、 培養できない土壌微生物の遺伝子解析ができな いことは明らかである （Rondon et al. 1999)。 土壌に含まれる DNAを直接抽出 し解析することができれば、 培養できないものも含め土壌にどのような微生物が いるのか知ることができ、 また新規の遺伝子の情報を塩基配列という形で入手す ることも可能になる。  Various microorganisms inhabit the environment such as soil, and have enormous diversity. However, at present, more than 99% of the microorganisms cannot be cultured or are extremely difficult to culture.Therefore, there is a limit in analyzing the community structure of soil microorganisms relying on culture methods, and cultivation is not possible as long as they rely on culture methods. It is clear that genetic analysis of soil microorganisms is not possible (Rondon et al. 1999). If DNA contained in soil can be directly extracted and analyzed, it is possible to know what kind of microorganisms are in the soil, including those that cannot be cultured, and to obtain information on new genes in the form of base sequences. Also becomes possible.
群集構造解析においては、 16S rRNA遺伝子や 18S rRNA遺伝子のように細菌 や真菌といった幅広い生物群が共通して保有する遺伝子について、 ユニバーサル プライマ—もしくは標的とする生物群 （属、 種レベル） のみに適合するようなプ ライマーを用いて PCR増幅し、 DGGE、 TGGEもしくはクロ一ニングを経て、 塩基配列を解読することにより解析が行われている。 この群集構造解析において 最も問題となるのが「バイアス」である。 PCRバイアスの問題も無視できないが、 それ以前に、 土壌からの抽出の段階であらゆる微生物からバイアスがかかること なく DNAが抽出されているのかという疑問がある。 より正確に微生物の群集構 造を解析しょうとするならば、 まずは土壌からの抽出の段階であらゆる微生物か ら偏りなく DNAが抽出されていることが求められる。  In community structure analysis, genes that are commonly held by a wide range of organisms such as bacteria and fungi, such as the 16S rRNA gene and 18S rRNA gene, are applicable only to universal primers or target organisms (genus, species level). Analysis is performed by PCR amplification using primers that perform the above steps, and decoding the nucleotide sequence through DGGE, TGGE, or cloning. The most important problem in this community structure analysis is "bias". The problem of PCR bias is not negligible, but before that, there is the question of whether DNA is extracted without bias from all microorganisms during the extraction from soil. In order to analyze the microbial community structure more accurately, it is first required that DNA be uniformly extracted from all microorganisms at the stage of extraction from soil.
また最近、 環境 DNA (=eDNA、 environmental DNA) という言葉が用いられ ることがある。 環境 （土壌や汚泥、 湖水、 海水など） から培養を経ずに直接抽出 された DNA (特に土壌から直接抽出された DNAを土壌 DNA(=sDNA、 soil DNA) と呼ぶ） を、 新規微生物の情報をも大量に保持している遺伝子のプールとして考 え、 これをもとにライブラリーを作成し、 クロ一ニングを行い、 遺伝子発現系に 導入することにより、 土壌微生物の遺伝子由来の有用物質 （抗生物質、 酵素など） を Escherichia coliなどの宿主に生産させることが試みられている （Seow 1997、 Handelsman et al. 1998) 。 培養困難な新規微生物は現在知られている種の少な くとも 100倍以上は存在すると考えられているため、 これらの遺伝子の情報を含 んでいる環境 DNAや土壌 DNAの潜在的利用価値は非常に高いと考えられる（植 田 2000、 長谷部 2003)。 これらを利用した研究はコンビナトリアル生物学 (Combinatorial Biology) もしくはコンビナトリアル遺伝学 （Combinatorial Genetics) と呼ばれ、 今後の発展が期待されている分野である。 このような研究 のためにも、 様々な土壌から、 そしてより多くの微生物から DNAを抽出する技 術は重要であろう。 Recently, the term environmental DNA (= eDNA) has sometimes been used. Direct extraction from the environment (soil, sludge, lake water, seawater, etc.) without culture DNA (especially DNA extracted directly from soil is called soil DNA (= sDNA, soil DNA)) is considered as a pool of genes that also retain a large amount of information on new microorganisms. By creating a library in Japan, performing cloning, and introducing it into a gene expression system, it has been attempted to produce useful substances (antibiotics, enzymes, etc.) derived from soil microorganism genes in hosts such as Escherichia coli. (Seow 1997, Handelsman et al. 1998). The potential use of environmental and soil DNA containing information on these genes is extremely high, as new microorganisms that are difficult to culture are thought to exist at least 100 times more than currently known species. It is considered high (Ueda 2000, Hasebe 2003). Research using these methods is called combinatorial biology or combinatorial genetics, and is an area where further development is expected. Techniques for extracting DNA from various soils and from more microorganisms will be important for such research.
土壌: DNA抽出法の開発の歩み  Soil: History of DNA extraction development
土壌から DNAを抽出する試みは Torsvik & Goksoyr (1978、 1980)によって初 めて行われた（Torsvik V L 、 Goksoyr J； Soil Biology and Biochemistry； 1978 > 10 : p.7-12) o 彼らの方法は、 土壌から微生物をピロリン酸緩衝液などにより分離 回収し、この微生物画分より DNAを抽出するものであり、こうして得られた DNA を彼らは 「土壌 DNA」 とした。 この方法は、 いったん土壌から微生物画分を回 収し、 ここから DNAを抽出するため間接抽出法 （indirect extraction method) と呼ばれている。 しかしながら、 この方法では、 緩衝液で土壌を洗浄しても回収 できない微生物の DNAを得ることはできない。 すなわち放線菌の一部など土壌 粒子に強固に吸着しているものや、 植物遺体内に生息している微生物、 また団粒 の内部に生息している微生物の DNAは、 この方法では抽出できなかった可能性 がある。 その後、 Ogram et al. (1987)や Tsai & Olson (1991)、 Zhou et al. (1996) などにより直接抽出法 （direct extraction method) が相次いで開発された。 これ らの方法は、 lysozymeや proteinase Kといった酵素や SDSを含むアル力リ性抽 出液で土壌を処理し、 タンパク質を変性させることにより、 菌体を土壌から分離 することなく、 すなわち土壌というマトリックスの存在する溶液中で、 溶菌させAttempts to extract DNA from soil was carried out Te first order by Torsvik & Goksoyr (1978, 1980) (Torsvik VL, Goksoyr J; Soil Biology and Biochemistry; 1978> 10: p.7-12) o Their method Microorganisms were separated and collected from soil with a pyrophosphate buffer and the like, and DNA was extracted from this microbial fraction. The DNA thus obtained was called "soil DNA." This method is called the indirect extraction method because the microbial fraction is collected from the soil and the DNA is extracted therefrom. However, this method does not provide DNA for microorganisms that cannot be recovered by washing the soil with a buffer. That is, DNA that is strongly adsorbed to soil particles, such as a portion of actinomycetes, microorganisms that live in plant remains, or microorganisms that live inside aggregates cannot be extracted by this method. May have been Later, direct extraction methods were successively developed by Ogram et al. (1987), Tsai & Olson (1991), Zhou et al. (1996), and others. In these methods, the bacteria are separated from the soil by treating the soil with an enzyme extract containing enzymes such as lysozyme and proteinase K and SDS, and denaturing the protein. Lysis in a solution where a matrix of soil exists.
DNA を抽出するものである。 この方法は、 原理的には間接抽出法よりもより実 際の土壌の微生物群集構造を反映した組成から成る DNAが得られると考えられ、 また収量の面で優れている。 しかしこの方法では、 緩衝液のアルカリ性条件下で 土壌を長時間加熱処理するため、 相当量の腐植物質の混入が問題となる。 It extracts DNA. This method is thought to yield DNA with a composition that reflects the actual microbial community structure of the soil more in principle than the indirect extraction method, and is superior in yield. However, in this method, since the soil is heated for a long time under the alkaline condition of the buffer solution, contamination of a considerable amount of humic substances becomes a problem.
間接抽出法は土壌の群集構造を正確に反映していない可能性があるが、 いったん 土壌から微生物を分離した後に DNAを抽出するので、 直接抽出法よりも腐植物 質の混入が少ないのみならず、 土壌粒子が DNAと衝突することによって生じる 物理的なせん断による DNAの断片化を抑えることが可能であり、 より高分子の DNAを得ることができる。 また近年、 クローニング効率が高い高分子 DNAを導 入することができるベクタ一が開発されたことから、 間接抽出法は、 群集構造解 祈よりも遺伝子探索の研究においてよく使用されるようになってきた （Bakken & Lindahl 1995、 Saano et al. 1995、 Berry et al. 2003、 Gabor et al. 2003)。 一方、 より短時間に、 より多くの土壌微生物から DNAを抽出することを目的 に、 beads beaterを用いて細胞を機械的に破壊する方法が新たに登場した (Kuske CR, Banton KL, Adoraaa DL, et al.； Applied and nvironmental Microbiology, 1998 、 64： (7) p .2463-2472)。 beads-beatingの条件検討は Burgmann et al.The indirect extraction method may not accurately reflect the community structure of the soil, but once the DNA is extracted after separating microorganisms from the soil, not only the humic substances are less contaminated than the direct extraction method, However, it is possible to suppress the fragmentation of the DNA due to physical shearing caused by the collision of the soil particles with the DNA, and it is possible to obtain higher-molecular-weight DNA. In recent years, vectors that can introduce high-molecular-weight DNA with high cloning efficiency have been developed, and the indirect extraction method has been used more often in gene search research than in community structure analysis. (Bakken & Lindahl 1995, Saano et al. 1995, Berry et al. 2003, Gabor et al. 2003). On the other hand, in order to extract DNA from more soil microorganisms in a shorter time, a new method of mechanically destroying cells using beads beater has appeared (Kuske CR, Banton KL, Adoraaa DL, et al .; Applied and nvironmental Microbiology, 1998, 64: (7) p.2463-2472). The examination of beads-beating conditions is described in Burgmann et al.
(2001)により極めて詳細に検討されている（Burgmann H., Pesaro M., Widmer F. and Zeyer J.； Journal of Microbiological Methods;2001、 45:(1) p.7"20) 。 この方法では、 細胞外多糖膜をもっため SDS などの界面活性剤の影響を受けに くいグラム陽性菌であっても機械的に破砕されるため、 極めて高収率で DNAが 抽出できるという利点がある。また beads-beatingは抽出が短時間で終わるため、 腐植物質の混入が少ない土壌 DNA試料を得ることができる。 しかし beads (ガ ラスビーズ、 シリカジルコニァビーズ、 アルミナビーズなど） による機械的な衝 撃破砕は菌体から抽出されてきた高分子である DNAにも作用し、 DNAが物理的 にせん断されてしまうこともある。このように様々な微生物群から偏りなく DNA が取れる一方、 DNA が低分子化することから比較的短い配列をターゲッ卜とす る DGGEなどの群集構造解析には、 この方法が多く用いられている。 また近年 BiolOl Fast DNA spin kit (Qbio, USA)や UltraClean Soil DNA kit (MoBio, USA) など、 試薬メーカーから独自の方法により短時間で土壌からの DNAを調製するキットも製品化されている。いずれもが beads beaterを使用し、 短時間で土壌 D N A抽出が可能になっているものである。 (2001) (Burgmann H., Pesaro M., Widmer F. and Zeyer J .; Journal of Microbiological Methods; 2001, 45: (1) p.7 "20). However, because it has an extracellular polysaccharide membrane and is hardly affected by detergents such as SDS, even gram-positive bacteria can be mechanically disrupted, so that there is an advantage that DNA can be extracted at an extremely high yield. Since beads-beating can be extracted in a short time, soil DNA samples with little humic substances can be obtained, but mechanical impact crushing with beads (glass beads, silica zirconia beads, alumina beads, etc.) is not possible. It also acts on high-molecular-weight DNA extracted from bacterial cells, which may cause physical shearing of the DNA, thus allowing DNA to be obtained from various microbial communities without bias. Target a relatively short sequence The community structure, such as DGGE shall be the Bok, this method is often used. In recent years, kits that prepare DNA from soil in a short time using a proprietary method, such as the BiolOl Fast DNA spin kit (Qbio, USA) and the UltraClean Soil DNA kit (MoBio, USA), have also been commercialized. All of them use beads beater and can extract soil DNA in a short time.
上記のように、 'これまで土壌 DNAの様々な抽出法が考案されたが、 用いる界 面活性剤や緩衝液の組成や濃度などは研究によってまちまちであり、 これらの条 件と DNA抽出効率、 あるいは抽出された DNAの質 （純度や断片化の程度など） との関係について体系的に検討した例はほとんどない。  As mentioned above, various extraction methods for soil DNA have been devised, but the composition and concentration of surfactants and buffers used vary depending on the research, and these conditions and DNA extraction efficiency, Also, there are few examples of systematically examining the relationship with the quality of the extracted DNA (purity, degree of fragmentation, etc.).
土壌 DNAの精製法に関する従来の知見  Previous knowledge on purification methods for soil DNA
DNA試料に混入した腐植物質は極めて微量でも PCR反応を阻害する （Tsai et al.l991)。 このため Zhou et al. (1996) らは抽出した土壌 DNAをァガ口一スゲ ルでいったん電気泳動することで腐植物質と DNA を分離し、 その後ゲルから DNAのみを回収するという方法をとっている （Zhou JZ,Bmns MA,Tiedje JM; Applied and environmental microbiology; 1996； 62:(2) p.316-322)。 また、 低融 点ァガロースゲルを利用した方法として agarose-embedded preparationがあげ られる （Moreim 1998)。 これは、 DNAが腐植物質より高分子であることを利用 し、 いったん腐植物質を含んだ土壌 DNA溶液を低融点ァガロースゲル中に閉じ 込めるように固化し、 そのゲル片を TE bufferで透析し、 低分子である腐植物質 を徐々に取り除くものである。 透析処理において高分子 DNAはァガロースゲル 中に留まり、低分子である腐植物質は拡散によりァガロースゲルから抜けてゆき、 腐植物質をゲル片から取り除いた後にァガロースを溶解させ DNAを回収するも のである。 このように DNAと腐植物質を分離できる物性の違いとしてとしてそ の分子量の差が上げられ、 多くの精製法がこの原理に基づいている。 分子量に基 づいた代表的分離法として、 ゲルろ過法があげられる。 Sephadexや Sepharose といった多孔質の樹脂を利用し、 サイズ分画により、 比較的大きな DNA分子を 小さな腐植物質から分離して回収しょうとするものである （e.g. Jackson et al. 1997， Miller 2001)。 その他には、 カオトロピック塩溶液中ではカオトロピック 効果により DNAがガラス表面に吸着しやすくなることを利用して、 ガラスパゥ ダー、シリ力ゲルパウダ一、 シリカメンブレンなどに DNAをまず吸着させ、 その 後エタノールなどで吸着していな夾雑物を洗浄して取り除き、 極性の高い水を用 いて DNAを再抽出する精製方法がある。 BiolOl Fast DNA spin kit (Qbio, USA) や UltraClean Soil DNA kit (MoBio, USA) などは抽出した土壌 DNAの精製に この原理を用いていると考えられる。 また、 磁性ビーズを利用した精製法も実用 化されている （製品名 .Wizard Magnetic, Promega, Madison WI, USA)。 一方、 ポリビニルポリピロリ ドン (PVPP)やセチルトリメチルアンモニゥムブロミ ド (CTAB) なども腐植物質の除去剤として使用されてきたが、 これらの使用によ つて土壌 DNA 試料への腐植物質の混入を軽減する効果は見られるものの、完全 な除去までには至っておらず、 PVPPについては DNAの収量が下がるなどの問 題が生じる場合もあった （Zhou et al. 1996)。 発明の開示 Humic substances contaminated in DNA samples can inhibit the PCR reaction even in very small amounts (Tsai et al. L991). For this reason, Zhou et al. (1996) et al. Used a method in which the extracted soil DNA was electrophoresed once with an agarose gel to separate humic substances and DNA, and then only the DNA was recovered from the gel. (Zhou JZ, Bmns MA, Tiedje JM; Applied and environmental microbiology; 1996; 62: (2) p.316-322). An agarose-embedded preparation is another method that uses low-melting-point agarose gel (Moreim 1998). This utilizes the fact that DNA is higher in molecular weight than humic substances, so that once a soil DNA solution containing humic substances is solidified in a low-melting agarose gel, the gel pieces are dialyzed against TE buffer, It gradually removes humic substances, which are molecules. In the dialysis treatment, the high-molecular-weight DNA remains in the agarose gel, and the low-molecular-weight humic substances escape from the agarose gel by diffusion. After the humic substances are removed from the gel pieces, the agarose is dissolved and the DNA is recovered. In this way, the difference in physical properties that can separate DNA and humic substances is attributed to the difference in molecular weight, and many purification methods are based on this principle. A typical separation method based on the molecular weight is a gel filtration method. It uses a porous resin such as Sephadex or Sepharose to separate and recover relatively large DNA molecules from small humic substances by size fractionation (eg Jackson et al. 1997, Miller 2001). In addition, utilizing the fact that DNA is easily adsorbed on the glass surface due to the chaotropic effect in a chaotropic salt solution, There is a purification method in which DNA is first adsorbed on a gel, silica gel powder, silica membrane, etc., and then unadsorbed impurities are removed by washing with ethanol, etc., and the DNA is re-extracted using highly polar water. . It is thought that BiolOl Fast DNA spin kit (Qbio, USA) and UltraClean Soil DNA kit (MoBio, USA) use this principle for purification of extracted soil DNA. A purification method using magnetic beads has also been put into practical use (product names: Wizard Magnetic, Promega, Madison WI, USA). On the other hand, polyvinylpolypyrrolidone (PVPP) and cetyltrimethylammonium bromide (CTAB) have also been used as humic substances removers, but their use has been considered to reduce the contamination of soil DNA samples with humic substances. Although the effect was reduced, it was not completely eliminated, and there were some problems with PVPP, such as a decrease in DNA yield (Zhou et al. 1996). Disclosure of the invention
本発明は、 高収量でかつ高純度の DNAを土壌から抽出するための方法を提供 することを目的とする。 ' 本発明者は、 高収量かつ高純度に土壌 DNAを抽出する手法を開発するため、 まず化学的な条件として、 土壌微生物細胞の溶解に適し、 土壌由来の腐植物質や 粘土など様々な物質が共存する条件下でも十分に微生物を溶菌させることが可能 な界面活性剤を検索した。 次に細胞破壊の物理的な条件として、 beads-beating による破砕や加熱の条件の検討を行った。 また、 抽出液に使用する Tris-HCl 緩 衝液、 EDTA溶液、 リン酸緩衝液の特性や、 得られた土壌 DNAの精製および沈 殿方法として陽ィォン界面活性剤である CTABによる精製およびポリエチレング リコール（PEG)による DNAの沈殿操作を最適な条件で組み合わせることなど、 性質の異なる様々な土壌からより簡便な操作でより高収量かつ高純度な DNAを 得る抽出—精製手法を確立するため、 鋭意検討を行なった。  An object of the present invention is to provide a method for extracting high yield and high purity DNA from soil. '' In order to develop a method for extracting soil DNA with high yield and purity, the present inventor first decided to use various substances such as soil-derived humic substances and clay as chemical conditions, which are suitable for lysing soil microbial cells. We searched for surfactants that can sufficiently lyse microorganisms even under coexisting conditions. Next, as physical conditions of cell destruction, the conditions of crushing and heating by beads-beating were examined. In addition, the properties of Tris-HCl buffer, EDTA solution, and phosphate buffer used in the extract, and the purification and precipitation of the obtained soil DNA were carried out by purification using CTAB, a positive surfactant, and polyethylene glycol. Investigating to obtain a high yield and high purity DNA by simpler operation from various soils with different properties, such as combining DNA precipitation operation with PEG under optimal conditions. Was performed.
その結果、 高収量かつ高純度な土壌 DNAを抽出する手法を開発することに成 功し、 本発明を完成するに至った。 すなわち、 本発明は以下の通りである。 (1) 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを処理す ることを特徴とする、 環境サンプルから DNAを抽出する方法。 As a result, they succeeded in developing a technique for extracting high-yield and high-purity soil DNA, and completed the present invention. That is, the present invention is as follows. (1) A method for extracting DNA from an environmental sample, comprising treating the environmental sample in the presence of a DNA extract containing 5% or less of a surfactant.
(2)界面活性剤が SDS、 CTAB、 Triton X-100及び Ν·ラウロイルサルコシンナト リゥムからなる群から選ばれるいずれかのものである（1)記載の方法。  (2) The method according to (1), wherein the surfactant is selected from the group consisting of SDS, CTAB, Triton X-100, and lauroyl sarcosine sodium.
(3) DNA抽出液が、 さらにリン酸緩衝液及び/又は EDTAを含むものである（1)記 載の方法。 (3) The method according to (1), wherein the DNA extract further contains a phosphate buffer and / or EDTA.
(4) DNA抽出液の ρΗが 7以上である（1)記載の方法。  (4) The method according to (1), wherein ρΗ of the DNA extract is 7 or more.
(5) リン酸緩衝液の濃度が 100mM〜： !500mMである (3)記載の方法。  (5) The method according to (3), wherein the concentration of the phosphate buffer is 100 mM or more:! 500 mM.
(6) EDTAの濃度が 50mM〜600mMである（3)記載の方法。  (6) The method according to (3), wherein the concentration of EDTA is 50 mM to 600 mM.
(7) リン酸緩衝液の濃度が 100ηιΜ〜750πιΜであり、かつ、 EDTAの濃度が 50mM 〜600mMである（3)記載の方法。 (7) The method according to (3), wherein the concentration of the phosphate buffer is from 100ηιΜ to 750πι 、 and the concentration of EDTA is from 50mM to 600mM.
(8)環境サンプルが、 土壌、 堆肥、 水系堆積物、 活性汚泥及び糞便からなる群か ら選ばれる少なくとも 1つである（1)記載の方法。  (8) The method according to (1), wherein the environmental sample is at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge, and feces.
(9)環境サンプルの処理が、 環境サンプルを beads-beating処理及び/又は加熱処 理するものである（1)記載の方法。 '  (9) The method according to (1), wherein the processing of the environmental sample is performed by beads-beating and / or heating the environmental sample. '
(10) 環境サンプルから DNAを抽出する方法であって、  (10) A method for extracting DNA from an environmental sample,
(a) 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 遠心後上清を採取し、  (c) Collect the supernatant after centrifugation,
(d) 上清の採取後の残存環境サンプルについて、 上記 (a)〜(c)工程を 1回〜 4回繰り返す  (d) Repeat steps (a) to (c) once to four times for the remaining environmental sample after collecting the supernatant.
工程を含む前記方法。  The method comprising:
(11) DNA抽出液が、 さらにリン酸緩衝液及び/又は EDTA を含むものである (10)記載の方法。  (11) The method according to (10), wherein the DNA extract further contains a phosphate buffer and / or EDTA.
(12) EDTAの濃度が 50〜600mMである（11)記載の方法。  (12) The method according to (11), wherein the concentration of EDTA is 50 to 600 mM.
(13) リン酸緩衝液の濃度が 100〜2000mMである (11)記載の方法。  (13) The method according to (11), wherein the concentration of the phosphate buffer is 100 to 2000 mM.
(14) 環境サンプルから DNAを抽出する方法であって、 (a) 5 %以下の界面活性剤及び 50〜600mMの EDTAを含む DNA抽出液 の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、 (14) A method for extracting DNA from an environmental sample, (a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 mM EDTA, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心して上清を採 取し、  (b) Centrifuge the extract after beads-beating treatment and / or heat treatment, collect the supernatant,
(c) 採取された上清と 600〜： LlOOmMの EDTAとを混合する  (c) Mix the collected supernatant with 600 ~: LlOOmM EDTA
工程を含む前記方法。  The method comprising:
(15) 環境サンプルから DNAを抽出する方法であって、  (15) A method for extracting DNA from an environmental sample,
(a) 5 %以下の界面活性剤及び 50〜600raMの EDTAを含む DNA抽出液 の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 raM of EDTA, subject the environmental sample to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清と 600〜： LlOOmMの EDTAとの混合物を加熱処理する 工程を含む前記方法。  (c) The above method comprising a step of heat-treating a mixture of the obtained supernatant and 600 to: LOOmM EDTA.
(16) 環境サンプルから DNAを抽出する方法であって、  (16) A method for extracting DNA from an environmental sample,
5 %以下の界面活性剤及び 250〜2000mMのリン酸緩衝液を含む DNA抽 出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理する 工程を含む前記方法。  The above method comprising the step of subjecting an environmental sample to beads-beating treatment and / or heat treatment in the presence of a DNA extract containing 5% or less of a surfactant and a phosphate buffer of 250 to 2000 mM.
(17) 環境サンプルから DNAを抽出する方法であって、  (17) A method for extracting DNA from an environmental sample,
5 %以下の界面活性剤、 100〜800mMの EDTA及び 250〜2000mMのリン 酸緩衝液を含む DNA抽出液の存在下で環境サンプルを beads-beating処理及 び/又は加熱処理する  Treat environmental samples with beads-beating and / or heat in the presence of a DNA extract containing 5% or less of surfactant, 100 to 800 mM EDTA and 250 to 2000 mM phosphate buffer.
工程を含む前記方法。  The method comprising:
(18) EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある (17)記載の方法。  (18) The method according to (17), wherein the concentration of EDTA is 400 mM and the concentration of the phosphate buffer is 750 mM.
(19) 環境サンプルから DNAを抽出する方法であって、  (19) A method for extracting DNA from an environmental sample,
(a) 5 %以下の界面活性剤を含む DNA抽出液 Iの存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of DNA extract I containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液 Iを、 75〜： l200mMの EDTA, 250〜3000mMのリン酸緩衝液、 又は前記 EDTAとリン酸緩衝液と の混合物と混合じて抽出液 IIを調製し、 (b) Extract solution I after beads-beating treatment and / or heat treatment was mixed with 75 to: l200 mM EDTA, 250 to 3000 mM phosphate buffer, or the above EDTA and phosphate buffer. Extract II was prepared by mixing with
(c) 前記抽出液 IIから DNAを抽出する  (c) extracting DNA from the extract II
工程を含む前記方法。  The method comprising:
(20) 抽出液 Iと混合する EDTAの濃度が 400〜800mMである（19)記載の方法。 (21) 抽出液 Iど混合するリン酸緩衝液の濃度が 750〜1500mMである（19)記載 の方法。 (20) The method according to (19), wherein the concentration of EDTA mixed with Extract I is 400 to 800 mM. (21) The method according to (19), wherein the concentration of the phosphate buffer mixed with the extract I is 750 to 1500 mM.
(22) 抽出液 Iと混合する EDTAの濃度が 400mMであり、 かつ、抽出液 Iと混 合するリン酸緩衝液の濃度が 750 mMである（19)記載の方法。  (22) The method according to (19), wherein the concentration of EDTA mixed with the extract I is 400 mM, and the concentration of the phosphate buffer mixed with the extract I is 750 mM.
(23) 環境サンプルから DNAを抽出する方法であって、  (23) A method for extracting DNA from an environmental sample,
(a) 5 %以下の界面活性剤、 400mM以下の EDTA及び 250mM以下のリン 酸緩衝液を含む DNA抽出液 IIIの存在下で土壌サンプルを beads-beating処 理し、  (a) beads-beating the soil sample in the presence of DNA extract III containing 5% or less surfactant, 400 mM or less EDTA and 250 mM or less phosphate buffer,
(b) beads-beating処理後の抽出液 IIIを、 400〜 OmMの EDTA、 750〜 2050mMのリン酸緩衝液、 又は前記 EDTAとリン酸緩衝液との混合物と混合 して抽出液 IVを調製し、  (b) Extract IV after beads-beating treatment is mixed with 400 to OmM EDTA, 750 to 2050 mM phosphate buffer, or a mixture of EDTA and phosphate buffer to prepare Extract IV. ,
(c) 前記抽出液 IVから DNAを抽出する  (c) extracting DNA from the extract IV
工程を含む前記方法。  The method comprising:
(24) DNAを抽出する工程が、抽出液 IVを加熱処理した後、遠心するものであ る（23)記載の方法。  (24) The method according to (23), wherein the step of extracting DNA comprises subjecting the extract IV to heat treatment and then centrifuging.
(25) 抽出液 IIIにおいて、 EDTAの濃度が 300mMであり、 かつ、 リン酸緩衝 液の濃度が lOOmMである (23)記載の方法。 (25) The method according to (23), wherein in the extract III, the concentration of EDTA is 300 mM, and the concentration of the phosphate buffer is 100 mM.
(26) 抽出液 IIIと混合する EDTAの濃度が 400mMであり、 かつ、 抽出液 III と混合するリン酸緩衝液の濃度が 750mMである (23)記載の方法。  (26) The method according to (23), wherein the concentration of EDTA mixed with Extract III is 400 mM, and the concentration of phosphate buffer mixed with Extract III is 750 mM.
(27) 環境サンプルから DNAを抽出する方法であって、  (27) A method for extracting DNA from an environmental sample,
(a) 100〜400mMの EDTA及び 250〜1500mMのリン酸緩衝液を含む DNA 抽出液 Vの存在下で環境サンプルを加熱処理し、  (a) heat-treating an environmental sample in the presence of a DNA extract V containing 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer,
(b) 加熱処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after the heat treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の界面活性剤、 400mM以下の EDTA 及び 250mM 以下のリン酸緩衝液を含む DNA 抽出液 III の存在下で beads-beating処理レ、 (c) Remaining environmental sample is 5% or less of surfactant and 400mM or less of EDTA. And beads-beating treatment in the presence of DNA extract III containing phosphate buffer of up to 250 mM
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取する  (d) Centrifuge extract III after beads-beating treatment and collect supernatant
工程を含む前記方法。  The method comprising:
(28) 環境サンプルから DNAを抽出する方法であって、 (28) A method for extracting DNA from an environmental sample,
(a) 100~400mMの EDTA及び 250〜： L500mMのリン酸緩衝液を含む DNA 抽出液 Vの存在下で環境サンプルを beads-beating処理し、  (a) 100 to 400 mM EDTA and 250 to: beads-beating treatment of environmental sample in the presence of DNA extract V containing 500 mM phosphate buffer,
(b) beads-beating処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after beads-beating treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の界面活性剤、 400mM以下の EDTA 及び 250mM 以下のリン酸緩衝液を含む DNA 抽出液 III の存在下で beads-beating処理し、  (c) Bead-beating the remaining environmental sample in the presence of DNA extract III containing 5% or less of a surfactant, 400 mM or less of EDTA, and 250 mM or less of a phosphate buffer,
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取する  (d) Centrifuge extract III after beads-beating treatment and collect supernatant
工程を含む前記方法。  The method comprising:
(29) 環境サンプルから DNAを抽出する方法であって、  (29) A method for extracting DNA from an environmental sample,
(a) 200〜800mMの EDTA及び 250〜2000mMのリン酸緩衝液を含む DNA 抽出液の存在下で環境サンプルを第一加熱処理し、  (a) first heat-treating the environmental sample in the presence of a DNA extract containing 200-800 mM EDTA and 250-2000 mM phosphate buffer,
(b) 第一加熱処理後の環境サンプルと、 5 %以下の界面活性剤とを混合して 当該混合物を第二加熱処理する  (b) Mix the environmental sample after the first heat treatment with 5% or less of surfactant and heat-treat the mixture with the second heat treatment
工程を含む前記方法。  The method comprising the steps of:
(30) 環境サンプルから DNAを抽出する方法であって、 (30) A method for extracting DNA from an environmental sample,
(a) 100〜400mMの EDTA及び 250〜： L500mMのリン酸緩衝液を含む DNA 抽出液の存在下で環境サンプルを beads-beating処理し、  (a) 100-400 mM EDTA and 250-: beads-beating treatment of environmental sample in the presence of DNA extract containing 500 mM phosphate buffer,
(b) beads-beating処理後の環境サンプルと 5 %以下の界面活性剤とを混合 して当該混合物を加熱処理する  (b) Mix the environmental sample after beads-beating treatment with 5% or less of surfactant and heat-treat the mixture
工程を含む前記方法。  The method comprising:
(31) 抽出液 Vにおいて、 EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝 液の濃度が 750mMである (27)又は (28)記載の方法。  (31) The method according to (27) or (28), wherein the extract V has an EDTA concentration of 400 mM and a phosphate buffer concentration of 750 mM.
(32) EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある (29)又は (30)言 3載の方法。 (32) When the EDTA concentration is 400 mM and the phosphate buffer concentration is 750 mM Yes (29) or (30) The method described in statement 3.
(33) 環境サンプル由来の DNAを、 陽イオン界面活性剤及び塩の存在下で精製 することを特徴とする DNAの精製方法。  (33) A method for purifying DNA, comprising purifying DNA derived from an environmental sample in the presence of a cationic surfactant and a salt.
(34) 環境サンプル由来の DNAが、（1)〜(32)のいずれか 1項に記載の方法によ り抽出された DNAである（33)記載の方法。  (34) The method according to (33), wherein the DNA from the environmental sample is DNA extracted by the method according to any one of (1) to (32).
(35) 陽イオン界面活性剤が CTABである (33)又は (34)記載の方法。  (35) The method according to (33) or (34), wherein the cationic surfactant is CTAB.
(36) 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモニゥ ム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからなる群 から選択される少なくとも 1つである (33)又は (34)記載の方法。  (36) The salt according to (33) or (34), wherein the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate. the method of.
(37) 陽イオン界面活性剤の濃度が 1〜3%である (33)又は (34)記載の方法。 (37) The method according to (33) or (34), wherein the concentration of the cationic surfactant is 1 to 3%.
(38) 塩の濃度が 0.7〜2.1Mである（33)又は (34)記載の方法。  (38) The method according to (33) or (34), wherein the salt concentration is 0.7 to 2.1M.
(39) 陽イオン界面活性剤の濃度が 2〜3%であり、 かつ、 塩の濃度が 1.0Mで ある（33)又は (34)記載の方法。  (39) The method according to (33) or (34), wherein the concentration of the cationic surfactant is 2 to 3%, and the concentration of the salt is 1.0 M.
(40) pH7.0未満の条件で精製することを特徴とする (33)又は (34)記載の方法。 (41) 上記 (33)〜(40)のいずれか 1項に記載の方法により精製された DNAを、 (40) The method according to (33) or (34), wherein the purification is performed under a condition of pH less than 7.0. (41) DNA purified by the method according to any one of (33) to (40) above,
2-プロパノール、 エタノール又はポリエチレングリコ一ルの存在下で沈殿さ せることを特徴とする DNAの回収方法。 A method for recovering DNA, comprising precipitating in the presence of 2-propanol, ethanol or polyethylene glycol.
(42) pH7.0以上の条件で沈殿させることを特徴とする (41)記載の方法。  (42) The method according to (41), wherein the precipitation is performed under a condition of pH 7.0 or more.
(43) ポリエチレングリコールの濃度が 5〜7.5%である (41)記載の方法。  (43) The method according to (41), wherein the concentration of the polyethylene glycol is 5 to 7.5%.
(44) 上記 (1)〜(9)のいずれか 1項に記載の方法により抽出された DNA を、 2- プロパノール、 エタノール又はポリエチレンダリコールの存在下で沈殿させ ることを特徴とする DNAの回収方法。 (44) A DNA, characterized in that the DNA extracted by the method according to any one of (1) to (9) above is precipitated in the presence of 2-propanol, ethanol or polyethylene dalicol. Collection method.
(45) 環境サンプルから DNAを回収する方法であって、 (45) A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清から DNAを回収する  (c) recover DNA from the resulting supernatant
工程を含む前記方法。 (46) DNA抽出液が'、さらにリン酸緩衝液及び/又は EDTAを含むものである (45) 記載の方法。 The method comprising: (46) The method according to (45), wherein the DNA extract further comprises' a phosphate buffer and / or EDTA.
(47) EDTAの濃度が 50〜600mMである (46)記載の方法。  (47) The method according to (46), wherein the concentration of EDTA is 50 to 600 mM.
(48) リン酸緩衝液の濃度が 100〜2000mMである (46)記載の方法。  (48) The method according to (46), wherein the concentration of the phosphate buffer is 100 to 2000 mM.
(49) 環境サンプルから DNAを回収する方法であって、 (49) A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを beads-beating処理又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment or heat treatment,
(b) beads-beating処理又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment or heat treatment,
(c) 得られる上清から DNAを回収し、 ，  (c) recovering DNA from the resulting supernatant,
(d) DNA回収後の残存環境サンプルについて、 上記 (a)〜(c)工程を 1回〜 4 回繰り返す  (d) Repeat steps (a) to (c) one to four times for the remaining environmental sample after DNA recovery
工程を含む前記方法。  The method comprising:
(50) DNA抽出液が、 さらにリン酸緩衝液及び EDTAを含むものである (49)記 ' 載の方法。 (50) The method according to (49), wherein the DNA extract further contains a phosphate buffer and EDTA.
(51) EDTAの濃度が 50 mM〜600mMである（50)記載の方法。 (51) The method according to (50), wherein the concentration of EDTA is 50 mM to 600 mM.
(52) リン酸緩衝液の濃度が 100〜2000mMである (50)記載の方法。  (52) The method according to (50), wherein the concentration of the phosphate buffer is 100 to 2000 mM.
(53) 環境サンプルから DNAを回収する方法であって、  (53) A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤及び 50〜600mMの EDTAを含む DNA抽出液 の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 mM EDTA, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心して上清を採 取し、  (b) The extract after beads-beating treatment and / or heat treatment is centrifuged and the supernatant is collected.
(c) 採取された上清と 600〜： llOOmMの EDTAとを混合して当該混合物を 、し、  (c) The collected supernatant is mixed with 600-: llOOmM EDTA to obtain the mixture,
(d) 得られる上清から DNAを回収する  (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising:
(54) 環境サンプルから DNAを回収する方法であって、  (54) A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤及び 50〜600mMの EDTAを含む DNA抽出液 の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、 (b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、(a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 mM EDTA, an environmental sample is subjected to beads-beating treatment and / or heat treatment, (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清と 600〜1100mMの EDTAとを混合して当該混合物を加 熱処理し、 (c) mixing the resulting supernatant with 600 to 1100 mM EDTA, heat treating the mixture,
(d) 加熱後の抽出液を遠心し、  (d) Centrifuge the heated extract,
(e) 得られる上清から DNAを回収する  (e) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising:
(55) 環境サンプルから DNAを回収する方法であって、  (55) A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤及び 250〜2000mMのリン酸緩衝液を含む DNA 抽出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、 (b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (a) beads-beating and / or heat treatment of the environmental sample in the presence of a DNA extract containing 5% or less surfactant and 250-2000 mM phosphate buffer; (b) beads-beating and / Or centrifuged extract after heat treatment,
(c) 得られる上清から DNAを回収する  (c) recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising:
(56) 環境サンプルから DNAを回収する方法であって、  (56) A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤、 100〜800mMの EDTA及び 250〜2000mMの リン酸緩衝液を含む DNA抽出液の存在下で環境サンプルを beads-beating処 理及び/又は加熱処理し、  (a) subjecting the environmental sample to beads-beating treatment and / or heat treatment in the presence of a DNA extract containing 5% or less of a surfactant, 100 to 800 mM EDTA and 250 to 2000 mM phosphate buffer,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清から DNAを回収する  (c) recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
(57) EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある（56)記載の方法。 (57) The method according to (56), wherein the concentration of EDTA is 400 mM and the concentration of the phosphate buffer is 750 mM.
(58) 環境サンプルから DNAを回収する方法であって、 (58) A method for recovering DNA from an environmental sample, comprising:
(a) 5 %以下の界面活性剤を含む DNA抽出液 Iの存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of DNA extract I containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液 Iを、 75〜： 1200mMの (b) Extract solution I after beads-beating treatment and / or heat treatment was 75-: 1200 mM
EDTA, 250〜3000mMのリン酸緩衝液、 又は前記 EDTAとリン酸緩衝液と の混合物と混合して抽出液 IIを調製し、 EDTA, 250-3000 mM phosphate buffer or a mixture of EDTA and phosphate buffer described above to prepare Extract II,
(c) 抽出液 IIを遠心し、 (d) 得られる上清から DNAを回収する (c) Centrifuge Extract II, (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
(59) 抽出液 Iと混合する EDTAの濃度が 400〜800mMである（58)記載の方法。 (59) The method according to (58), wherein the concentration of EDTA mixed with the extract I is 400 to 800 mM.
(60) 抽出液 Iと混合するリン酸緩衝液の濃度が 750〜1500mMである (58)記載 の方法。 ' (60) The method according to (58), wherein the concentration of the phosphate buffer mixed with the extract I is 750 to 1500 mM. '
(61) 抽出液 Iと混合する EDTAの濃度が 400mMであり、 かつ、抽出液 Iと混 合するリン酸緩衝液の濃度が 750 mMである (58)記載の方法。  (61) The method according to (58), wherein the concentration of EDTA mixed with Extract I is 400 mM, and the concentration of phosphate buffer mixed with Extract I is 750 mM.
(62) 環境サンプルから DNAを回収する方法であって、  (62) A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤、 400mM以下の EDTA及び 250rnM以下のリン 酸緩衝液を含む DNA抽出液 IIIの存在下で環境サンプルを beads-beating処 理し、  (a) Beads-beating treatment of the environmental sample in the presence of DNA extract III containing 5% or less surfactant, 400 mM or less EDTA and 250 rnM or less phosphate buffer,
(b) beads-beating処理後の抽出液 IIIを、 400〜1000mMの EDTA、 750〜 2050mMのリン酸緩衝液、又は前記 EDTAとリン酸緩衝液との混合物と混合 して抽出液 IVを調製し、  (b) Extract IV after beads-beating treatment was mixed with 400-1000 mM EDTA, 750-2050 mM phosphate buffer, or a mixture of the EDTA and phosphate buffer to prepare Extract IV. ,
(c) 抽出液 IVを遠心し、  (c) Centrifuge Extract IV,
(d) 得られる上清から DNAを回収する  (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
(63) 抽出液 IVを加熱処理した後、遠心することを特徴とする (62)記載の方法。 (63) The method according to (62), wherein the extract IV is heated and then centrifuged.
(64) 抽出液 IIIにおいて、 EDTAの濃度が 300mMであり、 かつ、 リン酸緩衝 液の濃度が lOOmMである (62)又は (63)記載の方法。 (64) The method according to (62) or (63), wherein in the extract III, the EDTA concentration is 300 mM, and the phosphate buffer concentration is 100 mM.
(65) 抽出液 IIIと混合する EDTAの濃度が 400mMであり、 かつ、 抽出液 III と混合するリン酸緩衝液の濃度が 750mMである (62)又は (63)記載の方法。 (65) The method according to (62) or (63), wherein the concentration of EDTA mixed with Extract III is 400 mM, and the concentration of phosphate buffer mixed with Extract III is 750 mM.
(66) 環境サンプルから DNAを回収する方法であって、 (66) A method for recovering DNA from an environmental sample,
(a) 100〜400mMの EDTA及び 250〜1500mMのリン酸緩衝液を含む DNA 抽出液 Vの存在下で環境サンプルを加熱処理し、  (a) heat-treating an environmental sample in the presence of a DNA extract V containing 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer,
(b) 加熱処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after the heat treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の界面活性剤、 400mM以下の EDTA 及び 250mM 以下のリン酸緩衝液を含む DNA 抽出液 III の存在下で beads-beatin 処 ίΐし、 (c) Residual environmental samples should be prepared in the presence of DNA extract III containing 5% or less of detergent, 400 mM or less of EDTA, and 250 mM or less of phosphate buffer. beads-beatin processing
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取し、  (d) Centrifuge Extract III after beads-beating treatment and collect supernatant,
(e) 工程 (b)及び/又は (d)において得られた上清から DNAを回収する 工程を含む前記方法。  (e) The above method, comprising the step of collecting DNA from the supernatant obtained in step (b) and / or (d).
(67) 環境サンプルから DNAを回収する方法であって、 (67) A method for recovering DNA from an environmental sample,
(a) 100〜400mMの EDTA及び 250〜： L500mMのリン酸緩衝液を含む DNA 抽出液 Vの存在下で環境サンプルを beads-beating処理し、  (a) EDTA of 100 to 400 mM and 250 to: beads-beating treatment of environmental sample in the presence of DNA extract V containing phosphate buffer of L500 mM,
(b) beads-beating処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after beads-beating treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の SDS、 400mM以下の EDTA及 び 250mM 以下のリ ン酸緩衝液を含む DNA 抽出液 III の存在下で beads-beating処 レ、  (c) The remaining environmental sample was subjected to beads-beating treatment in the presence of DNA Extract III containing 5% or less of SDS, 400 mM or less of EDTA, and 250 mM or less of phosphate buffer.
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取し、  (d) Centrifuge Extract III after beads-beating treatment and collect supernatant,
(e) 工程 (b)及び/又は (d)において得られた上清から DNAを回収する 工程を含む前記方法。  (e) The above method, comprising the step of collecting DNA from the supernatant obtained in step (b) and / or (d).
(68) 環境サンプルから DNAを回収する方法であって、 (68) A method for recovering DNA from an environmental sample,
(a) 200〜800mM の EDTA 及び 250〜2000mM のリン酸緩衝液を含む DNA抽出液の存在下で環境サンプルを第一加熱処理し、  (a) first heat treating the environmental sample in the presence of a DNA extract containing 200-800 mM EDTA and 250-2000 mM phosphate buffer,
(b) 5 %以下の界面活性剤と環境サンプルとを混合して当該混合物を第二 加熱処理し、  (b) 5% or less of a surfactant and an environmental sample are mixed and the mixture is subjected to a second heat treatment,
(c) 第二加熱処理後の抽出液を遠心し、  (c) centrifuging the extract after the second heat treatment,
(d) 得られる上清から DNAを回収する  (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
(69) 環境サンプルから DNAを回収する方法であって、 (69) A method for recovering DNA from an environmental sample,
(a) 100〜400mM の EDTA 及び 250〜： 1500mM のリン酸緩衝液を含む DNA抽出液の存在下で環境サンプルを beads-beating処理し、  (a) 100-400 mM EDTA and 250-: beads-beating of environmental sample in the presence of DNA extract containing 1500 mM phosphate buffer,
(b) beads-beating処理後の環境サンプルと 5 %以下の界面活性剤とを混合 して当該混合物を加熱処理し、  (b) Mix the environmental sample after beads-beating treatment with 5% or less of surfactant, heat-treat the mixture,
(c) 加熱処理後の抽出液を遠心し、 (d) 得られる上 ¾から DNAを回収する (c) centrifuging the extract after the heat treatment, (d) Recover DNA from the obtained 上
工程を含む前記方法。  The method comprising the steps of:
(70) 抽出液 Vにおいて、 EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝 液の濃度が 750ηιΜである (66)又は (67)記載の方法。 (70) The method according to (66) or (67), wherein in the extract V, the EDTA concentration is 400 mM, and the phosphate buffer concentration is 750ηιΜ.
(71) EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある (68)又は (69)記載の方法。 (71) The method according to (68) or (69), wherein the EDTA concentration is 400 mM and the phosphate buffer concentration is 750 mM.
(72) DNAの回収が、 遠心後の上清と陽イオン界面活性剤及び塩とを混合して DNAを精製する工程を含む (45)〜(57)のいずれか 1項に記載の方法。  (72) The method according to any one of (45) to (57), wherein recovering the DNA comprises a step of mixing the supernatant after centrifugation with a cationic surfactant and a salt to purify the DNA.
(73) DNAの回収が、抽出液 IIと陽イオン界面活性剤及び塩とを混合して DNA を精製する工程を含む (58)〜(61)のいずれか 1項に記載の方法。  (73) The method according to any one of (58) to (61), wherein recovering the DNA comprises a step of purifying the DNA by mixing the extract II with a cationic surfactant and a salt.
(74) DNA の回収が、 抽出液 IV と陽イオン界面活性剤及び塩とを混合して DNAを精製する工程を含む (62)〜(65)のいずれか 1項に記載の方法。  (74) The method according to any one of (62) to (65), wherein recovering the DNA comprises a step of purifying the DNA by mixing the extract IV with a cationic surfactant and a salt.
(75) DNAの回収が、加熱処理後の抽出液 V及び/又は beads-beating後の抽出 液 IIIと陽ィォン界面活性剤及び塩とを混合して DNAを精製する工程を含む (66)記載の方法。  (75) The step of recovering DNA includes a step of purifying DNA by mixing extract V after heat treatment and / or extract III after beads-beating with a cationic surfactant and a salt. the method of.
(76) DNAの回収が、 beads-beating後の抽出液 III及び/又は beads-beating 後の抽出液 Vと陽イオン界面活性剤及び塩とを混合して DNAを精製するェ 程を含む (67)記載の方法。  (76) Recovering DNA includes purifying DNA by mixing Extract III after beads-beating and / or Extract V after beads-beating with cationic surfactant and salt (67 ) Described method.
(77) DNAの回収が、 第二加熱後の抽出液と陽イオン界面活性剤及び塩とを混 合して DNAを精製する工程を含む (68)記載の方法。  (77) The method according to (68), wherein the step of recovering the DNA comprises a step of mixing the extract after the second heating with a cationic surfactant and a salt to purify the DNA.
(78) DNAの回収が、 加熱処理後の抽出液と陽イオン界面活性剤及び塩とを混 合して DNAを精製する工程を含む (69)記載の方法。  (78) The method according to (69), wherein recovering the DNA comprises a step of mixing the extract after the heat treatment, a cationic surfactant and a salt to purify the DNA.
(79) 陽イオン界面活性剤が CTABである（72)〜(78)のいずれか 1項に記載の方 法。  (79) The method according to any one of (72) to (78), wherein the cationic surfactant is CTAB.
(80) 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモニゥ ム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからなる群 から選択される少なくとも 1 つである（72)〜(78)のいずれか 1項に記載の方 法。 (81) 混合液中における陽イオン界面活性剤の濃度が 1〜3 %である (72)〜(78) のいずれか 1項に記載の方法。 (80) The salt according to (72) to (78), wherein the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate. The method described in any one of the above items. (81) The method according to any one of (72) to (78), wherein the concentration of the cationic surfactant in the mixture is 1 to 3%.
(82) 混合液中における塩の濃度が 0.7〜2·1Μである（72)〜(78)のいずれか 1項 に記載の方法。  (82) The method according to any one of (72) to (78), wherein the salt concentration in the mixed solution is 0.7 to 2.1Μ.
(83) 混合液中における陽イオン界面活性剤の濃度が 2〜3 %であり、 かつ、 混 合液中における塩の濃度が 1.0Mである（72)〜(78)のいずれか 1項に記載の方 法。 (83) The method according to any one of (72) to (78), wherein the concentration of the cationic surfactant in the mixed solution is 2 to 3%, and the concentration of the salt in the mixed solution is 1.0 M. The method described.
(84) ρΗ7.0未満の条件で精製することを特徴とする (72)〜(78)のいずれか 1項 に記載の方法。  (84) The method according to any one of (72) to (78), wherein the purification is performed under a condition of ρ 未 満 7.0 or less.
(85) DNA の回収が、 2-プロパノール、 エタノール又はポリエチレングリコー ルの存在下で DNAを沈殿させる工程を含む (45)〜(84)のいずれか 1項に記載 の方法。 (85) The method according to any one of (45) to (84), wherein recovering the DNA comprises a step of precipitating the DNA in the presence of 2-propanol, ethanol or polyethylene glycol.
(86) ポリエチレンダリコールの濃度が 5.0〜7.5 %である (85)記載の方法。  (86) The method according to (85), wherein the concentration of polyethylene dalicol is 5.0 to 7.5%.
(87) ρΗ7.0以上の条件で DNAを沈殿させることを特徴とする (85)記載の方法。 (88) 環境サンプルから DNAを回収する方法であって、  (87) The method according to (85), wherein the DNA is precipitated under a condition of ρΗ7.0 or more. (88) A method for recovering DNA from an environmental sample,
(a) 1 %の界面活性剤、 400mMの EDTA及び 750mMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを処理して DNAを抽出し、  (a) extracting the DNA by treating the environmental sample in the presence of a DNA extract containing 1% surfactant, 400 mM EDTA and 750 mM phosphate buffer,
(b) DNA抽出液と 2%の CTAB及び 1 Mの塩とを混合して DNAを精製し、 (d) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記方法。  (b) mixing the DNA extract with 2% CTAB and 1 M salt to purify the DNA, and (d) precipitating the purified DNA in the presence of polyethylene glycol.
(89) 環境サンプルから DNAを回収する方法であって、  (89) A method for recovering DNA from an environmental sample,
(a) 1 %の界面活性剤、 300mMの EDTA及び 100mMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを beads-beating処理し、  (a) beads-beating the environmental sample in the presence of a DNA extract containing 1% surfactant, 300 mM EDTA and 100 mM phosphate buffer,
(b) beads-beating処理後の抽出液と、 600mMの EDTA及び 2050mMのリ ン酸緩衝液とを混合し、  (b) Mix the extract after beads-beating treatment with 600 mM EDTA and 2050 mM phosphate buffer,
(c) 工程 (b)により得られる抽出液を遠心し、  (c) centrifuging the extract obtained in step (b),
(d) 得られる上清と、 2%の CTAB及び 1 Mの塩とを混合して DNAを精製 し、 (e) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記方法。 (d) Purify the DNA by mixing the resulting supernatant with 2% CTAB and 1 M salt, (e) the method comprising a step of precipitating the purified DNA in the presence of polyethylene glycol;
(90) 環境サンプルから DNAを回収する方法であって、  (90) A method for recovering DNA from an environmental sample,
(a) 1 %の界面活性剤、 300mMの EDTA及び lOOmMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを beads-beating処理し、  (a) beads-beating the environmental sample in the presence of a DNA extract containing 1% surfactant, 300 mM EDTA and 100 mM phosphate buffer,
(b) beads-beating処理後の抽出液と、 600mMの EDTA及び 2050mMのリ ン酸緩衝液とを混合して加熱処理し、  (b) Mix the extract after beads-beating treatment with 600 mM EDTA and 2050 mM phosphate buffer, heat-treat,
(c) 加熱処理後の抽出液を遠心し、  (c) centrifuging the extract after the heat treatment,
(d) 得られる上清と、 2%の CTAB及び 1 Mの塩とを混合して DNAを精製 し、  (d) Purify the DNA by mixing the resulting supernatant with 2% CTAB and 1 M salt,
(e) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記方法  (e) precipitating the purified DNA in the presence of polyethylene glycol.
(91) 環境サンプルから DNAを回収する方法であって、  (91) A method for recovering DNA from an environmental sample,
(a) 1%の界面活性剤、 300mMの EDTA及び lOOmMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを beads-beating処理し、  (a) beads-beating the environmental sample in the presence of a DNA extract containing 1% surfactant, 300 mM EDTA and 100 mM phosphate buffer,
(b) beads-beating処理後の抽出液を遠心し、  (b) Centrifuge the extract after beads-beating treatment,
(c) 得られる上清と、 2%CTAB及び 1 Mの塩とを混合して DNAを精製し、 (c) The resulting supernatant is mixed with 2% CTAB and 1 M salt to purify DNA,
(d) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記方法。 (d) The method comprising a step of precipitating the purified DNA in the presence of polyethylene glycol.
(92) 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモニゥ ム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからなる群 から選択される少なくとも 1つである（88)〜(91)のいずれか 1 項に記載の方 法。 (92) The salt according to (88) to (91), wherein the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate. The method described in any one of the above.
(93) ポリエチレンダリコールの濃度が 5.0〜7.5%である（88)〜(91)のいずれか 1項に記載の方法。  (93) The method according to any one of (88) to (91), wherein the concentration of polyethylene dalicol is 5.0 to 7.5%.
(94) pH7.0以上の条件で DNAを沈殿させることを特徵とする (88)〜(91)のい ずれか 1項に記載の方法。  (94) The method according to any one of (88) to (91), wherein the DNA is precipitated under conditions of pH 7.0 or more.
(95) 5 %以下の界面活性剤、又は前記界面活性剤と beads-beating用ビーズと の組合せを含む、 '環境サンプルからの DNA抽出用キット。 (95) 5% or less surfactant, or the surfactant and beads for beads-beating Kit for DNA extraction from environmental samples, including combinations of
(96) アル力リ性緩衝液並びに EDTA及び/又はリン酸緩衝液をさらに含む (95) 記載のキット。  (96) The kit according to (95), further comprising an alkaline buffer and an EDTA and / or phosphate buffer.
(97) アル力リ性緩衝液が Tris緩衝液である (96)記載のキット。  (97) The kit according to (96), wherein the alkaline buffer is a Tris buffer.
(98) EDTAの濃度が 50mM〜1200mMの範囲から選ばれるいずれかのもので ある (96)記載のキット。 (98) The kit according to (96), wherein the EDTA concentration is any one selected from the range of 50 mM to 1200 mM.
(99) リン酸緩衝液の濃度が 50mM〜3000mMの範囲から選ばれるいずれかの ものである（96)記載のキット。  (99) The kit according to (96), wherein the concentration of the phosphate buffer is selected from the range of 50 mM to 3000 mM.
(100) DNA抽出液の pHを 7.0以上に調整することができる（96)記載のキット。 (101) 酸性側の pKaを有する pH緩衝液を含む塩溶液、 陽イオン界面活性剤、 又は当該塩溶液と陽イオン界面活性剤との混合物を含む、 環境サンプルから の DNA精製用キット。  (100) The kit according to (96), wherein the pH of the DNA extract can be adjusted to 7.0 or more. (101) A kit for purifying DNA from an environmental sample, comprising a salt solution containing a pH buffer having a pKa on the acidic side, a cationic surfactant, or a mixture of the salt solution and a cationic surfactant.
(102) 酸性側の pKaを有する pH緩衝液が酢酸緩衝液、 リン酸緩衝液、 塩酸緩 衝液又は硫酸緩衝液である (101)記載のキット。 (102) The kit according to (101), wherein the pH buffer having a pKa on the acidic side is an acetate buffer, a phosphate buffer, a hydrochloric acid buffer, or a sulfate buffer.
(103) 陽イオン界面活性剤が CTABである（101)記載のキット。 (103) The kit according to (101), wherein the cationic surfactant is CTAB.
(104) 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモニゥ ム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからなる群 から選択される少なくとも 1つである（101)記載のキット。  (104) The kit according to (101), wherein the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate.
(105) DNA含有溶液の pHを 7.0未満に調整することができる（101)記載のキッ 卜。  (105) The kit according to (101), wherein the pH of the DNA-containing solution can be adjusted to less than 7.0.
(106) アルカリ性緩衝液を含む、 環境サンプルからの DNA回収用キット。  (106) A kit for recovering DNA from an environmental sample, comprising an alkaline buffer.
(107) アル力リ性の緩衝液が Tris緩衝液である（106)記載のキット。  (107) The kit according to (106), wherein the buffer is a Tris buffer.
(108) さらに 2-プロパノ一ル、エタノール又はポリエチレンダリコールを含む、 (106)記載のキット。  (108) The kit according to (106), further comprising 2-propanol, ethanol or polyethylene dalicol.
(109) DNA含有溶液の pHを 7.0以上に調整することができる（106)記載のキッ 卜。 (109) The kit according to (106), wherein the pH of the DNA-containing solution can be adjusted to 7.0 or more.
(110) 上記 (95)〜(100)のいずれか 1項に記載の DNA抽出用キット、 上記 (101) 〜(105)のいずれか 1項に記載の DNA精製用キット、 及び上記 (106)〜(： 109) のいずれか 1項に ί3載の DNA 回収用キットからなる群から選ばれる少なく とも 2組のキットを含む、 環境サンプルからの DNA取得用キッ卜セット。 図面の簡単な説明 (110) The DNA extraction kit according to any one of (95) to (100), the DNA purification kit according to any one of (101) to (105), and (106) ~ (： 109) A kit for obtaining DNA from an environmental sample, comprising at least two kits selected from the group consisting of the kits for DNA recovery described in any one of the above-mentioned ί3. Brief Description of Drawings
図 1は、 土壌 Dl iAの定量例を示す図である。  FIG. 1 is a diagram showing an example of quantification of soil DliA.
図 2は、 各種界面活性剤による土壌 DNAの抽出量を示す図である。  FIG. 2 is a diagram showing the amount of soil DNA extracted by various surfactants.
図 3は、弥生圃場堆肥区土壌からの DNA抽出に SDS濃度が与える影響を示す 図である。  Figure 3 shows the effect of SDS concentration on DNA extraction from compost plotted soil in Yayoi field.
図 4は、 SDS濃度が土壌 DNA抽出に与える影響を示す図である。  FIG. 4 shows the effect of SDS concentration on soil DNA extraction.
図 5は、 抽出液の pHが土壌 DNAに与える影響を示す図である。 各バーの上 に記載の数字は beads-beating後の抽出液の pHを表す。  FIG. 5 is a diagram showing the effect of the pH of the extract on soil DNA. The numbers above each bar represent the pH of the extract after beads-beating.
図 6は、 EDTA濃度が火山灰土壌からの DNA抽出に与える影響を示す図であ る。  Figure 6 shows the effect of EDTA concentration on DNA extraction from volcanic ash soil.
図 7は、 EDTA濃度が DNA抽出量に与える影響を示す図である。  FIG. 7 is a graph showing the effect of EDTA concentration on DNA extraction.
図 8 Aは、 抽出液の EDTA濃度と金属元素の抽出量との関係を示す図である。 図 8 Bは、 抽出液の EDTA濃度と金属元素の抽出量との関係を示す図である。 図 8 Cは、 抽出液の EDTA濃度と金属元素の抽出量との関係を示す図である。 図 9 Aは、 抽出液の EDTA濃度と EDTA-金属元素複合体形成率との関係を示 す図である。  FIG. 8A is a diagram showing the relationship between the EDTA concentration of the extract and the amount of extracted metal element. FIG. 8B is a diagram showing the relationship between the EDTA concentration of the extract and the amount of extraction of the metal element. FIG. 8C is a diagram showing the relationship between the EDTA concentration of the extract and the amount of extraction of the metal element. FIG. 9A is a diagram showing the relationship between the EDTA concentration of the extract and the EDTA-metal element complex formation rate.
図 9 Bは、 抽出液の EDTA濃度と EDTA-金属元素複合体形成率との関係を示 す図である。  FIG. 9B is a diagram showing the relationship between the EDTA concentration of the extract and the EDTA-metal element complex formation rate.
図 9 Cは、 抽出液の EDTA濃度と EDTA-金属元素複合体形成率との関係を示 す図である。  FIG. 9C is a diagram showing the relationship between the EDTA concentration of the extract and the EDTA-metal element complex formation rate.
図 1 0 Aは、 土壌から DNAを繰返し抽出したときの DNA収量を示す図であ る。  FIG. 10A shows the DNA yield when DNA is repeatedly extracted from soil.
図 1 0 Bは、 土壌から DNAを繰返し抽出したときの DNA収量を示す図であ る。  FIG. 10B is a view showing DNA yield when DNA is repeatedly extracted from soil.
図 1 0 Cは、 土壌から DNAを繰返し抽出したときの DNA収量を示す図であ る。 Figure 10C shows the DNA yield when DNA was repeatedly extracted from soil. The
図 1 1は、 再添加した EDTA溶液の新たな土壌 DNAの抽出量の計算結果を示 す図である。  FIG. 11 is a diagram showing the calculation results of the amount of fresh soil DNA extracted from the re-added EDTA solution.
図 1 2は、 新たに添加した EDTAにより抽出できたと考えられる土壌 DNA量 を示す図である。 '  FIG. 12 is a diagram showing the amount of soil DNA considered to have been able to be extracted by newly added EDTA. '
図 1 3 Aは、 新たに添加した EDTAにより土壌から抽出された金属量 (A1及び Fe)を示す図である。  FIG. 13A shows the amounts of metals (A1 and Fe) extracted from soil by newly added EDTA.
図 1 3 Bは、 新たに添加した EDTAにより土壌から抽出された金属量 (Ca及び Mg)を示す図である。  FIG. 13B is a diagram showing the amounts of metals (Ca and Mg) extracted from soil by newly added EDTA.
図 1 4は、高濃度 EDTA溶液を beads-beating後に添加した弥生圃場対照区土 壌からの土壌. DNA抽出結果を示す図である。  FIG. 14 is a diagram showing the results of extracting soil and DNA from the Yayoi field control plot soil to which a high-concentration EDTA solution was added after beads-beating.
図 1 5 Aは、 抽出液の EDTA濃度が土壌 DNA抽出量に及ぼす影響を示す図で ある。  FIG. 15A shows the effect of the EDTA concentration of the extract on the amount of soil DNA extracted.
図 1 5 Bは、 抽出液の P043-濃度が土壌 DNA抽出量に及ぼす影響を示す図で ある。 FIG. 15B shows the effect of the P04 3 -concentration of the extract on the amount of soil DNA extracted.
図 1 6 Αは、 火山灰土壌における抽出液中の EDTA-リン酸濃度が土壌 DNA抽 出に及ぼす影響を示す図である。  Figure 16Α shows the effect of EDTA-phosphate concentration in the extract of volcanic ash soil on soil DNA extraction.
図 1 6 Bは、 非火山灰土壌における抽出液中の EDTA-リン酸濃度が土壌 DNA 抽出に及ぼす影響を示す図である。  Figure 16B shows the effect of EDTA-phosphate concentration in the extract on non-volcanic ash soil on soil DNA extraction.
図 1 7は、 2 stepによる DNA抽出における抽出溶液のリン酸イオン濃度と土 壌 DNAの収量との関係を示す図である。  FIG. 17 is a diagram showing the relationship between the phosphate ion concentration of the extraction solution and the yield of soil DNA in the two-step DNA extraction.
図 1 8 Aは、高濃度の EDTA及びリン酸緩衝液を用いて 2段階の操作で抽出し た土壌 DNAの収量を示す図である。  FIG. 18A is a diagram showing the yield of soil DNA extracted by a two-step operation using a high concentration of EDTA and a phosphate buffer.
図 1 8 Bは、 EDTA、 リン酸緩衝液を用いて改良 2 step法で弥生圃場対照区土 壌から抽出した土壌 DNAの収量を示す図である。 図中に示した EDTA濃度およ び P04 3 -濃度は beads-beating時の抽出液における濃度であり、 beads-beating 後にそれぞれ調製した溶液添加後の終濃度はすべて 4 0 0 mMEDTA/750mM PO4 3 _である。 図 1 9は、 改良 2 stepによる DNA抽出結果を示す電気泳動写真である。 FIG. 18B shows the yield of soil DNA extracted from Yayoi field control plot soil by the improved 2-step method using EDTA and phosphate buffer. Beauty P0 4 Oyo EDTA concentrations shown in Figure 3 - concentration is the concentration in the extraction solution at the time of beads-beating, beads-beating final concentration after addition of the solution prepared respectively after every 4 0 0 mMEDTA / 750mM PO 4 3 _. Figure 19 is an electrophoresis photograph showing the results of DNA extraction by the improved 2 steps.
図 2 O Aは、 各沈殿剤を使用したときの DNAの回収結果を示す図である。 図 2 0 Bは、 土壌 DNAの沈殿方法の違いによる腐植の除去効果を示す図であ る。  FIG. 2 OA shows the results of DNA recovery when each precipitant was used. FIG. 20B is a diagram showing the effect of removing humus by the difference in the method of precipitating soil DNA.
図 2 1は、 PEG濃度が土壌 DNAの回収に与える影響を示す図である。  FIG. 21 shows the effect of PEG concentration on soil DNA recovery.
図 2 2 Aは、 CTABによる簡易精製を行った場合の DNAの回収量を示す図で ある。  FIG. 22A shows the amount of DNA recovered when simple purification was performed using CTAB.
図 2 2 Bは、 CTAB処理による腐植物質の除去効果を示す図である。  FIG. 22B shows the effect of removing humic substances by CTAB treatment.
図 2 2 Cは、 CTAB処理による腐植物質の除去効果を示す図である。  FIG. 22C is a diagram showing the effect of removing humic substances by CTAB treatment.
図 2 3は、オリジナルの 4条件による土壌 DNAの収量の比較を示す図である。 図 2 4は、 オリジナルの 4条件による抽出 DNAのサイズを示す電気泳動写真 である。  FIG. 23 shows a comparison of soil DNA yields under the four original conditions. Figure 24 is an electrophoresis photograph showing the size of the extracted DNA under the original four conditions.
図 2 5は、 土壌 DNA収量における本発明の方法と既往の方法との比較を示す 図である。  FIG. 25 is a diagram showing a comparison between the method of the present invention and a conventional method in soil DNA yield.
図 2 6 Aは、 オリジナル 1 step法により抽出した土壌 DNAの PCR反応を用 いた純度検定結果を示す電気泳動写真である。 上 2つのパネルは鍀型濃度 100ng/50 L 中 2つのパネルは铸型濃度 50ng/50 l、 した 2つのパネルは鍀型 濃度 10ng/50 lである。  Fig. 26A is an electrophoresis photograph showing the results of a purity test using a PCR reaction of soil DNA extracted by the original one-step method. The upper two panels have a type I concentration of 100ng / 50l, the two panels have a type I concentration of 50ng / 50l, and the other two panels have a type I concentration of 10ng / 50l.
図 2 6 Bは、 オリジナル 2 step加熱法により抽出した土壌 DNAの PCR反応 を用いた純度検定結果を示す電気泳動写真である。各パネルにおける踌型濃度は、 図 26Aと同様である。  Figure 26B is an electrophoresis photograph showing the results of a purity assay using PCR reaction of soil DNA extracted by the original two-step heating method. The Δ type concentration in each panel is the same as in FIG. 26A.
図 2 7 Aは、 抽出した DNAの評価結果を示す電気泳動写真である。 レーン 1 〜5は弥生圃場対照区土壌、 レーン 6〜10は千葉農試森林土壌、 レーン 11〜： L5 は茨城農試森林土壌、 レーン 16〜20 は田無農場牧草地土壌から抽出した DNA の泳動図である。 各土壌におけるレーンは、 左から順にオリジナル IStep 法、 2Step法、 2Step加熱法、 LCB法及び Zhou et al.(1996)により抽出した土壌 DNA の PCR産物を表す。  FIG. 27A is an electrophoresis photograph showing the evaluation results of the extracted DNA. Lanes 1-5 are the soils of the Yayoi field control plot, Lanes 6-10 are the Chiba agricultural trial forest soil, Lane 11-: L5 is the Ibaraki agricultural trial forest soil, and Lane 16-20 is the migration of DNA extracted from Tanashi farm pasture soil FIG. Lanes in each soil represent the original IStep method, 2Step method, 2Step heating method, LCB method, and PCR products of soil DNA extracted by Zhou et al. (1996) in order from the left.
図 2 7 Bは、 抽出した DNAの評価結果を示す電気泳動写真である。 レーン 1 〜 8は群馬畜試牧草地、 レーン 9〜： L6は栃木森林土壌から抽出した DNAの泳動 図である。各土壌におけるレーンは、左から順にオリジナル IStep法、 2Step法、 2Step加熱法、 LCB法、 Zhou et al.(1996)の方法、 Cullen&Hirsch (1996) の方 法、 UltraClean Soil DNA kit、 Bio 101 Fast DNA spin kitによつて抽出した土 壌 DNAの PCR産物である。 FIG. 27B is an electrophoresis photograph showing the evaluation results of the extracted DNA. Lane 1 8 to 8 are the Gunma animal pastures, and lanes 9 to: L6 is the electrophoretic diagram of DNA extracted from Tochigi forest soil. The lanes in each soil are, in order from the left, the original IStep method, 2Step method, 2Step heating method, LCB method, the method of Zhou et al. (1996), the method of Cullen & Hirsch (1996), UltraClean Soil DNA kit, Bio 101 Fast DNA PCR product of soil DNA extracted by spin kit.
図 2 7 Cは、 抽出した DNAの評価結果を示す電気泳動写真である。 レーン 1 〜 8は東北大学森林土壌、 レーン 9〜16は草地試験場永年採草地土壌から抽出し た DNAの泳動図である。 各土壌におけるレーンは図 27Aと同様である。  FIG. 27C is an electrophoresis photograph showing the evaluation results of the extracted DNA. Lanes 1 to 8 are the migration plots of DNA extracted from Tohoku University forest soil, and lanes 9 to 16 are the grass extractive soil from the permanent grass field. Lanes for each soil are similar to FIG. 27A.
図 2 7 Dは、 抽出した DNAの評価結果を示す電気泳動写真である。 レーン 1 〜8は埼玉農試畑土壌、 レーン 9〜： 16は大阪農試畑土壌から抽出した DNAの泳 動図である。 各土壌におけるレーンは図 27Aと同様である。  FIG. 27D is an electrophoresis photograph showing the evaluation results of the extracted DNA. Lanes 1 to 8 are the Saitama agricultural test field soil, and lanes 9 to 16 are swimming diagrams of DNA extracted from the Osaka agricultural test field soil. Lanes for each soil are similar to FIG. 27A.
図 2 7 Eは、 抽出した DNAの評価結果を示す電気泳動写真である。 レーン 1 〜 8は兵庫農試畑土壌、 レーン 9〜16は奈良農試畑土壌から抽出した DNAの泳 動図である。 各土壌におけるレーンは図 27Aと同様である。  FIG. 27E is an electrophoresis photograph showing the evaluation results of the extracted DNA. Lanes 1 to 8 are the Hyogo Agricultural Experiment Station soils, and Lanes 9 to 16 are the swimming diagrams of DNA extracted from the Nara Agricultural Experiment Station soil. Lanes for each soil are similar to FIG. 27A.
図 2 8は、 CTABによる DNA精製時に塩溶液の酸性緩衝能により pHを低下 させることが DNA抽出液の純度に与える影響を示す図である。  FIG. 28 shows the effect of lowering the pH due to the acidic buffering capacity of the salt solution on the purity of the DNA extract during the purification of DNA by CTAB.
図 2 9は、 CTABによる DNA精製時に塩溶液の酸性緩衝能により pHを低下 させることが沈殿回収後の DNA溶液の純度に与える影響を示す図である。  FIG. 29 is a diagram showing the effect of lowering the pH due to the acidic buffering capacity of the salt solution during DNA purification by CTAB on the purity of the DNA solution after precipitation and recovery.
図 3 0は、 CTABによる DNA精製時に塩溶液の酸性緩衝能により pHを低下 させることが DNAの回収量に与える影響影響を示す図である（PEG溶液を DNA の回収に使用）。  FIG. 30 shows the effect of lowering the pH due to the acidic buffering capacity of the salt solution on the amount of DNA recovered during the purification of DNA by CTAB (PEG solution was used for DNA recovery).
図 3 1は、 CTABによる精製時に塩溶液の酸性緩衝能により pHを低下させる ことが DNA抽出液の純度に与える影響を示す図である （塩溶液に CH3COONa および NaClの混合液を使用した場合）。 Figure 31 shows the effect of lowering the pH due to the acidic buffer capacity of the salt solution on the purity of the DNA extract during the purification by CTAB. (A mixed solution of CH 3 COONa and NaCl was used for the salt solution.) Case).
図 3 2は、 CTABによる精製時に塩溶液の酸性緩衝能による pHを低下させる ことが PEG溶液により回収した DNAの純度に与える影響を示す図である（塩溶 液に CH3COONaおよび NaClの混合液を使用した場合）。 図 3 3は、 CTAB ίこよる精製時に塩溶液の酸性緩衝能により ρΗを低下させる ことが DNAの回収量に与える影響を示す図である （塩溶液に CH3COONaおよ び NaClの混合液を使用、 DNAの回収に PEG溶液を使用）。 Figure 32 shows the effect of lowering the pH due to the acidic buffering capacity of the salt solution during purification by CTAB on the purity of the DNA recovered with the PEG solution (a mixed solution of CH3COONa and NaCl was added to the salt solution). If used). Figure 33 shows the effect of reducing ρΗ due to the acidic buffering capacity of the salt solution on the amount of DNA recovered during purification by CTAB (using a mixture of CH3COONa and NaCl for the salt solution). Use PEG solution for DNA recovery).
図 3 4は、 PEG溶液のアル力リ緩衝能の有無が回収する DNAの純度に与える 影響を示す図である。  FIG. 34 shows the effect of the presence or absence of the buffering capacity of the PEG solution on the purity of the recovered DNA.
図 3 5は、 PEG溶液のアルカリ緩衝能の有無が DNAの回収量に与える影響を 示す図である。 .  FIG. 35 shows the effect of the presence or absence of the alkaline buffering capacity of the PEG solution on the amount of DNA recovered. .
図 3 6は、 DNAを回収するときに使用する PEG溶液のアルカリ緩衝能が回収 した DNAの純度に与える影響を示す図である （EDTA 200mM I Na2HP04 375mMの抽出液で得られた土壌 DNA抽出液を対象として）。  Figure 36 shows the effect of the alkaline buffering capacity of the PEG solution used to recover DNA on the purity of the recovered DNA (EDTA 200 mM I Na2HP04 Soil DNA extract obtained with 375 mM extract) Targeting).
図 3 7は、 CTABによる精製時の塩溶液組成と DNA回収時に使用するの PEG 溶液のアル力リ緩衝能が DNAの回収量に与える影響を示す図である （EDTA 200mM I Na2HP04 375mMの抽出液で得られた土壌 DNA抽出液を対象として）。 図 3 8は、 加熱抽出による高分子土壌 DNAの抽出条件の検討結果を示す図で ある （弥生対照区土壌について）。  Figure 37 shows the effect of the salt solution composition during purification by CTAB and the buffering capacity of the PEG solution used for DNA recovery on the amount of DNA recovered (EDTA 200 mM I Na2HP04 375 mM extract). For the soil DNA extract obtained in the above). Figure 38 shows the results of examining the extraction conditions for polymer soil DNA by heat extraction (for the Yayoi control plot soil).
図 3 9は、 加熱抽出による高分子土壌 DNAの抽出条件の検討結果を示す図で ある。  Figure 39 is a diagram showing the results of examining the conditions for extracting polymer soil DNA by heat extraction.
図 4 0は、 抽出液組成と物理処理が糞便からの DNA抽出量に与える影響を示 す図である。  FIG. 40 shows the effects of the composition of the extract and the physical treatment on the amount of DNA extracted from feces.
図 4 1は、 糞便から抽出した DNAの電気泳動写真である。  Figure 41 is an electrophoresis photograph of DNA extracted from feces.
図 4 2は、 糞便からの DNA抽出法の検討結果を示す図である。  FIG. 42 is a diagram showing the results of studies on a DNA extraction method from feces.
'図 4 3は、 糞便から抽出した DNAの純度を示す図である。  FIG. 43 shows the purity of DNA extracted from feces.
図 4 4は、 抽出液組成と物理処理が堆肥からの DNA抽出量に与える影響を示 す図である。  Figure 44 shows the effect of extract composition and physical treatment on the amount of DNA extracted from compost.
図 4 5は、 堆肥及び活性汚泥から抽出した DNAの電気泳動写真である。  Figure 45 is an electrophoretic photograph of DNA extracted from compost and activated sludge.
図 4 6は、 堆肥からの DNA抽出法の検討結果を示す図である。  Figure 46 is a diagram showing the results of studies on a method for extracting DNA from compost.
図 4 7は、 堆肥から抽出した DNAの純度を示す図である。  FIG. 47 is a diagram showing the purity of DNA extracted from compost.
図 4 8は、 活性汚泥からの DNA抽出法の検討結果を示す図である。 図 4 9は、 活性汚泥から抽出した DNAの純度を示す図である。 FIG. 48 is a diagram showing the results of a study on a method for extracting DNA from activated sludge. FIG. 49 shows the purity of DNA extracted from activated sludge.
図 5 0は、 湖底堆積物から抽出した DNAの電気泳動写真である。  Figure 50 is an electrophoretic photograph of DNA extracted from lake sediments.
図 5 1は、 湖底堆積物からの DNA抽出法の検討結果を示す図である。  Figure 51 shows the results of a study on DNA extraction from lake sediments.
図 5 2は、 湖底堆積物から抽出した DNAの純度を示す図である。  Figure 52 shows the purity of DNA extracted from lake sediments.
図 5 3は、 土壌: DNAの DGGE解析結果を示す図である。  FIG. 53 is a diagram showing a result of DGGE analysis of soil: DNA.
図 5 4は、 糞便 DNAの DGGE解析結果を示す図である。  FIG. 54 is a diagram showing a DGGE analysis result of fecal DNA.
図 5 5は、 堆肥 DNA及び活性汚泥 DNAの DGGE解析結果を示す図である。 図 5 6は、 湖底堆積物 DNAの DGGE解析結果を示す図である。 発明を実施するための最良の形態  FIG. 55 shows DGGE analysis results of compost DNA and activated sludge DNA. Figure 56 shows the results of DGGE analysis of lake bottom sediment DNA. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
1 . 概要  1. Overview
本発明は、 環境サンプルから DNAを効率良く抽出、 精製又は回収する方法で あり、 DNAを抽出するステップと、 DNA抽出液中から DNA以外の夾雑物質を 除去する精製するステップが含まれる。  The present invention is a method for efficiently extracting, purifying, or recovering DNA from an environmental sample, and includes a step of extracting DNA and a step of removing contaminants other than DNA from a DNA extract.
本発明においては、 まず環境サンプルからの DNAの抽出に適した条件の 1つ として、 使用する界面活性剤の種類及びその濃度を検討した。 次に、 DNA の抽 出が困難とされる火山灰土壌であっても高収量の DNAを得るための EDTA及び リン酸緩衝液およびその混合液の濃度条件を検討した。 さらに、 DNA を回収す るにあたり、 最適な沈殿条件、 及び夾雑物を含まない最適な精製条件を検討し、 DNAを土壌から効率よく高純度で回収する方法を見出した。  In the present invention, first, as one of the conditions suitable for extracting DNA from an environmental sample, the type of surfactant used and its concentration were examined. Next, the concentration conditions of EDTA, phosphate buffer, and a mixture thereof to obtain a high yield of DNA were examined even in a volcanic ash soil where DNA extraction would be difficult. Furthermore, when recovering DNA, we examined the optimal precipitation conditions and the optimal purification conditions that do not contain contaminants, and found a method for efficiently recovering DNA from soil with high purity.
本発明においては、 界面活性剤を含む DNA抽出液による処理を DNA抽出の 基本操作として採用する。 そして、 抽出の際に beads-beating又は加熱処理を組 合せることによって、 効率良く抽出することを可能とする。 例えば、 環境サンプ ルと、 微小粒子と、 所定濃度の界面活性剤が含まれる DNA抽出液とを混合し、 これらの混合物を beads-beating処理することにより DNAを抽出する。 あるい は、 上記土壌サンプルと所定濃度の界面活性剤との混合物を加熱処理することに より DNAを抽出する。 この DNA抽出操作に、 抽出液の pH、 EDTA又はリン酸 緩衝液および両者の湟合液の濃度条件、 加熱条件、 CTABなどの陽イオン界面活 性剤の濃度条件、 CTAB処理時に添加する塩の種類や濃度、 ポリエチレングリコ ールの濃度条件、 およびポリエチレングリコールによる DNA沈殿操作時の pH などの条件を種々検討することで、 土壌の種類や性質に応じた適切な抽出条件、 回収条件及び精製条件を設定することが可能となる。 . In the present invention, treatment with a DNA extract containing a surfactant is employed as a basic operation of DNA extraction. Combining beads-beating or heat treatment at the time of extraction enables efficient extraction. For example, an environmental sample, microparticles, and a DNA extract containing a surfactant at a predetermined concentration are mixed, and the mixture is subjected to beads-beating treatment to extract DNA. Alternatively, DNA is extracted by heat-treating a mixture of the above soil sample and a surfactant at a predetermined concentration. The pH of the extract, EDTA or phosphate Concentration conditions of buffer solution and Huangwei solution, heating condition, concentration condition of cationic surfactant such as CTAB, kind and concentration of salt added during CTAB treatment, polyethylene glycol concentration condition, and polyethylene glycol By examining various conditions such as pH during the DNA precipitation operation by, it is possible to set appropriate extraction conditions, recovery conditions, and purification conditions according to the type and properties of the soil. .
サンプルから DNAを抽出するときに使用する抽出液全体の pHは Ί以上に調 製し、 その最適 pHは 8.6である （詳細は後述する）。  The pH of the whole extract used to extract DNA from the sample is adjusted to Ί or more, and its optimum pH is 8.6 (details will be described later).
それぞれの環境サンプルに応じて界面活性剤と混合する基本的抽出液組成は、 EDTA溶液及びリン酸緩衝液を含むものである。  The basic extract composition to be mixed with a surfactant for each environmental sample includes an EDTA solution and a phosphate buffer.
環境サンプルの代表例である土壌から DNAを抽出しよゔとした場合、 関東口 —ム層等の火山灰土壌からの DNA抽出においては土壌に含まれる非晶質のアル ミニゥムにより、 DNA が吸着されて回収率が非常に悪くなる。 この土壌による 吸着は EDTA、 リン酸、 又はこれらの両者を高濃度で含む溶液を使用することで 解決することができる。 また、 通常の土壌及びその他の微生物を含む環境サンプ ル、すなわち堆肥や水系の堆積物、活性汚泥や糞便からの DNA抽出においても、 EDTA、 リン酸、 又はこれらの両者を使用することが有効である。  When DNA is extracted from soil, which is a representative example of environmental samples, the DNA is adsorbed by amorphous aluminum contained in the soil in the extraction of DNA from volcanic ash soil such as the Kanto-guchi layer. And the recovery rate is very poor. This adsorption by soil can be solved by using a solution containing high concentrations of EDTA, phosphoric acid, or both. EDTA, phosphoric acid, or both are also effective in extracting DNA from normal soil and other environmental samples containing microorganisms, such as compost and water-based sediments, activated sludge and feces. is there.
この場合の EDTA及びリン酸の濃度は、 それぞれ 50mM〜600mM、 100mM〜 1500mMであり、 EDTAとリン酸との混合液では EDTAが 50mM〜600mM、 リ ン酸が 100mM〜750mMである。  In this case, the concentrations of EDTA and phosphoric acid are 50 mM to 600 mM and 100 mM to 1500 mM, respectively. In a mixture of EDTA and phosphoric acid, EDTA is 50 mM to 600 mM, and phosphoric acid is 100 mM to 750 mM.
但し、 上記濃度は一例であって、 抽出操作の際に行う抽出液添加後の物理的処 理の違いにより、 濃度を適宜変えることが可能である。  However, the above concentration is an example, and the concentration can be appropriately changed depending on a difference in physical treatment after the addition of the extract during the extraction operation.
環境サンプルより得た DNAの精製には、 CTABを初めとする 4級アンモニゥ ム塩 （陽イオン界面活性剤） を用いることができる。 4級アンモニゥム塩として は、 例えば陽イオン界面活性剤である CTAB、 DTABなどが挙げられる。  Quaternary ammonium salts (cationic surfactants) such as CTAB can be used to purify DNA obtained from environmental samples. Examples of the quaternary ammonium salt include cationic surfactants such as CTAB and DTAB.
本発明において、 CTABを用いて DNAを精製するには塩の存在下で行うこと が好ましい。 この塩としては、 例えば塩化ナトリウム、'酢酸ナトリウム等のナト リゥム塩、 塩化力リゥム等の力リゥム塩が挙げられる。  In the present invention, the purification of DNA using CTAB is preferably performed in the presence of a salt. Examples of the salt include sodium salts such as sodium chloride and sodium acetate, and potassium salts such as chloride chloride.
本発明は、 抽出液に陽イオン界面活性剤及び塩溶液を添加することにより、 精 製時の pHを抽出時の pHよりも下げることを特徴とする。 The present invention provides a purification method comprising adding a cationic surfactant and a salt solution to an extract. It is characterized in that the pH during production is lower than the pH during extraction.
ここで、 本発明において 「添加」 とは、 一方の溶液に他方の溶液を添加するこ とを意味するほか、 一方の溶液と他方の溶液とを混合する意味も包含する。  Here, “addition” in the present invention means not only adding one solution to the other solution, but also includes mixing one solution and the other solution.
抽出操作を行った後の DNAを含む抽出溶液は、 抽出液に含まれている緩衝液 により pHは 7.0 '以上、 好ましくは 8.0以上に調整されているが、 CTAB等の陽 イオン界面活性剤と pHを低下させる緩衝能をもつ塩溶液とを抽出液に混合する ことにより、 pH を 7.0未満に低下させる。 従って、 塩溶液には酢酸などの酸性 側の pKaを持つ pH緩衝液を含有させることが好ましい。 この場合の最適な塩溶 液は、 3.33M酢酸ナトリウム /1.67M塩化ナトリウム （pH5.2) の溶液である。 こ の 2種類のナトリウム塩を混合することにより、 抽出液の pHは 6.0以下に低下 させることができる。 なお、 陽イオン界面活性剤及び塩溶液は、 両者を混合した 後に混合液を抽出液に添加することも可能である。  The pH of the extraction solution containing DNA after the extraction operation is adjusted to 7.0 'or more, preferably 8.0 or more by the buffer solution contained in the extraction solution, but the pH is adjusted with a cationic surfactant such as CTAB. The pH is lowered to less than 7.0 by mixing the extract with a salt solution that has a buffering capacity to lower the pH. Accordingly, the salt solution preferably contains a pH buffer having an acidic pKa such as acetic acid. The optimal salt solution in this case is a solution of 3.33M sodium acetate / 1.67M sodium chloride (pH 5.2). By mixing these two kinds of sodium salts, the pH of the extract can be lowered to 6.0 or less. In addition, the cationic surfactant and the salt solution can be added to the extract after mixing the two.
DNA を回収するには、 CTAB 等と塩溶液を添加し、 攪拌後クロ口ホルムを添 加してさらに攪拌し、 遠心後の水相 （上清） を DNA溶液として採取する。  To recover DNA, add CTAB, etc. and a salt solution, stir, add chromate form, further stir, and collect the aqueous phase (supernatant) after centrifugation as a DNA solution.
上記採取された溶液に対し、 ポリエチレングリコール (PEG)等の溶液を添加す ることにより、 DNAのみを選択的に沈殿させる。 PEGを含む溶液は、 精製時に 下げた pHを再びアルカリ性に上昇させる緩衝能を有するものである。 精製時に 酸性側に下げた pHを、 回収時にアルカリ側に pHを上昇させることで、 より選 択的な DNAの沈殿 ·回収が可能であり、 腐植ゃ糖などの夾雑物の共沈を防ぐこ とができる。 アル力.リ性の緩衝能を持つ PEG溶液としては、 例えば Tris-HCl緩 衝系などにより pHが 8.0以上の PEG溶液を作製し、使用することができる。最 適な条件は、 12%PEG/1.5M Tris-HCl(pH8.6)の溶液を DNA抽出液と混合して遠 心を行う。この溶液の混合により、 DNAを回収するときの溶液の pHは 7.5以上、 好ましくは 8.0以上に上昇する。 PEG溶液と Tris-HCl溶液とは、 それぞれ別々 に調製し、 最終の pHが 7.5以上となるように混合してもよい。 ここで、 本明細書において使用する略号の意味は以下の通りである。  By adding a solution such as polyethylene glycol (PEG) to the collected solution, only DNA is selectively precipitated. The solution containing PEG has a buffer capacity to raise the pH lowered during purification to alkaline again. Increasing the pH to the acidic side during purification and increasing the pH to the alkaline side during recovery enables more selective precipitation and recovery of DNA, and prevents co-precipitation of humic sugars and other contaminants. Can be. As a PEG solution having an alkaline buffering capacity, for example, a PEG solution having a pH of 8.0 or more can be prepared and used by a Tris-HCl buffer system or the like. The optimal condition is to mix a solution of 12% PEG / 1.5M Tris-HCl (pH 8.6) with the DNA extract and centrifuge. By mixing this solution, the pH of the solution when recovering DNA rises to 7.5 or more, preferably 8.0 or more. The PEG solution and the Tris-HCl solution may be separately prepared and mixed so that the final pH is 7.5 or more. Here, the meanings of the abbreviations used in the present specification are as follows.
CTAB:セチルトリメチルアンモニゥムブロミ ド（cetyltrimethylammonium bromide) CTAB: Cetyltrimethylammonium bromide bromide)
DTAB: ド デ シ ル ト リ メ チ ル ア ン モ ニ ゥ ム ブ ロ ミ ド
Figure imgf000028_0001
bromide) DTAB: dodecyl trimethyl ammonium bromide
Figure imgf000028_0001
bromide)
EDTA:エチレンジァミン四酢酸 (ethylenediaminetetraacetic acid)  EDTA: ethylenediaminetetraacetic acid
PEG: ポリェチレングリコ一ル (polyethylene glycol)  PEG: polyethylene glycol
SDS: 硫酸ドデシルナトリゥム (sodium dodecyl sulfate)  SDS: sodium dodecyl sulfate
2 . 環境サンプル 2. Environmental samples
本発明において、 DNA抽出の対象となる環境サンプルは、 環境中に存在する 固形又は液体成分であれば特に限定されるものではない。 例えば土壌、 堆肥、 水 系堆積物、 活性汚泥、 糞便などが挙げられ、 これらの環境サンプルを使用目的に 応じて適宜選択することができる。  In the present invention, the environmental sample to be subjected to DNA extraction is not particularly limited as long as it is a solid or liquid component existing in the environment. For example, soil, compost, water-based sediment, activated sludge, feces and the like can be mentioned, and these environmental samples can be appropriately selected according to the purpose of use.
( 1 ) 土壌  (1) Soil
本発明において、 回収の対象となる土壌サンプルは特に限定されるものではな く、 あらゆる土壌を使用することができる。 わが国は火山灰土壌が比較的広い範 囲で分布しており、 火山灰土壌が DNA抽出の対象土壌となることが多いが、 対 象土壌は非火山灰土壌であつてもよい。  In the present invention, the soil sample to be collected is not particularly limited, and any soil can be used. In Japan, volcanic ash soil is distributed over a relatively wide area, and volcanic ash soil is often the target soil for DNA extraction. However, the target soil may be non-volcanic ash soil.
火山灰土壌としては関東地方に主に分布する富士火山灰の堆積物である関東口 —ム層や、 各地の火山噴出物および降灰した火山灰を母材とするァロフェン質黒 ポク土および東北地方の一部に見られるような非ァロフェン質黑ポク土などが挙 げられ、 非火山灰土壌としては日本各地の平野部の灰色低地土、 低地水田土、 火 山灰の影響を受けていない褐色森林土、 赤色土、 黄色土などが挙げられる。  The volcanic ash soils include the Kanto-guchi layer, which is a deposit of Fuji volcanic ash mainly distributed in the Kanto region, as well as the alofen black-pok soil and the Tohoku region, which are based on volcanic eruption products and tephra fallen ash. Non-arofenic soils such as those found in the southern part of the country are listed. The non-volcanic ash soils include gray lowland soil, lowland paddy soil, and brown forest soil unaffected by fire ash in plains throughout Japan. , Red soil, yellow soil and the like.
( 2 ) 堆肥 (2) Compost
堆肥は作物の栽培および土壌の生産性の維持のためには欠く事のできない有機 質肥料である。 堆肥は通常否わらや麦わらなどの畑作物の植物残渣もしくは落ち 葉や木材チップのような木質系の材料に無機肥料や家畜糞を窒素源として添加し て、 堆積し、 微生物による分解作用 （醱酵作用） により、 その一部が分解される ことで作られるもの ある。 また、牛糞や馬糞、豚糞、鶏糞などの家畜糞を堆積、 醱酵させることによつても作られる。 これらの堆肥の特徴は醱酵前と比較すると 堆肥化後のサンプルでは有機物の分解作用によって増殖した微生物菌体が多量に 含まれることが挙げられる。 これらの微生物は土壌に添加された後、 肥料源とし て有用のみならず'、作物の生育を促進する有用微生物としても重要なものである。 また近年、 農薬や PCB などの難分解性有機物の処理にこのような堆肥由来の微 生物が利用されることも多い。 Compost is an essential organic fertilizer for growing crops and maintaining soil productivity. Compost is usually added to plant residues of field crops such as straw and straw, or wood-based materials such as leaves and wood chips, by adding inorganic fertilizers and livestock dung as a nitrogen source, depositing, and decomposing by microorganisms (醱A part of it is decomposed Some are made by things. It is also produced by depositing and fermenting livestock dung such as cow dung, horse dung, pig dung, and chicken dung. The characteristic of these composts is that compared to before fermentation, the samples after composting contain a large amount of microbial cells grown by the decomposition of organic matter. After being added to the soil, these microorganisms are not only useful as fertilizer sources, but also important as useful microorganisms that promote crop growth. In recent years, compost-derived microorganisms are often used to treat persistent chemicals such as pesticides and PCBs.
( 3 ) 水系堆積物 (3) Water-based sediments
水系堆積物は湖、 池、 河川や海などのそこに沈殿した土壌や有機物、 微生物な どの堆積物を指す。 これらのサンプル中には水中で増殖した植物および動物ブラ ンクトンやこれらの死骸が大量に堆積し、 これらを分解する微生物も多く存在し ている。 微生物によってこれらの死骸は分解され、 そこに含まれる窒素などの養 分は無機化され、 再び水中に放出される。 このようにして堆積物に含まれる微生 物は栄養分を循環させる役割を果たしている。 その微生物の数や種類を明らかに することは水系での物質循環を明らかにする上で極めて重要であると考えられて いる。  Aqueous sediment refers to sediment, such as lakes, ponds, rivers and oceans, and sediment, such as soil, organic matter, and microorganisms. In these samples, plant and animal blanktons grown in water and their carcasses are deposited in large quantities, and there are many microorganisms that degrade them. These dead bodies are broken down by microorganisms, and nutrients such as nitrogen contained therein are mineralized and released back into the water. In this way, the microorganisms in the sediment play a role in circulating nutrients. It is considered that clarifying the number and type of such microorganisms is extremely important in clarifying the material cycle in water systems.
( 4 ) 活性汚泥 (4) Activated sludge
活性汚泥法は汚水を処理する最も一般的な方法の一つである。 汚水には一般的 な都市生活廃水および家畜のし尿などが代表的なものとして挙げられる。 これら の汚水に空気によるばつ気処理を施し、 微生物により有機物を分解し、 その分解 産物および分解により増殖した微生物菌体を回収することで汚水を浄化するもの である。 この処理で増殖した微生物すなわち活性汚泥は処理する汚水の水質ゃ処 理条件によってその構成が異なり、 汚水の浄化処理においてこれらの微生物の分 析は極めて重要である。  Activated sludge is one of the most common methods of treating wastewater. Typical examples of sewage include general urban wastewater and livestock night soil. The wastewater is purified by subjecting these wastewaters to aeration treatment with air, decomposing organic substances by microorganisms, and collecting the decomposition products and microbial cells grown by the decomposition. The composition of the microorganisms grown in this treatment, that is, activated sludge, differs depending on the quality of the wastewater to be treated and the treatment conditions, and the analysis of these microorganisms is extremely important in the purification treatment of wastewater.
( 5 ) 糞便 人や家畜、 昆虫な の糞便は非常に多量の微生物が含まれている。 人の糞便に は腸内で増殖した大腸菌や乳酸菌などを初めとして極めて多くの微生物が含まれ ている。 その中には有用な微生物が含まれていることが現在までに明らかにされ ている。 また食中毒などの原因となる有害微生物も含まれることからこれら糞便 に含まれる微生物の分析は極めて重要である。 また反芻動物である牛などの草食 動物は摂取した植物を腸内の微生物を利用して一部を分解しており、 糞便の分析 により、 これらの微生物群集を明らかにすることは極めて重要である。 (5) Feces Feces, such as humans, livestock, and insects, contain very large amounts of microorganisms. Human feces contain an extremely large number of microorganisms, including Escherichia coli and lactic acid bacteria grown in the intestine. It has been clarified to date that useful microorganisms are included. In addition, the analysis of microbes contained in these feces is extremely important because they include harmful microorganisms that cause food poisoning. Also, herbivore animals such as cattle, which are ruminants, partially degrade ingested plants using intestinal microorganisms, and it is extremely important to clarify these microbial communities by analyzing feces. .
3 . beads-beating又は加熱処理 3. Beads-beating or heat treatment
ビーズ打破（beads-beating) とは、 スクリユーキャップチューブに土壌と D N A抽出液を添加し、 微小粒子 （ガラスビーズ、 シリカジルコ二アビ一ズ、 アルミ ナビーズなど） を加えて物理的に細胞を破壊する方法であり、 beads組成等も含 め Bな rgmannら (2001)により詳しく検討されている。 この方法では、 細胞外多糖 膜をもっため SDS などの界面活性剤の影響を受けにくいグラム陽性菌であって も機械的に破砕されるため、 極めて高収率で DNAが抽出できる。 また、 抽出も 短時間で終わるため、 腐植物質の混入が少ない土壌 DNA試料を得ることができ る。  Beads-beating is the process of adding soil and DNA extract to a screw cap tube and adding microparticles (glass beads, silica zirconium abs, aluminum beads, etc.) to physically destroy cells. It is a method, including the composition of beads, etc., which has been studied in detail by Rgmann et al. In this method, even gram-positive bacteria that have an extracellular polysaccharide membrane and are not easily affected by detergents such as SDS can be mechanically disrupted, so that DNA can be extracted in extremely high yield. In addition, since extraction is completed in a short time, a soil DNA sample with little humic substance contamination can be obtained.
具体的には、 スクリューキャップチューブに土壌、 ビーズ及び DNA抽出液を 添加して激しく攪拌する。 beads-beating時には、最も強く物理的衝撃が DNAに 加わるため、 beads-beatingの強度を適宜調節する。  Specifically, add soil, beads and DNA extract to a screw cap tube and mix vigorously. At the time of beads-beating, since the strongest physical impact is applied to DNA, the intensity of beads-beating is adjusted appropriately.
本発明においては、 DNA の抽出の際に加熱処理をすることができる。 加熱条 件は、 45°C〜70°Cで 0.5〜24時間であり、 好ましくは 6(TCで 1時間である。  In the present invention, a heat treatment can be performed during DNA extraction. The heating conditions are 45 to 70 ° C. for 0.5 to 24 hours, preferably 6 (1 hour at TC).
4 . DNA抽出液 4. DNA extract
(4-1) 界面活性剤 (4-1) surfactant
細菌や真菌などからの DNA抽出には、 まず SDSや CTABをはじめとする界 面活性剤により細胞のタンパク質を変性させ細胞構造を破壊することが必要とな る。 またフエノールや塩化べンジルなど強力なタンパク質の変性能力を持ち、 細 胞壁ゃ細胞膜も破壊する有機溶媒も使用されることがある。 Extraction of DNA from bacteria, fungi, etc. first requires the denaturation of cellular proteins with surfactants such as SDS and CTAB to destroy cell structures. It also has the ability to denature powerful proteins such as phenol and benzyl chloride. Organic solvents that also destroy the cell wall and cell membrane may be used.
SDS は分子生物学においてよく使用される界面活性剤であり、 微生物、 動物、 植物を問わず DNA抽出に広く使用されている （Marniur 1961)。 CTABを用い る手法は、 当初は植物から DNA抽出する方法として開発されたが （Murray & Thompson 1980)'、 微生物などからの DNA の抽出にも一部で使用されている (Velegraki et al. 1999)。 CTABには、 界面活性剤としてタンパク質を変性させる 作用の他に、 低塩濃度において DNAと選択的に結合して沈殿させる特性もある (Murray & Thompson 1980)。 また CTABは溶液中の多糖の除去 （Sambrook 1989) や腐植物質の除去 （Wilstrom et al. 1996、 Zhou et al. 1996) にも効果的 であることが知られていている。  SDS is a commonly used surfactant in molecular biology and is widely used for DNA extraction in microorganisms, animals and plants (Marniur 1961). The method using CTAB was originally developed as a method for extracting DNA from plants (Murray & Thompson 1980) ', but it has also been partially used for extracting DNA from microorganisms (Velegraki et al. 1999). ). In addition to denaturing proteins as a surfactant, CTAB also has the property of selectively binding and precipitating DNA at low salt concentrations (Murray & Thompson 1980). CTAB is also known to be effective in removing polysaccharides from solutions (Sambrook 1989) and humic substances (Wilstrom et al. 1996, Zhou et al. 1996).
グアジ二ジンチオシァネートは強力なタンパク質変性剤であり、 DNA の抽出 だけではなく RNAの抽出にもよく使用されている （e.g. Chirgwin et al. 1979、 Pitcher et al. 1989、 Chomczynski & Sacchi 1987、 Logemann et al. 1987、 Ausubel et al. 2000)。 この方法ではサルコシルを界面活性剤として添加している。 ベンジルクロライド法 （Zhu et al. 1993) は、 塩化べンジルを菌体破壊に使う簡 易で迅速な方法として開発された。 塩化ベンジルは細菌や糸状菌、 植物が細胞壁 の構成成分として合成するポリサッカライドすなわちセルロース、 へミセル口一 スの OH基と反応し、 細胞を破壊する。 これにより水溶性分子である DNAは塩 化ベンジルの有機層から水層に抽出され、 またこの方法はその後の遠心分離によ り塩化べンジルからなる有機層と水層の二層界面で除タンパク質も同時に行える 利点がある。 この方法を応用したキットも販売されている （製品名 Isoplant、 Nippon Gene、 Japan)。  Guadizidine thiosinate is a powerful protein denaturant and is commonly used for RNA as well as DNA extraction (eg Chirgwin et al. 1979, Pitcher et al. 1989, Chomczynski & Sacchi 1987, Logemann et al. 1987, Ausubel et al. 2000). In this method, sarkosyl is added as a surfactant. The benzyl chloride method (Zhu et al. 1993) was developed as a simple and rapid method of using benzyl chloride to destroy cells. Benzyl chloride reacts with polysaccharides, ie, cellulose, synthesized by bacteria, filamentous fungi, and plants as components of the cell wall, and OH groups in the micelle mouth to destroy cells. As a result, DNA, which is a water-soluble molecule, is extracted from the organic layer of benzyl chloride to the aqueous layer, and this method uses centrifugation to remove protein at the interface between the organic layer consisting of benzyl chloride and the aqueous layer. Has the advantage that it can be performed simultaneously. Kits that use this method are also available (product names Isoplant, Nippon Gene, Japan).
Triton X100は非イオン系の界面活性剤であり、 SDSや CTABなどイオン系の 界面活性剤よりも穏やかな界面活性作用を持つており、 夕ンパク質変性作用は弱 · レ^ そのため、 活性を維持したまま調製したい膜タンパク質の抽出や、 PCRなど の酵素反応液中に酵素の活性保持、 増強の目的で用いられることがある。 Triton X100は 1 %の濃度までなら PCR反応を阻害しないので、コロニー PCRや酵素反 応と組み合わせた簡便な DNA解析に用いられている （Agei'sborg et al. 1997)。 本発明においては： DNA を抽出することができる限り、 界面活性剤の種類に 限定されるものではない。 例えば SDS、 TritonX-100, N-ラウロイルサルコシン ナトリウム等が好ましく、 SDSがさらに好ましい。 Triton X100 is a nonionic surfactant that has a milder surface activity than ionic surfactants such as SDS and CTAB, and has a weak protein denaturing effect, thus maintaining its activity. It may be used for extraction of membrane proteins to be prepared as-is, or for maintaining or enhancing enzyme activity in enzyme reaction solutions such as PCR. Triton X100 does not inhibit the PCR reaction at concentrations up to 1%, and is used for simple DNA analysis in combination with colony PCR and enzyme reaction (Agei'sborg et al. 1997). In the present invention: The type of surfactant is not limited as long as DNA can be extracted. For example, SDS, TritonX-100, N-lauroylsarcosine sodium and the like are preferable, and SDS is more preferable.
使用する界面活性剤の濃度は、 SDS の場合は 5.0%以下であり、 0.：!〜 2.0%が 好ましく、 0.5%〜2.0%がより好ましく、 0.5〜1.0%がさらに好ましい。 また、 TritonX-100の場合は 5.0%以下であり、 0.1〜2.0%が好ましく、 0.5%〜2.0%が より好ましく、 0.5〜： L.0%がさらに好ましい。  The concentration of the surfactant used is 5.0% or less in the case of SDS, and is preferably from 0 ::! To 2.0%, more preferably from 0.5% to 2.0%, even more preferably from 0.5 to 1.0%. In the case of TritonX-100, the content is 5.0% or less, preferably 0.1% to 2.0%, more preferably 0.5% to 2.0%, and still more preferably 0.5% to L.0%.
(4-2) 抽出液の pH (4-2) Extract pH
DNAの抽出には通常 pH 8.0〜8.3程度の弱アル力リ性の緩衝液が使用されて いる。この pH条件は、 DNAの安定性ゆえ抽出液にも適用されていると考えられ、 抽出後の DNAを溶液として保存するために用いる TE 緩衝液と同じ pHである。 一方、 日本の大部分の土壌が pH 5.5〜6.5程度の弱酸性であり、 一部の火山灰土 壌、 非ァロフェン質黒ポク土壌には pH 4.5〜pH 5.5という酸性土壌も存在して いる。 そこで、 土壌から DNAを抽出する際には、 抽出液自体の pHが抽出量に 与える影響のみならず、 土壌の酸度により抽出液の pHが影響を受け、 抽出時の pHが変化してしまうことも考慮する必要がある。  For extraction of DNA, a weak buffer having a pH of about 8.0 to 8.3 is usually used. This pH condition is considered to be applied to the extract because of the stability of DNA, and is the same pH as the TE buffer used to store the extracted DNA as a solution. On the other hand, most soils in Japan are slightly acidic with a pH of about 5.5 to 6.5, and some volcanic ash soils and non-arofenic black pork soils have acidic soils with a pH of 4.5 to 5.5. Therefore, when extracting DNA from soil, not only the pH of the extract itself affects the amount of extraction, but also the pH of the extract is affected by the acidity of the soil, and the pH during extraction changes. Also need to be considered.
本発明においては、抽出液全体の pHは、 7以上であり、好ましくは 8.0〜9.0、 より好ましくは 8.6付近とすることができる。  In the present invention, the pH of the whole extract is 7 or more, preferably 8.0 to 9.0, and more preferably around 8.6.
(4-3) EDTA濃度、 ris-HCl濃度及び加熱処理 (4-3) EDTA concentration, ris-HCl concentration and heat treatment
一般に生物体からの DNA抽出の際には、 細胞から放出される DNase によつ て DNAが分解されることを防ぐために EDTAが用いられ、 その濃度は 1〜： L00 mMである。 土壌からの DNA抽出においては、 分子生物学分野でもよく使用さ れている Tris-EDTAの組み合わせが同じく使われる。多くの土壌 DNA抽出法に おいて、 抽出液中の EDTAは主に DNaseの不活性化という目的で使用されてい ると考えられ、その濃度は使用目的を果たすのに十分な量である lOO mM 以下と なってレ る (e.g. Kuske et al. 1998、 Arlene Porteous et al. 1994、 Bell et al. 1999、 Miller et al. 1999、 Watson & Blackwell 2000、 Burgmann et al. 2001) 。  Generally, when DNA is extracted from an organism, EDTA is used to prevent the DNA from being degraded by DNase released from cells, and its concentration is 1 to: L00 mM. For extraction of DNA from soil, the combination of Tris-EDTA, which is often used in the field of molecular biology, is also used. In many soil DNA extraction methods, EDTA in the extract is considered to be used mainly for the purpose of inactivating DNase, and its concentration is lOO mM, which is sufficient to achieve the intended purpose. (Eg Kuske et al. 1998, Arlene Porteous et al. 1994, Bell et al. 1999, Miller et al. 1999, Watson & Blackwell 2000, Burgmann et al. 2001).
現在のところ代表的な土壌 DNA抽出法である Zhou et al. (1996) の手法にお いても、 lOO mM EDTA (および 100 mM Tris、 100 mM Na2HP04 (pH 8.0) ) が使用されている。 EDTA濃度を高くすると、 DNAの酵素分解が抑制されるの と同時に土壌粒子への吸着も減少するため、 DNA の収量は増加するものの、 そ の収量増加と同時に腐植物質の混入も多くなるため、 EDTAは低濃度の方が好ま しいとの報告もある （Krsek & Wellington 1999) 。 すなわち、 高濃度 EDTA の使用は、 DNA の収量増加の利点よりも、 腐植物質の混入という欠点ゆえに敬 遠されてきており、 しかもその場合の 「高濃度」 というレベルは lOO mM 程度の ものである。 At present, the method of Zhou et al. (1996), which is a typical soil DNA extraction method, is used. You can have, lOO mM EDTA (and 100 mM Tris, 100 mM Na 2 HP0 4 (pH 8.0)) is used. When the EDTA concentration is increased, the enzymatic degradation of DNA is suppressed, and at the same time, the adsorption to soil particles is reduced.Therefore, the DNA yield is increased, but the humic substances are also increased at the same time as the yield is increased. It has been reported that low concentrations of EDTA are preferred (Krsek & Wellington 1999). In other words, the use of high-concentration EDTA has been shunned by the disadvantage of humic substances rather than by the benefit of increased DNA yields, and the level of "high concentration" in that case is on the order of 100 mM. .
日本の代表的な土壌である火山灰土壌は、 前述の通り DNAが吸着されやすい 土壌であると考えられる。 日本の火山灰土壌のように非晶質アルミニウムに富ん だ土壌は、 海外ではニュージーランドなど限られた国や地方に点在しているのみ であり、 世界の広い範囲には分布していない。 多くの海外の研究者にとって、 ァ 口フェンを多く含む土壌は研究対象とならず、 それゆえ、 従来の土壌 DNA抽出 法のほとんどが、 火山灰土壌を考慮せずに開発されてきたと思われる。 よって、 EDTA濃度の設定も、 火山灰土壌以外の土壌で検討されたものである。 しかしな がら、 多くの多価イオンを形成する金属イオンに対し強力なキレ一ト剤である EDTAは火山灰土壌において DNA吸着の原因である活性アルミニウムと錯体を 形成すると考えられ、 火山灰土壌では従来検討されてきた範囲外の濃度の EDTA が効果を発揮する可能性が考えられた。また、火山灰土壌以外の土壌であっても、 土壌への DNA吸着が起きているならば、 EDTA濃度を高めることにより多くの DNAが抽出できる可能性も考えられた。  Volcanic ash soil, a typical soil in Japan, is considered to be a soil to which DNA is easily adsorbed as described above. Soil rich in amorphous aluminum, such as volcanic ash soil in Japan, is scattered only in limited countries and regions, such as New Zealand, and is not distributed over a wide area of the world. For many overseas researchers, soils that are high in a-mouth fins were not considered, and therefore most conventional soil DNA extraction methods would have been developed without considering volcanic ash soils. Therefore, the setting of the EDTA concentration was also studied for soils other than volcanic ash soil. However, EDTA, which is a strong chelating agent for metal ions that form many polyvalent ions, is thought to form a complex with active aluminum, which causes DNA adsorption, in volcanic ash soil. It was suggested that concentrations of EDTA outside the specified range could be effective. In addition, it was considered that more DNA could be extracted by increasing the EDTA concentration even in soils other than volcanic ash soil if DNA adsorption to the soil occurred.
火山灰土壌の非晶質成分の分析には通常、 クェン酸ゃシユウ酸、 ピロリン酸な ど土壌中の A1や Feの形態により錯形成能が異なるキレーターが使用される。 こ れらの反応性の異なるキレーターを使用することにより、 土壌の非晶質成分を選 択的に融解し、 定量分析を行っている。 一方、 EDTAはキレート剤の中でもほと んどの金属イオンに対して極めて優れたキレート安定定数を持つ化合物である。 DNAの抽出や保存において EDTAが使用されるのは DNaseがその活性保持に必 要としている Mgイオンをキレートして DNaseを失活させるためである。 そこで、 選択融解法におけるキレーターと同様に、 土壌中から DNA吸着の原 因となる非晶質 A1を EDTAを用いて除去できるのではないか、 また EDTAが力 ルポキシル基により土壌に吸着することで DNAの吸着を解除することができる のではないかと推測し、 抽出液中の EDTA濃度について検討した。 EDTAにより キレートされた金属イオンは溶液中に金属イオン- EDTA 複合体として抽出され るため、 この定量を通して、 吸着の原因がどの金属元素であるかも推定すること ができると考えられた。 For the analysis of amorphous components of volcanic ash soil, chelators with different complexing ability depending on the form of A1 and Fe in the soil, such as citrate-oxalic acid and pyrophosphate, are usually used. By using these chelators with different reactivities, the amorphous components of soil are selectively melted and quantitatively analyzed. EDTA, on the other hand, is a compound having an extremely excellent chelate stability constant for most metal ions among chelating agents. EDTA is used in DNA extraction and storage because DNase inactivates DNase by chelating the Mg ions required to maintain its activity. Therefore, similar to the chelator in the selective melting method, it may be possible to remove the amorphous A1, which causes DNA adsorption, from the soil using EDTA. We speculated that the DNA adsorption could be released, and examined the EDTA concentration in the extract. Since the metal ion chelated by EDTA is extracted as a metal ion-EDTA complex in the solution, it was thought that it was possible to estimate which metal element caused the adsorption through this quantification.
本発明において抽出液として使用される EDTAの濃度は、 例えば 20mM以上 である。  The concentration of EDTA used as an extract in the present invention is, for example, 20 mM or more.
火山灰土壌を beads-beating 処理により土壌 DNA抽出しようとする場合の EDTA濃度は、 50mM以上であり、 好ましくは 100mM〜600mM、 好ましくは 200〜400mM、 さらに好ましくは 300mM〜400mMである。  When attempting to extract soil DNA from the volcanic ash soil by beads-beating treatment, the EDTA concentration is 50 mM or more, preferably 100 mM to 600 mM, preferably 200 to 400 mM, and more preferably 300 mM to 400 mM.
本発明においては、 上記濃度の EDTA を含む抽出液と土壌とを混合し、 beads-beating処理後、さらに高濃度 EDTA溶液を含有させて、濃度を例えば 600 〜1100mM、 好ましくは 600〜800 mMに高めた状態で加熱処理を併用すること ができる。 加熱条件は、 45°C〜70°Cで 0.5〜24時間であり、 好ましくは 60°Cで 1時間である。 これにより、 土壌から DNAをより高収量で抽出することができ る。  In the present invention, the extract containing the above-mentioned concentration of EDTA is mixed with soil, and after beads-beating treatment, a high-concentration EDTA solution is further contained to bring the concentration to, for example, 600 to 1100 mM, preferably 600 to 800 mM. Heat treatment can be used together in the elevated state. The heating conditions are 45 ° C to 70 ° C for 0.5 to 24 hours, preferably 60 ° C for 1 hour. This allows higher yields of DNA to be extracted from soil.
また、 本発明においては、 抽出液に Tris-HCl等の緩衝液を含めることもでき. る。 Tris-HClの濃度は、 例えば lOOmMである。 なお、 Tris-HClは、 本発明全 体を通して、 DNA抽出液に含めることができる。  In the present invention, the extract may also contain a buffer such as Tris-HCl. The concentration of Tris-HCl is, for example, 100 mM. Tris-HCl can be included in the DNA extract throughout the present invention.
(4-4) 抽出回数 (4-4) Number of extractions
本発明においては、 DNAの抽出回数は 1回に限定されるものではない。 EDTA を含む DNA抽出液について beads-beatingを行った後に遠心し、 その上清を採 取して DNA抽出液とする。 上清を採取した後のチューブには土壌が残存する。 この土壌中には 1回の抽出作業ではすべて抽出できずに土壌に吸着したまま残さ れた DNAも含まれていると考えられる。  In the present invention, the number of times of DNA extraction is not limited to one. After performing beads-beating on the DNA extract containing EDTA, centrifuge and collect the supernatant to obtain the DNA extract. The soil remains in the tube after collecting the supernatant. It is considered that this soil contains DNA that could not be extracted in one extraction operation and remained adsorbed on the soil.
そこで本発明において、 上記抽出液の beads-beating工程、 遠心工程、 上清の 採取工程を複数回繰 0返すことができる。 繰り返す回数は、 例えば 1〜4回であ る （回数は全部で 2 ~ 5回となる）。 Therefore, in the present invention, in the beads-beating step, centrifugation step, The sampling process can be repeated multiple times. The number of repetitions is, for example, 1 to 4 (the total number of times is 2 to 5).
(4-5) リン酸緩衝液 (4-5) phosphate buffer
リン酸緩衝液は生物学的な実験で使用される代表的な緩衝液の一つである。 土 壌からの DNA抽出においては、 間接抽出法の多くがリン酸緩衝液を使用して土 壌からの微生物の分離を行っており （e.g. Torsvik et al. 1980)、 また直接抽出法 でもリン酸緩衝液が使用されることは少なくない （e.g. Ogram et al. 1987、 Cullen & Hirsch 1998)。 直接抽出法、 間接抽出法を問わず、 DNA抽出にリン 酸緩衝液が使用される場合のリン酸濃度は 100〜120 mMに設定されている。 し かし、 Bell et al. (1999) が報告しているように、 リン酸緩衝液は Tris-EDTA緩 衝液よりも多くの腐植物質の溶出を引き起こし、 その除去が極めて困難となるた め、 DNA抽出に関してはリン酸緩衝液よりも Tris-EDTA緩衝液を使用したほう が良いとされている。 しかし、 EDTA と土壌中のァロフェン態の A1 とは錯体形 成の反応速度が遅いため、加熱処理によりその反応を加速させない限り EDTA緩 衝液のみを用いても土壌 DNAを十分に抽出することはできない。 一方、 リン酸 イオンは火山灰土壌に添加すると極めて短時間で土壌の A1や Feに吸着され不動 態化することが知られている。 非晶質 A1すなわち活性 A1に富む火山灰土壌では このリン酸の不動態化が作物生産上大きな制限となっているため、「リン酸吸収係 数」 が重視きれている。 '  Phosphate buffer is one of the representative buffers used in biological experiments. In DNA extraction from soil, many of the indirect extraction methods use phosphate buffer to separate microorganisms from soil (eg Torsvik et al. 1980). Buffers are often used (eg Ogram et al. 1987, Cullen & Hirsch 1998). Regardless of the direct extraction method or the indirect extraction method, the phosphate concentration is set to 100 to 120 mM when a phosphate buffer is used for DNA extraction. However, as reported by Bell et al. (1999), phosphate buffers cause more humic substances to elute than Tris-EDTA buffers, making their removal extremely difficult. It is said that Tris-EDTA buffer is better than phosphate buffer for DNA extraction. However, since the reaction rate of complex formation between EDTA and arophenic A1 in soil is slow, soil DNA cannot be sufficiently extracted using only EDTA buffer unless the reaction is accelerated by heat treatment. . On the other hand, phosphate ions are known to be absorbed and immobilized by A1 and Fe in soil in a very short time when added to volcanic ash soil. In volcanic ash soils rich in amorphous A1, that is, active A1, this passivation of phosphate is a major limitation on crop production, so the “phosphate absorption coefficient” has been emphasized. '
これらのことから、 リン酸イオンを土壌に添加することにより、 DNA の吸着 に関わるァロフェン態 A1をマスキングし、 DNAの吸着を抑制できるのではない かと考えられる。  From these facts, it is suggested that adding phosphate ions to soil could mask the Arophen-form A1 involved in DNA adsorption and suppress DNA adsorption.
本発明において使用することができるリン酸緩衝液は特に限定されるものでは なく、 リン酸カリウム緩衝液、 リン酸ナトリウム緩衝液などのリン酸塩緩衝液な どが挙げられる。  The phosphate buffer that can be used in the present invention is not particularly limited, and examples thereof include a phosphate buffer such as a potassium phosphate buffer and a sodium phosphate buffer.
DNA抽出液に使用されるリン酸緩衝液の濃度は、 250〜2000mMである。 火山灰土壌の場合は、 例えば 250〜2000mMであり、 非火山灰土壌の場合は例 えば 250〜1000mMである。 (4-6) 高濃度 EDTAとリン酸緩衝液との併用 The concentration of the phosphate buffer used in the DNA extract is 250-2000 mM. In the case of volcanic ash soil, for example, it is 250 to 2000 mM, and in the case of non-volcanic ash soil, it is, for example, 250 to 1000 mM. (4-6) Combined use of high concentration EDTA and phosphate buffer
DNAの抽出液は、 EDTA及びリン酸緩衝液のいずれを使用しても、 DNAの最 大収量が得られる条件 （EDTA、 リン酸、 SDSの濃度など） が土壌によって異な る。 また、 沈殿精製操作の際に高濃度の緩衝液は溶液が混ざり合わないなど不都 合が生じたため、 EDTAとリン酸緩衝液とを併用することを試みた。  Regardless of whether EDTA or phosphate buffer is used as the DNA extract, the conditions (maximum concentrations of EDTA, phosphate, SDS, etc.) at which the maximum yield of DNA is obtained differ depending on the soil. In addition, during the precipitation purification operation, there was a problem such that the solution was not mixed with the high-concentration buffer, and therefore, an attempt was made to use EDTA and a phosphate buffer together.
本発明においては、 EDTAの濃度は 100〜800mM、 例えば 200〜800mMであ り、 リン酸緩衝液 （例えばリン酸カリウム緩衝液） の濃度は 250〜2000mM、 例 えば 250〜1250mMの範囲で使用することができる。 EDTAとリン酸緩衝液とを 併用することにより、それぞれを単独で使用するよりも EDTA及びリン酸緩衝液 の濃度を低く抑えることができる。  In the present invention, the concentration of EDTA is 100 to 800 mM, for example, 200 to 800 mM, and the concentration of the phosphate buffer (for example, potassium phosphate buffer) is 250 to 2000 mM, for example, 250 to 1250 mM. be able to. By using EDTA and phosphate buffer together, the concentrations of EDTA and phosphate buffer can be kept lower than when each is used alone.
火山灰土壌について使用するときの組合せは、 EDTAが 100〜600mMであつ てリン酸緩衝液が 250〜1500mM (例えば 250〜1250mM) であることが好まし く、 非火山灰土壌について使用するときの組合せは、 EDTAが 100〜400ηιΜで あってリン酸緩衝液が 250〜： !250mMであることが好ましい。火山灰土壌及び非 火山灰土壌のいずれの場合も、 EDTAが 400mM、 リン酸緩衝液が 750mMの組 合せが最も好ましい。 上記 400mM EDTAと 750mMリン酸緩衝液との組合せに より、 ほとんどすべての土壌に対してほぼ最大収量の DNAを得ることができる ため、 この組合せはいわゆる 「ユニバーサルな緩衝液組成」 であるといえる。 但し、 上記ユニバーサルな緩衝液組成は、. 土壌によっては土壌 DNAの低分子 化 (20〜7kbp)が生じることがある。 微生物群集構造解析をする上では、 一般に比 較的短い DNA領域 (200〜1500bp)しか解析対象としないことなどから、多少低分 子化された DNAであっても十分に目的は達成できる。 しかし、 クロ一ニングな ど断片化した DNAでは不都合な操作や、 群集構造解析であってもより正確に解 祈したい場合には、 より高分子の DNAが得られる抽出条件 （EDTA及びリン酸 緩衝液の濃度） を適宜設定する。  The combination to be used for volcanic ash soil is preferably 100 to 600 mM EDTA and the phosphate buffer is 250 to 1500 mM (for example, 250 to 1250 mM), and the combination to be used for non-volcanic ash soil is Preferably, the EDTA is 100-400ηιΜ and the phosphate buffer is 250-:! 250mM. For both volcanic ash soils and non-volcanic ash soils, a combination of 400 mM EDTA and 750 mM phosphate buffer is most preferred. Since the combination of the above 400 mM EDTA and 750 mM phosphate buffer can provide almost the maximum yield of DNA for almost all soils, this combination can be said to be a so-called “universal buffer composition”. However, the above-mentioned universal buffer composition may cause low molecular weight of soil DNA (20 to 7 kbp) depending on the soil. In the analysis of microbial community structure, generally only relatively short DNA regions (200-1500 bp) are to be analyzed. Therefore, the purpose can be sufficiently achieved even with a somewhat low molecular weight DNA. However, if inconvenient procedures are used for fragmented DNA such as cloning, or if it is desired to perform more accurate prayer even in community structure analysis, extraction conditions (EDTA and phosphate buffer Solution concentration) as appropriate.
本発明においては、 土壌サンプルを、 SDS又は TritonX-100、 EDTA及びリン 酸緩衝液を含む抽出液に混合し、 これを加熱処理することもできる。 SDS 又は ritonX-100 の濃度は 5 %以下であり、 EDTA の濃度は 100〜800mM、 例えば 200〜800mM であり'、 リン酸緩衝液 （例えばリン酸カリウム緩衝液） の濃度は 250〜2000mM、 例えば 250〜1250mMの範囲で使用することができる。 加熱条 件は、 45°C〜70°Cで 0.5〜24時間であり、 好ましくは 60でで 1時間である。 (4-7) 2ステップ法 In the present invention, a soil sample can be mixed with an extract containing SDS or TritonX-100, EDTA and a phosphate buffer, and then heat-treated. The concentration of SDS or ritonX-100 is 5% or less, and the concentration of EDTA is 100-800 mM, for example The concentration of the phosphate buffer (for example, potassium phosphate buffer) can be used in the range of 250 to 2000 mM, for example, 250 to 1250 mM. The heating condition is 45 to 70 ° C for 0.5 to 24 hours, preferably 60 to 1 hour. (4-7) Two-step method
本発明においては、 土壌から DNAを回収するに際し、 beads-beating (第 1段 階） では低濃度の EDTA/リン酸緩衝液を使用し、 抽出の段階 （第 2段階） では高 濃度の EDTA/リン酸緩衝液を使用することにより、 さらに効率に DNAを抽出す ることができる。 このように、 濃度を 2段階に設定して抽出する操作を 「2ステ ップ法」 という。 2ステップ法を採用することにより、 DNA を効率よく抽出す ることができるとともに、 DNAの低分子化を回避することができる。  In the present invention, when recovering DNA from soil, low concentration EDTA / phosphate buffer is used in beads-beating (first stage), and high concentration EDTA / phosphate buffer is used in the extraction stage (second stage). By using a phosphate buffer, DNA can be extracted more efficiently. In this way, the operation of setting the concentration in two stages and extracting is called "two-step method". By employing the two-step method, it is possible to efficiently extract DNA and to avoid the reduction of the molecular weight of DNA.
第 1段階では、 5 %以下の SDS (好ましくは 1 % SDS) 又は 5 %以下の TritonX-100 と Tris'HCl緩衝液との混合物 （これを 「抽出液 I」 という） を使用 する。  In the first step, 5% or less of SDS (preferably 1% SDS) or a mixture of 5% or less of TritonX-100 and Tris'HCl buffer (hereinafter referred to as "extract I") is used.
上記 DNA抽出液 Iの存在下で土壌サンプルを beads-beating処理した後、 数 秒の簡易高速遠心を行って、土壌溶液をチューブ下部に集め、次に高濃度の EDTA 及びリン酸緩衝液を含む抽出液（「抽出液 II」 という） を調製してよく攪拌する。 抽出液 IIの組成は、 上記抽出液 Iに、 例えば、  After bead-beating the soil sample in the presence of DNA Extract I above, perform a simple high-speed centrifugation for a few seconds to collect the soil solution at the bottom of the tube, and then contain a high concentration of EDTA and phosphate buffer Prepare the extract (referred to as “Extract II”) and mix well. The composition of Extract II is the same as that of Extract I above, for example,
(0 75〜： L200mM、 好ましくは 600〜1200mMの EDTA、  (0 75-: L200 mM, preferably 600-1200 mM EDTA,
(ii) 250〜3000mM、 好ましくは 750〜2250mMのリン酸緩衝液、 又は  (ii) 250 to 3000 mM, preferably 750 to 2250 mM phosphate buffer, or
(iii) 上記 EDTA (好ましくは 100〜600mM) とリン酸緩衝液（好ましくは 250 〜1250mM) との混合物  (iii) a mixture of the above EDTA (preferably 100 to 600 mM) and a phosphate buffer (preferably 250 to 1250 mM)
を混合することにより調製することができる。抽出液 IIを作製するために抽出液 Iと混合する EDTA及びリン酸緩衝液については、 EDTAの濃度が 400〜800mM であり、 リン酸緩衝液の濃度が 750〜： 1500mM であることがさらに好ましく、 EDTAの濃度が 400mMであり、 リン酸緩衝液の濃度が 750mMであることが最 も好ましい。 Can be prepared by mixing For EDTA and phosphate buffer mixed with extract I to prepare extract II, the concentration of EDTA is preferably 400 to 800 mM, and more preferably the concentration of phosphate buffer is 750 to 1500 mM. Most preferably, the concentration of EDTA is 400 mM and the concentration of the phosphate buffer is 750 mM.
その後は、 上記抽出液 IIを遠心して DNAを抽出させ、 上清から DNAを回収 する。 本発明においては、 '上記 2ステップ法にさらに改良を加えて、 DNA の抽出効 率を高めたうえで、 DNA の低分子化を抑えることができる。 この改良法を 「2 ステップ改良法」 という。 Thereafter, DNA is extracted by centrifuging the extract II, and DNA is recovered from the supernatant. In the present invention, it is possible to further improve the above two-step method to increase the DNA extraction efficiency and to suppress the reduction of the molecular weight of DNA. This improved method is called the “two-step improved method”.
この場合は、 第 1段階に使用する抽出液は、 5 %以下の SDS (好ましくは 1 % SDS)又は 5 %以下の TritonX-100、 Tris-HCl緩衝液、 400mM以下の EDTA及び 250mM以下のリン酸緩衝液を含むものである （「抽出液 III」 という）。抽出液 III において、 EDTAは 300mMが好ましく、 リン酸緩衝液は lOOmMが好ましい。 そして、 上記抽出液 IIIの存在下で土壌サンプルを beads-beating処理する。 次に、 beads-beating処理後の抽出液 IIIに、 400〜： !OOOmM (好ましくは 400 〜600mM) の EDTA、 750〜2050mMのリン酸緩衝液、 又は前記 EDTAとリン 酸緩衝液との混合物を混合して抽出液を調製する。 このようにして調製された抽 出液を 「抽出液 IV」 という。  In this case, the extract used in the first step should contain 5% or less of SDS (preferably 1% SDS) or 5% or less of Triton X-100, Tris-HCl buffer, 400 mM or less of EDTA, and 250 mM or less of phosphorus. It contains an acid buffer (referred to as "Extract III"). In Extract III, EDTA is preferably 300 mM, and phosphate buffer is preferably 100 mM. Then, the soil sample is subjected to beads-beating treatment in the presence of the extract III. Next, the extract III after the beads-beating treatment is mixed with 400 to:! OOmM (preferably 400 to 600 mM) EDTA, 750 to 2050 mM phosphate buffer, or a mixture of the EDTA and the phosphate buffer. Mix to prepare an extract. The extract thus prepared is referred to as “extract IV”.
抽出液 IVを作製するために抽出液 IIIに混合する EDTA及びリン酸緩衝液に ついては、 EDTAの濃度が 400raM、 かつ、 リン酸緩衝液の濃度が 750mMであ ることが好ましい。  Regarding EDTA and phosphate buffer mixed with extract III to prepare extract IV, it is preferable that the concentration of EDTA is 400 raM and the concentration of phosphate buffer is 750 mM.
その後は、 抽出液 IVを遠心し、 上清から DNAを回収する。  After that, centrifuge Extract IV and collect DNA from the supernatant.
本発明においては、 上記 2ステップ法及び 2ステップ改良法を行う際、 2 ステ ップ法においては抽出液 IIを調製した後に、 2ステップ改良法においては抽出液 IVを調製した後に、それらの抽出液の存在下で土壌サンプルを加熱（例えば 60°C で 1時間） 処理し、 土壌 DNAの土壌からの回収率を高めて土壌 DNAを抽出す ることも可能である。 また、 本発明においては、 上記 2ステップ法及び 2ステ ップ改良法を行う前に、 100〜400mM EDTA及び 250〜1500mMリン酸緩衝液 を含む抽出液（「抽出液 V」 という） の存在下で土壌サンプルを加熱（例えば 60°C で 1時間） 処理又は beads-beating処理し、 処理後の抽出液 Vを遠心し、 上清を 回収した後 （「上清 I」 とする） に、 抽出液 I 又は抽出液 III を加えてさらに beads-beating処理し、 処理後の溶液を遠心して上清を得 （上清 II とする）、 上 清 I及び上清 IIから土壌 DNAを回収することもできる。  In the present invention, when performing the above two-step method and the two-step improvement method, extract the extract II after preparing the extract II in the two-step method, and extract the extract IV after preparing the extract IV in the two-step method. It is also possible to extract soil DNA by heating the soil sample in the presence of liquid (eg, 60 ° C for 1 hour) to increase the recovery of soil DNA from the soil. Further, in the present invention, before performing the two-step method and the two-step improvement method, an extract containing 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer (hereinafter referred to as “extract V”) is used. The soil sample is heated (eg, at 60 ° C for 1 hour) or beads-beating, centrifuged after the extraction, and the supernatant is collected (referred to as “supernatant I”). Add solution I or extract solution III, perform beads-beating treatment, centrifuge the treated solution to obtain a supernatant (referred to as supernatant II), and collect soil DNA from supernatant I and supernatant II. it can.
また、 100〜800mM EDTA及び 250〜2000mMリン酸緩衝液を含む溶液の存 在下で土壌サンプルを加熱 （例えば 60 で 1時間）処理または beads-beating処 理を行い、処理後の溶液に、 SDS又は抽出液 IIIを加えてさらに加熱処理を行い、 その後遠心してこの上清から土壌 DNAを回収することも可能である。 In addition, a solution containing 100 to 800 mM EDTA and 250 to 2000 mM phosphate buffer was used. The soil sample is heated (eg, 60 hours for 1 hour) or beads-beating in the presence of SDS, and SDS or Extract III is added to the treated solution, heat-treated, centrifuged, and centrifuged. It is also possible to recover soil DNA.
さらに、 本発明においては、 最初に SDS が存在しない状態で加熱処理及び beads-beating を組み合わせることで、 DNA を抽出することもできる（下記 (i)〜  Furthermore, in the present invention, DNA can be extracted by combining heat treatment and beads-beating in the absence of SDS first (see (i) to (i) below).
(i) SDS Freeカロ熱 +beads_beating法 (i) SDS Free calorie heat + beads_beating method
まず、 100〜400mMの EDTA及び 250〜1500mMのリン酸緩衝液を含む DNA 抽出液 （「抽出液 V」 という） の存在下で土壌サンプルを加熱処理する。 加熱処理 条件は前記と同様に、 45°C〜70°Cで 0.5〜24時間、 好ましくは 60°Cで 1時間で ある。 この場合、 抽出液 Vは EDTAが 400mM、 リン酸緩衝液が 750mMである ことが好ましい。  First, a soil sample is heat-treated in the presence of a DNA extract containing 100-400 mM EDTA and 250-1500 mM phosphate buffer (referred to as “extract V”). The heat treatment conditions are, as described above, 0.5 to 24 hours at 45 ° C to 70 ° C, preferably 1 hour at 60 ° C. In this case, the extract V is preferably 400 mM in EDTA and 750 mM in phosphate buffer.
次に、加熱処理後の抽出液 Vを遠心して上清を採取する。上清を採取した後は、 チューブ中に土壌サンプルが残存しているので、 これを、 5 %以下の SDS、 Tris-HCl緩衝液、 400mM以下の EDTA及び 250mM以下のリン酸緩衝液を含む DNA抽出液 IIIの存在下で beads-beating処理する。 その後、 抽出液 IIIを遠心 して上清を採取し、 上記 2つの採取された上清から DNAを回収する。  Next, the extract V after the heat treatment is centrifuged to collect the supernatant. After collecting the supernatant, a soil sample remains in the tube, and it is necessary to remove the DNA containing 5% or less of SDS, Tris-HCl buffer, 400 mM or less of EDTA, and 250 mM or less of phosphate buffer. Perform beads-beating treatment in the presence of Extract III. Then, extract III is centrifuged to collect the supernatant, and DNA is recovered from the two collected supernatants.
(ii) SDS Free beads-beating + beads-beatmg法  (ii) SDS Free beads-beating + beads-beatmg method
100〜400mMの EDTA及び 250〜1500mMのリン酸緩衝液を含む DNA抽出 液 Vの存在下で土壌サンプルを beads-beating処理する。 次に、 beads-beating 処理後の抽出液 Vを遠心して上清を採取する。そして、残存した土壌サンプルを、 5 %以下の SDS、 Tris-HCl緩衝液、 400mM以下の EDTA及び 250mM以下のリ ン酸緩衝液を含む DNA抽出液 IVの存在下で beads-beating処理する。 その後、 抽出液 IVを遠心して上清を採取し、上記 2つの採取された上清から DNAを回収 する。  The soil sample is beads-beated in the presence of DNA extract V containing 100-400 mM EDTA and 250-1500 mM phosphate buffer. Next, the extract V after beads-beating treatment is centrifuged to collect the supernatant. Then, the remaining soil sample is subjected to beads-beating treatment in the presence of a DNA extract IV containing 5% or less of SDS, Tris-HCl buffer, 400 mM or less of EDTA, and 250 mM or less of a phosphate buffer. Then, extract IV is centrifuged to collect the supernatant, and DNA is recovered from the two collected supernatants.
(iii) SDS Free加熱 +加熱法  (iii) SDS Free heating + heating method
200〜800mM EDTA及び 250〜2000mMリン酸緩衝液を含む DNA抽出液の存 在下で土壌サンプルを第一加熱処理する。 これと 5 %以下の SDS とを混合して 土壌サンプルを第二加熱処理する。 そして、 第二加熱処理後の抽出液を遠心した のち、 上清から DNAを回収する。 この場合、 抽出液は EDTAが 400mM、 リン 酸緩衝液が 750mMであることが好ましい。 The first heat treatment of the soil sample is performed in the presence of a DNA extract containing 200-800 mM EDTA and 250-2000 mM phosphate buffer. Mix this with less than 5% SDS A second heat treatment of the soil sample. After centrifuging the extract after the second heat treatment, DNA is recovered from the supernatant. In this case, the extract is preferably 400 mM EDTA and 750 mM phosphate buffer.
(iv) SDS Free beads-beating +加熱法  (iv) SDS Free beads-beating + heating method
100〜400mM EDTA及び 250〜： l500mMリン酸緩衝液を含む DNA抽出液の存 在下で土壌サンプルを beads-beating処理する。これと 5 %以下の SDSとを混合 して土壌サンプルを加熱処理する。 その後抽出液を遠心し、 上清から DNAを回 収する。  Beads-beating the soil sample in the presence of a DNA extract containing 100-400 mM EDTA and 250-: 500 mM phosphate buffer. Heat the soil sample by mixing it with less than 5% SDS. Then, centrifuge the extract and collect the DNA from the supernatant.
(4-8) まとめ -土壌 DNAの抽出に関して- 本発明においては、 DNA抽出に関して種々検討した結果、 以下のことがいえ る。  (4-8) Summary-Regarding extraction of soil DNA-In the present invention, as a result of various studies on DNA extraction, the following can be said.
(i) 土壌からの DNA抽出において、 抽出時の抽出液 pHは 8.6付近で安定して いる必要がある。  (i) In extracting DNA from soil, the pH of the extract during extraction must be stable at around 8.6.
(ii) 火山灰土壌からの DNA抽出を困難にしていた最大の原因は、 土壌による DNAの吸着である。  (ii) The biggest factor that made DNA extraction from volcanic ash soil difficult was the adsorption of DNA by the soil.
(iii) 火山灰土壌からの DNA抽出には高濃度の EDTA、 リン酸を用い、 非晶質 A1による DNAの吸着を解消すると効果的である。  (iii) It is effective to use a high concentration of EDTA and phosphoric acid to extract DNA from volcanic ash soil and to eliminate the adsorption of DNA by amorphous A1.
(iv) 土壌 DNAの最大収量が得られる抽出液中の EDTAおよびリン酸の濃度は、 土壌により異なる。  (iv) Soil The concentrations of EDTA and phosphate in the extract that yield the maximum yield of DNA vary depending on the soil.
(V) リン酸は DNAの抽出効果が高い。但し、 火山灰土壌に用いる場合は、 極め て高濃度で使用する必要があるため、 DNAの低分子化に注意する。  (V) Phosphate has a high DNA extraction effect. However, when used in volcanic ash soil, it is necessary to use extremely high concentrations, so care must be taken to reduce the molecular weight of DNA.
(vi) EDTAとリン酸を共存させることにより、 土壌 DNA抽出への補完効果が 見られ、 比較的低濃度での使用でも DNA抽出効果が高い。  (vi) The coexistence of EDTA and phosphoric acid has a complementary effect on soil DNA extraction, and the DNA extraction effect is high even at relatively low concentrations.
(vii) 400 mM EDTA/ 750 mM リン酸緩衝液 1 1 % SDS抽出液は、火山灰土壌、 非火山灰土壌を問わず、 ほとんどの土壌 DNAを抽出できるユニバーサルな抽出 液組成であるといえる。  (vii) 400 mM EDTA / 750 mM phosphate buffer 11% SDS extract has a universal extract composition that can extract most soil DNA regardless of volcanic ash soil or non-volcanic ash soil.
(viii) DNAの低分子化を阻止するためには beads-beating時の塩濃度に留意す る必要があり、 操作時の塩濃度を低くすることにより DNAの低分子化は防ぐこ とができる。 (viii) It is necessary to pay attention to the salt concentration during beads-beating in order to prevent the molecular weight reduction of DNA, and it is possible to prevent the molecular weight reduction of DNA by lowering the salt concentration during operation. You can.
(ix) 低濃度緩衝液で beads-beatingを行い、その後高濃度 EDTA-リン酸緩衝液 で DNAを剥離させることにより、 高分子 DNAも得ることが可能である。 この 場合も、400
Figure imgf000041_0001
リン酸による土壌 DNAの回収率が最もよい。 (ix) High-molecular-weight DNA can be obtained by performing beads-beating with a low-concentration buffer solution and then exfoliating the DNA with a high-concentration EDTA-phosphate buffer solution. Again, 400
Figure imgf000041_0001
Best recovery of soil DNA by phosphoric acid.
(X) 高濃度 DTA-リン酸緩衝液で beads-beating又は加熱処理を行つても界面 活性剤である SDS及び TritonXlOOを含んでいなければ、 土壌 DNAの低分子化 は抑えることができる。 beads-beating 又は加熱処理に界面活性剤を含む抽出液 を添加して抽出することで高分子 DNAを抽出することが可能である。 5 . DNAの精製 ·回収  (X) Even if beads-beating or heat treatment is performed with a high-concentration DTA-phosphate buffer solution, if the surfactants SDS and TritonXlOO are not included, the low molecular weight of soil DNA can be suppressed. Polymer DNA can be extracted by adding an extract containing a surfactant to beads-beating or heat treatment for extraction. 5. Purification and recovery of DNA
遺伝子の解析によく使用される技術としては、 PCR反応、 クローニング、 シー クェンシング、 ハイブリダィゼーシヨン、 遺伝子発現試験などがあげられる。 中 でも PCR反応は、 多くの遺伝子解析にとって重要で欠くことができない要素技 術であり、 遺伝情報に基づく微生物群集構造解析にとつても必須の操作である。 土壌からの DNA抽出の場合にも、 注目されるのは抽出した DNAを錡型として PCR産物が得られるかどうかであり、 その成否は土壌 DNAの純度を判定する一 つの指標として用いられることもある（Tsai & Olson 1992、 Watson & Blackwell 2000)  Techniques often used for gene analysis include PCR, cloning, sequencing, hybridization, and gene expression tests. Among them, the PCR reaction is an important and indispensable elemental technology for many gene analyses, and is also an essential operation for analyzing the microbial community structure based on genetic information. In the case of DNA extraction from soil, it is important to note whether the PCR product can be obtained using the extracted DNA as type III, and its success or failure can be used as an index to determine the purity of soil DNA. Yes (Tsai & Olson 1992, Watson & Blackwell 2000)
土壌からの DNA抽出液には、 微生物や植物などの細胞膜や細胞壁といった破 砕物、 界面活性剤などにより変性したタンパク、 土壌自体に蓄積していた腐植物 質を初めとする土壌有機物や重金属などが含まれており、 その後の解析のために はこれらの夾雑物をできるだけ取り除くのが望ましい。  DNA extracts from soil include debris such as cell membranes and cell walls of microorganisms and plants, proteins denatured by surfactants, soil organic matter such as humic substances accumulated in soil itself, and heavy metals. It is included, and it is desirable to remove these contaminants as much as possible for subsequent analysis.
微生物菌体由来の細胞構成成分の混入は、 少量の場合は PCR反応の阻害は著 しくないとされている。 その理由は、 これら菌体構成成分は、 本来同じ菌体内で 機能する DNAポリメラ一ゼの働きを阻害するものではないからである。 事実、 E. coRやその他の培養菌株を材料としたコロニー PCRは頻繁に行われており、こ れらは菌体自体を PCR反応液に添加して PCR反応の加熱により菌タンパクを変 性させて DNAを反応液中に放出させ、 PCR反応を行っているものである。 菌体 自体が PGR反応液中に存在しているにもかかわらず、 PCRは阻害されていない。 しかし、 土壌から DNAを抽出すると、 このような細胞構成成分以外の様々な物 質が多量に存在し、 これらが抽出操作に伴い、 DNA試料に混入する。 とりわけ 腐植物質はナノグラム単位の微量な混入であっても PCR反応を強く阻害するこ とが知られている' (Tsai & Olson 1992、 Boon et al. 2002、 Watson & Blackwell 2000)。よってこの腐植物質を DNA溶液の中からできるだけ取り除くことが重要 となる。 It is said that contamination of cell components derived from microbial cells does not significantly inhibit the PCR reaction in small amounts. The reason is that these cell components do not inhibit the function of DNA polymerase, which originally functions in the same cell. In fact, colony PCR using E.coR and other cultured strains as materials is frequently performed, and these cells are added to the PCR reaction solution, and the bacterial protein is denatured by heating the PCR reaction. To release the DNA into the reaction solution and perform the PCR reaction. Fungi PCR is not inhibited even though it is present in the PGR reaction. However, when DNA is extracted from soil, various substances other than such cell components are present in large amounts, and these are mixed into the DNA sample during the extraction operation. In particular, humic substances are known to strongly inhibit the PCR reaction even in trace amounts of nanograms' (Tsai & Olson 1992, Boon et al. 2002, Watson & Blackwell 2000). Therefore, it is important to remove this humic substance from the DNA solution as much as possible.
土壌 DNAの精製操作に関しては、 これまでにいくつもの研究がなされてきた。 なかでもゲルろ過ゃァガロース電気泳動切り出し、 ハイドロキシァパタイトカラ ム、 カオトロピック効果を利用したガラスパウダーもしくはシリカメンブレン精 製、 磁性ビーズによる分離精製などがよく行われている。 ゲルろ過による DNA の精製は、 土壌 DNA と腐植物質の分離に最もよく使用されているものである (Miller et al. 1999、 Howeler et al. 2003) カラムに充填した樹脂の間に試料を 通じ、 遠心するのみで精製作業が行えるスピンカラムも製品化されている。 ゲル ろ過による土壌 DNAの精製の原理については Miller (2000) によって詳しく検 討されている。 ゲルろ過に使用される樹脂製品には Sephadex、 Sepharose , Bio-GeL Toyopearl などがあるが、 その分子ふるい効果や特性は異なっており、 腐植物質と DNAの分離の目的では Sepharose 4Bを最も高く評価する報告があ る (Miller 2001、 Jackson et al 1997)。  A number of studies have been performed on the purification of soil DNA. Above all, gel filtration agarose electrophoresis, hydroxyapatite column, glass powder or silica membrane using the chaotropic effect, purification by magnetic beads, and separation and purification by magnetic beads are often used. Purification of DNA by gel filtration is the most commonly used method for separating soil DNA and humic substances (Miller et al. 1999, Howeler et al. 2003). Spin columns that can be purified only by centrifugation have been commercialized. The principle of purification of soil DNA by gel filtration has been discussed in detail by Miller (2000). Resin products used for gel filtration include Sephadex, Sepharose, Bio-GeL Toyopearl, etc., but their molecular sieving effects and properties are different. There are reports (Miller 2001, Jackson et al 1997).
しかし、 ゲルろ過による精製はカラムへの樹脂の充填、 DNA画分の分取が煩 雑であること（充填されている樹脂量と溶出液量の条件がかなり厳密であるため）、 またすベての腐植物質が取り除けるわけではなく、 一部の腐植物質が DNA画分 にも混入することが問題点としてあげられる。土壌に蓄積されている腐植物質は、 その性質、 形態、 分子量がそれぞれすべて異なっており、 数種類の土壌試料に含 まれる腐植物質について得られた知見を元に精製を行おうとしても、 土壌の種類 が異なれば混入してくる腐植物質の特性も異なるためであると考えられる。 また 通常使用される 2 ml以下の容量のカラムでは、 おのずと試料負荷量が小さくな り、 ごく少量の DNA溶液しか精製できない。 ァガロースゲル電 泳動後の DNA切り出し操作は、 巨大分子である DNAと より低分子である腐植物質を分離して DNAのみを回収しょうというものである (Zhou et al. 1996、 Kurien et al. 2001、 Kurien & Scofield 2002、 Chandler et al. 1997) が、 ァガロースゲルからの切り出し操作は極めて煩雑である。 However, purification by gel filtration requires complicated packing of the resin into the column and fractionation of the DNA fraction (because the conditions for the amount of loaded resin and the amount of eluate are quite strict). Not all humic substances can be removed, and the problem is that some humic substances are also mixed into the DNA fraction. Humic substances accumulated in soil have different properties, morphologies, and molecular weights.Even if you try to purify based on knowledge obtained about humic substances contained in several types of soil samples, This is probably because different types of humic substances mixed in have different characteristics. In addition, a column with a volume of 2 ml or less, which is commonly used, naturally reduces the sample load and can purify only a small amount of DNA solution. The DNA excision operation after agarose gel electrophoresis is to separate the macromolecule DNA and the lower molecular humic substance and recover only the DNA (Zhou et al. 1996, Kurien et al. 2001, Kurien & Scofield 2002, Chandler et al. 1997), but the cutting operation from agarose gel is extremely complicated.
これら以外に、 DNA 抽出の際に腐植物質のコン夕ミネ一シヨンをできるだけ 減らすため、 緩衝液の組成などを検討した研究も多く、 また、 土壌 DNA抽出液 からのおおまかな腐植物質除去のために、 CTABや P VPPなど腐植物質の除去剤 が使用されることも多い。  In addition to these, many studies have examined the composition of buffers to minimize humic substances during DNA extraction as much as possible, and to remove humic substances roughly from soil DNA extracts. Humic substances such as CTAB and PVPP are often used.
(5-1) 精製 (5-1) Purification
DNA の精製工程は、 前述の beads-beating 処理又は加熱処理後の抽出液と CTAB及び塩とを混合して DNAを精製する工程、 あるいは前記 beads-beating 処理又は加熱処理後の抽出液を遠心し、遠心後の上清と CTAB及び塩とを混合し て DNAを精製する工程を含むものである。 DNAの精製とは、 DNAの抽出液に 含まれる、 DNA以外の夾雑物を除去することを意味する。 また、 本発明の精製 方法は、 上記抽出工程における抽出液又は抽出された DNAを精製の対象とする のみならず、上記抽出方法以外の方法で抽出された DNA (又は DNAを含む溶液） に対しても精製することが可能である。  In the DNA purification step, the extract after beads-beating or heat treatment described above is mixed with CTAB and salt to purify DNA, or the extract after beads-beating or heat treatment is centrifuged. And mixing the supernatant after centrifugation with CTAB and salt to purify DNA. Purification of DNA means removing contaminants other than DNA contained in the DNA extract. In addition, the purification method of the present invention not only targets the extraction solution or the extracted DNA in the above-mentioned extraction step, but also purifies the DNA (or a solution containing DNA) extracted by a method other than the above-mentioned extraction method. Can also be purified.
CTAB及び塩を用いた精製は、 CTAB及びいずれかの塩溶液を土壌 DNAの抽 出液と混合し （酸性側に緩衝能を持つ緩衝液で弱酸性側にし）、 45^〜70 （例 えば 60°C)でのィンキュベート及びクロ口ホルムによる除タンパク操作を行うと いうものである。  For purification using CTAB and salt, the CTAB and any salt solution are mixed with an extract of soil DNA (made weakly acidic with a buffer having a buffering capacity on the acidic side), and then 45 ^ -70 (for example, (60 ° C) to remove protein by incubate and black mouth form.
CTABの濃度は 1〜3%であり、 2〜3%が好ましい。  The concentration of CTAB is 1-3%, preferably 2-3%.
一般に、 DNA はアルカリ条件下のほうが土壌から抽出されやすいため、 抽出 液には Tris-HCl、 EDTA、 リン酸緩衝液といった緩衝能の高いものが含まれてお り、 抽出液はアルカリ性に維持される。 従って、 これらの緩衝能を打ち消した上 で pHを弱酸性にする必要がある。 そこで、 塩は弱酸性側で緩衝能を有するもの を使用することが好ましい。  In general, DNA is more easily extracted from soil under alkaline conditions.Therefore, the extract contains highly buffered substances such as Tris-HCl, EDTA, and phosphate buffer, and the extract is kept alkaline. You. Therefore, it is necessary to neutralize these buffer capacities and make the pH weakly acidic. Therefore, it is preferable to use a salt having a buffer capacity on the weakly acidic side.
「塩」 とは、 抽出液に 1.0M以上の 1価のカチオンを添加でき、 かつ pHを 5.0 〜6.5の弱酸性に調節できるような物質をいう。 "Salt" means that a monovalent cation of 1.0 M or more can be added to the extract and the pH is 5.0 A substance that can be adjusted to a weak acidity of ~ 6.5.
このような塩として、 例えば、 塩化ナトリウム （NaCl)、 酢酸ナトリウム、 酢 酸カリウム、 酢酸アンモニゥム、 リン酸ナトリウム、 リン酸カリウムおよびリン 酸アンモニゥムがある。 この場合、 NaCl、 酢酸ナトリウム、 酢酸カリウム、 酢酸 アンモニゥム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムの濃 度は 0.7〜2.1Mである。 そして、 NaCl、 酢酸ナトリウム、 酢酸カリウム、 酢酸 アンモニゥムについては 1.0M以上が、 リン酸ナトリウム、 リン酸カリウム及び リン酸アンモニゥムについては 0.7 M以上が好ましく、 いずれの塩も 1.0〜： 1.4M であることがより好ましい。酢酸ナトリゥム、酢酸力リゥム、酢酸アンモニゥム、 リン酸ナトリウム、 リン酸カリウム、 リン酸アンモニゥムの pHは 5.0〜6.0が好 ましい。 なお、 1価のカチオンとは Na+、 K+、 ΝΗ4+などをいう。 Such salts include, for example, sodium chloride (NaCl), sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate. In this case, the concentrations of NaCl, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate and ammonium phosphate are 0.7 to 2.1M. For NaCl, sodium acetate, potassium acetate and ammonium acetate, 1.0M or more is preferable, and for sodium phosphate, potassium phosphate and ammonium phosphate, 0.7M or more is preferable. All salts are 1.0 to 1.4M. Is more preferred. The pH of sodium acetate, acetate acetic acid, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate is preferably 5.0 to 6.0. Incidentally, Na + and monovalent cations, K +, ΝΗ 4 + refers to such.
但し、 塩は上記物質に限定されるものではなく、 抽出液に 1.0 Μ以上の 1価力 チオンを添加でき、 pHを 5.0〜6.5に調製することが可能な塩はすべて本発明に However, the salts are not limited to the above substances, and all salts that can add 1.0% or more of monovalent thione to the extract and can adjust the pH to 5.0 to 6.5 are included in the present invention.
( まれる。 (Come on.
本発明においては、 抽出液への CTAB添加により、 土壌 DNA溶液から PCR が阻害されないレベルにまで腐植物質を除去することができる。 そして、 土壌 D Ν Αの沈殿に適した手法を検索し、上記開発した土壌 D Ν Α抽出法と組み合わせ て最適な精製条件を決定することができる。  In the present invention, humic substances can be removed from a soil DNA solution to a level that does not inhibit PCR by adding CTAB to the extract. Then, it is possible to search for a method suitable for sedimentation of the soil D Ν 、 and determine the optimal purification conditions in combination with the soil D Ν Α extraction method developed above.
本発明においては、 CTABの濃度が 2〜3%であり、 かつ、 塩の濃度が 1.4Mで あることがさらに好ましい。  In the present invention, more preferably, the concentration of CTAB is 2-3% and the concentration of salt is 1.4M.
(5-2) 沈殿  (5-2) Settling
DNAを溶液から分離回収する方法には、 DNAを沈殿させる方法、 カオトロピ ック効果によりシリカ表面に吸着させる方法、 磁性ビーズなどで溶液から分離す る方法などがある。 この中で最もよく使われる方法は DNAを沈殿させる方法で あり、 一定以上の濃度のエタノールや 2-プロパノールの存在下で DNAが沈殿す ることを利用している。ェタノールは、 70%程度の濃度で DNAが沈殿するため、 抽出液の 2〜2.5倍量のエタノールを添加する必要があり、マイク口チューブを用 いたミニスケールでの抽出時には、 エタノール沈殿に供試できる抽出液量が少な く効率が悪い。 Methods for separating and recovering DNA from a solution include a method of precipitating DNA, a method of adsorbing on a silica surface by a chaotropic effect, and a method of separating from a solution using magnetic beads or the like. The most commonly used method is DNA precipitation, which utilizes the precipitation of DNA in the presence of a certain concentration of ethanol or 2-propanol. Since ethanol precipitates DNA at a concentration of about 70%, it is necessary to add 2-2.5 times the amount of ethanol to the extract, and when extracting on a mini-scale using a microphone-mouth tube, test ethanol precipitation. Low extractable volume Poor efficiency.
それに対し 2-プロパノールはエタノールよりも DNAを沈殿させる作用が強く、 抽出液の 6/10等量で DNAの沈殿が得られるため、 ミニスケールでの DNA抽出 には最もよく使用されている。  On the other hand, 2-propanol has a stronger effect of precipitating DNA than ethanol, and can be used for miniscale DNA extraction because it can precipitate DNA in 6/10 equivalent of the extract.
本発明においては、 DNA の回収は、 上記の通り酸性側に緩衝能を持つ酢酸や リン酸緩衝液で弱酸性側にした後、 土壌 DNAの沈殿を行う。 本発明において、 沈殿は、 （i)上記抽出及び精製工程を経た後のサンプルを沈殿させる工程、 並びに (ii) 精製工程を経た後のサンプルを沈殿させる工程のいずれをも含むものである。 沈殿に使用される物質は、 2-プロパノール、 エタノール又はポリエチレンダリ コールが挙げられるが、 ポリエチレングリコール （PEG)が好ましい。 PEGの濃 度は、 10〜15%であり、 12%であることが好ましい （溶液中の PEG 濃度： 5〜 7.5% )。 その他、 ポリエチレングリコール 8000 (PEG) も使用される。 PEGは シークェンス反応前に、 PCR産物からプライマ一を取り除くのによく使用されて いる （Kusukawa et al.l990)。 PEG溶液は沈殿させる物質の選択性が高く、 構 造上 DNAと非常によく似ている RNAも沈殿しない。また同じ DNAであっても 短鎖であるプライマーは沈殿せず、 そのサイズにも選択性があることが示されて レ る （Paithanker & Prasad 1991、 Lis 1980、 Sambrook et al 1989)。 PEG【ま、 他のアルコール類よりも夾雑物質を沈殿させることが少ないため、 土壌ゃコンポ ストなど、 夾雑物が混入しやすい試料から. DNAを抽出する研究によく使用され ている （Ogram et al 1987、 Porteous et al 1997、 Howeler et al 2003、 LaMontagne et al 2002)。  In the present invention, as described above, the DNA is recovered by weakening the acid side with an acetic acid or phosphate buffer having a buffering capacity on the acidic side, and then precipitating the soil DNA. In the present invention, the precipitation includes both (i) the step of precipitating the sample after the extraction and purification steps, and (ii) the step of precipitating the sample after the purification step. The substance used for the precipitation includes 2-propanol, ethanol or polyethylene dalicol, but polyethylene glycol (PEG) is preferred. The concentration of PEG is 10-15%, preferably 12% (PEG concentration in solution: 5-7.5%). In addition, polyethylene glycol 8000 (PEG) is also used. PEG is commonly used to remove primers from PCR products before the sequence reaction (Kusukawa et al. L990). The PEG solution has high selectivity for the substance to be precipitated, and does not precipitate RNA that is structurally very similar to DNA. Even with the same DNA, short-chain primers did not precipitate, indicating their selectivity in size (Paithanker & Prasad 1991, Lis 1980, Sambrook et al 1989). PEG [Because it is less likely to precipitate contaminants than other alcohols, PEG is often used in research to extract DNA from samples that are easily contaminated with soil or compost. (Ogram et al.) 1987, Porteous et al 1997, Howeler et al 2003, LaMontagne et al 2002).
ところで、 弱酸性条件下で PEG沈殿を行うと、 完全に除去しきれなかった腐 植物質又は夾雑物が DNAとともに沈殿する可能性がある。  By the way, if PEG precipitation is performed under weakly acidic conditions, humic substances or impurities that could not be completely removed may precipitate together with DNA.
そこで本発明は、 好ましくは以下の 2つの手段のいずれかを採用することがで きる。  Therefore, the present invention can preferably employ one of the following two means.
(a) CTAB精製後、 クロロホルム等により除夕ンパクを行って回収した上清（い わゆる水層部分） と Tris緩衝液 （pH8.0以上） 等とを混合し、 pHを再びアル力 リ側 （pH8.0〜8.6) に戻し、 PEGを添加し、 高速遠心して DNAを回収する。 (b) PEG溶液の組成を改良する。 すなわち、 PEG / Tris-HClを調製し （この溶 液の pHは 8.0〜8.6)、 これにより PEGによる沈殿を行う。伹し、 PEGと組み合 わせる緩衝液は、 pHを上昇させることができる限り上記 Tris-HClに限定される ものではなく、 リン酸緩衝液などその他の緩衝液も使用することができる。 (a) After purification of CTAB, the supernatant (so-called aqueous layer portion) collected by removing the protein with chloroform or the like is mixed with Tris buffer (pH 8.0 or more), etc., and the pH is again adjusted to the alkaline side. (PH 8.0-8.6), add PEG, and centrifuge at high speed to collect DNA. (b) Improve the composition of the PEG solution. That is, PEG / Tris-HCl is prepared (pH of this solution is 8.0 to 8.6), and thereby precipitation with PEG is performed. However, the buffer to be combined with PEG is not limited to the above Tris-HCl as long as the pH can be raised, and other buffers such as a phosphate buffer can also be used.
(5-3) まとめ (5-3) Summary
(i) CTAB精製 （弱酸性側での精製） 及び PEG沈殿 （アルカリ側での沈殿） の 組み合わせは、 土壌 DNAの精製に関してのみでも使用可能である。  (i) Combination of CTAB purification (purification on weak acid side) and PEG precipitation (precipitation on alkaline side) can be used only for purification of soil DNA.
(ii) CTAB精製の際に NaCl以外の緩衝能を持つ塩を用い、弱酸性側で精製処理 を行うことも可能である。  (ii) When purifying CTAB, it is also possible to use a salt with a buffering capacity other than NaCl and carry out purification on a weakly acidic side.
(iii) 抽出、 精製、 沈殿といった一連のプロトコールは、 全てを実施しなくとも それぞれの工程をを独立で使用しても、 土壌 DNAを得ることができ、 あるいは 上記抽出方法以外の方法で抽出した土壌 DNAの精製が可能である。 6 . DNAの抽出、 精製及び/又は回収用キット  (iii) In a series of protocols such as extraction, purification, and precipitation, soil DNA can be obtained even if each step is used independently without performing all of them, or extracted by a method other than the above extraction method. Purification of soil DNA is possible. 6. DNA extraction, purification and / or recovery kit
本発明は、 上記土壌 DNAを抽出、 精製、 又は回収するためのキットを提供す る。  The present invention provides a kit for extracting, purifying, or recovering the above soil DNA.
環境サンプルからの DNA抽出用キットは、 5 %以下の界面活性剤、 又は前記 界面活性剤と beads-beating 用ビーズとの組合せを含む。 すなわち、 本発明の DNA抽出用キットは、 界面活性剤 （SDS、 CTAB, Triton X- 100又は Ν·ラウ口 ィルサルコシンナトリゥム）を基本構成とし、 これに beads-beatingに使用するビ ーズ、 EDTA、 リン酸緩衝液、 アルカリ性緩衝液 （Tris-HCl等の Tris緩衝液）、 pH調整剤等を含めることが可能である。本発明の DNA抽出用キットは、 アル力 リ性緩衝液により pHを 7.0以上に調整することが可能である。 '  The kit for extracting DNA from environmental samples contains 5% or less of a surfactant or a combination of the surfactant and beads for beads-beating. That is, the DNA extraction kit of the present invention has a basic composition of a surfactant (SDS, CTAB, Triton X-100 or E. laurel sarcosine sodium), and the beads used for beads-beating. , EDTA, phosphate buffer, alkaline buffer (Tris buffer such as Tris-HCl), pH adjuster, etc. The DNA extraction kit of the present invention can adjust the pH to 7.0 or more with an alkaline buffer. '
本発明に使用する抽出液において、 EDTAの濃度は、 50mM〜： L200mMの範囲 から任意に選択することができる。 また、 リン酸緩衝液の濃度は、 50mM〜 3000mMの範囲から任意に選択することができる。  In the extract used in the present invention, the concentration of EDTA can be arbitrarily selected from the range of 50 mM to: L200 mM. The concentration of the phosphate buffer can be arbitrarily selected from the range of 50 mM to 3000 mM.
また、 DNA抽出液は、 使用する土壌に応じて適宜濃度設定ができるように、 各濃度に段階的に区分けしておくことができる。例えば、 SDSは 0.1%から 2.0% の範囲で数段階に調整されていてもよく、 実験者が希望する濃度調整ができるよ うに、 高濃度の SDS を 1種類用意しておいて希釈液によって希釈できるように しておくこともできる。 EDTAやリン酸緩衝液の場合も、 50mM、 100 mM、 200 mM、 300mM、 400mMのように段階的に濃度調整したものを含めておくことも、 高濃度 （例えば 1 M) のものを用意しておいて希釈液によって任意の濃度に希釈 できるようにしておくこともできる。さらに、ユニバーサルな緩衝液組成として、 400mM EDTAと 750mMリン酸緩衝液との組合せをキットに含めておくことも 可能である。 In addition, the DNA extract should be prepared so that the concentration can be set appropriately according to the soil used. Each concentration can be divided stepwise. For example, the SDS may be adjusted in several steps from 0.1% to 2.0%, and one type of high-concentration SDS is prepared and diluted with a diluent so that the experimenter can adjust the concentration as desired. You can also make it available. In the case of EDTA or phosphate buffer, be sure to include those whose concentration is adjusted stepwise such as 50 mM, 100 mM, 200 mM, 300 mM, or 400 mM, or prepare high concentration (for example, 1 M). It can be prepared so that it can be diluted to any concentration with a diluent. Furthermore, a combination of 400 mM EDTA and 750 mM phosphate buffer can be included in the kit as a universal buffer composition.
さらに、 本発明は、 酸性側の pKaを有する pH緩衝液を含む塩溶液、 陽イオン 界面活性剤、 又は当該塩溶液と陽イオン界面活性剤との混合物を含む、 環境サン プルからの DNA精製用キットを提供する。 酸性側の pKaを有する pH緩衝液と しては、 例えば酢酸緩衝液、 リン酸緩衝液、 塩酸緩衝液又は硫酸緩衝液が挙げら れる。 このキットには、 DNA精製用の陽イオン界面活性剤 （CTAB 等） 及び前 記塩を含めておくことが可能である。上記酸性側の pKaを有する pH緩衝液を塩 と混合して使用することにより、 精製時の pHを 7.0未満に調整することが可能 である。  Furthermore, the present invention relates to a method for purifying DNA from an environmental sample, comprising a salt solution containing a pH buffer having an acidic pKa, a cationic surfactant, or a mixture of the salt solution and a cationic surfactant. Provide a kit. Examples of the pH buffer having a pKa on the acidic side include an acetate buffer, a phosphate buffer, a hydrochloric acid buffer and a sulfate buffer. This kit can contain a cationic surfactant (such as CTAB) for DNA purification and the above salt. It is possible to adjust the pH at the time of purification to less than 7.0 by using the pH buffer having the pKa on the acidic side mixed with a salt.
さらに、 本発明は、 アルカリ性緩衝液 （例えば Tris緩衝液） を含む、 環境サン プルからの DNA回収用キットを提供する。.このキットには、 沈殿に使用するた めの 2-プロパノール、 エタノール又は PEGなどを含めておくこともできる。 上 記緩衝液により、 回収時の DNA溶液の pHは 7.0以上に調整することが可能で ある。  Further, the present invention provides a kit for recovering DNA from an environmental sample, which contains an alkaline buffer (for example, Tris buffer). The kit can also include 2-propanol, ethanol or PEG for use in precipitation. With the above buffer, the pH of the DNA solution at the time of recovery can be adjusted to 7.0 or more.
さらに、 本発明は、 上記 DNA抽出用キット、 DNA精製用キット、 DNA回収 用キットから適宜 2組以上を選択し、 本発明の DNA抽出と精製、 DNA抽出と回 収、 DNA精製と回収、 あるいは DNA抽出、 精製及び回収を行うためのキットセ ットをも提供する。このような DNA抽出、精製及び回収の 2つ以上の組合せを、 本発明においては DNAの取得という。 従って、 各キットの組合せを用いること により、 DNA 取得 （抽出、 精製、 回収、 又はこれらの組合せ） を行うことがで きる。 以下、 実施例により本発明をさらに具体的に説明する。 但し、 本発明はこれら 実施例に限定されるものではない。 Further, the present invention provides a DNA extraction and purification, a DNA extraction and recovery, a DNA purification and recovery, A kit set for DNA extraction, purification and recovery is also provided. In the present invention, such a combination of two or more of DNA extraction, purification and recovery is referred to as obtaining DNA. Therefore, DNA acquisition (extraction, purification, recovery, or a combination thereof) can be performed by using a combination of each kit. Wear. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
本発明において詳細な濃度範囲は実施例に記載するが、 当該濃度範囲は例示で あって、 本発明の目的を逸脱しない限り何ら限定されるものではない。  Although a detailed concentration range in the present invention is described in Examples, the concentration range is an example and is not limited at all without departing from the object of the present invention.
〔実施例 1〕 界面活性剤の使用条件の検討 [Example 1] Examination of use conditions of surfactant
本実施例は、 代表的な界面活性剤および塩化ベンジルを用いて、 さらに細胞破 壌の処理として加熱および beads-beatingによる 2種類の処理による DNA抽出 実験を行い、 土壌からの DNA抽出に最も適した界面活性剤とその使用条件の検 討を行ったものである。  In this example, using a typical surfactant and benzyl chloride, a DNA extraction experiment was performed by heating and beads-beating as two treatments for cell breakage, and it was most suitable for DNA extraction from soil. This study examined the surfactants used and their conditions of use.
(1) 材料と方法 (1) Materials and methods
抽出試験には、 東京大学農学部構内弥生圃場に設置された土壌圏科学研究室農 薬連用圃塲の堆肥区の土壌、 埼玉県農業試験場の水田転換畑土壌 （かって長野の 水田土壌であった土壌を客土として移入したもの、以下鴻巣水田土壌と表記する）、 および東北大学附属農場 （川渡） の森林土壌の 3種類を用いた。 それぞれァロフ ェン質黒ポク土、 灰色低地土、 非ァロフェン質黒ポク土であり、 互いに理化学性 が大きく異なる （表 1 )。  In the extraction test, the soil in the compost area of the field of agricultural chemicals used at the Yayoi field in the campus of the Faculty of Agriculture of the University of Tokyo was used. (Hereinafter referred to as Konosu paddy soil) and forest soil from Tohoku University farm (Kawatari). These are arophenic black pork soil, gray lowland soil, and non-arofenic black pork soil, respectively, which have greatly different physicochemical properties (Table 1).
表 1 供試土壌の理化学性 酸性シユウ酸塩抽出 ピロリン酸抽出ァロフェン pH pH(KCI)全窒素全灰茶 (mg/g soil) (mg/g soil) (mg/g soil)  Table 1 Physicochemical properties of test soils Acid oxalate extraction Pyrophosphate extraction alofen pH pH (KCI) Total nitrogen total ash tea (mg / g soil) (mg / g soil) (mg / g soil) (mg / g soil)
(¾) (¾) Si Fe Al Fe Al Al 弥生圃場堆肥連用区土壌 ァロフェン質黒ポク土 6.88 5.76 0.38 4.23 20.89 20.85 37.76 0.77 2.03 35.73 鴻巣水田転換畑土壌 灰色低地土 4.96 4.27 0.21 1.95 4.78 16.78 10.91 3.42 2.88 8.03 東北大学森林土壌 非ァロフェン質黒ポク ± 4.97 4.24 0.45 9.19 4.07 16.39 38.62 8.13 17.80 20.82 各土壌の理化学性の測定は、 pH (H20) および pH (KC1) はそれぞれ 1:5水お よび 1 N KC1抽出法により、 全窒素および全炭素は N/C アナライザ一により、 酸性シユウ酸塩およびピロリン酸抽出 Al、 Fe は土壌環境分析法 （土壌環境分析 法編集委員会編 199^7) および Blackmoi'e et al. ( 1981) の方法により行った。 本研究全体を通じて使用した土壌はすべて、 サンプリング後すぐに実験に使用し ない場合は 4°Cの低温室において湿潤状態で保存し、 使用する際には 2mmのふ るいを通し、 最大容水量の 70%に水分含量を調製し、 2週間程度室温でプレイン キュベ一ションしたものを供試土壌とした。 (¾) (¾) Si Fe Al Fe Al Al Yayoi field compost continuous use soil Arofen black soil 6.88 5.76 0.38 4.23 20.89 20.85 37.76 0.77 2.03 35.73 Konosu paddy converted field soil Gray lowland 4.96 4.27 0.21 1.95 4.78 16.78 10.91 3.42 2.88 8.03 Tohoku forest soil non Arofen quality black Pok ± 4.97 4.24 0.45 9.19 4.07 16.39 38.62 8.13 17.80 20.82 measurement of Physical and Chemical properties of the soil, pH (H 2 0) and pH (KC1) respectively 1: 5 water Contact And 1 N KC1 extraction method, total nitrogen and total carbon were extracted by N / C analyzer, and acid oxalate and pyrophosphate were extracted by Al / Fe. The method was edited by the Law Editing Committee, 199 ^ 7) and by Blackmoi'e et al. (1981). All soils used throughout this study should be stored wet in a low-temperature room at 4 ° C if not used immediately in the experiment after sampling, and should be passed through a 2 mm sieve when used for maximum water capacity. The soil content was adjusted to 70%, and pre-incubated at room temperature for about 2 weeks was used as the test soil.
これらの土壌 0.5 gに 2% SDS抽出液、 2% CTAB/1.4 M NaCl抽出液、 8 M グ ァニジンチオシァネート抽出液、 2% SDS および塩化べンジル抽出液、 2% TritonXlOO抽出液の 5種類の抽出液を 1.2 mlずつ添加し、 beads-beating処理 (強度 5 m/secで 30秒間処理） もしくは 65°C 1時間の加熱処理を行った。 その 後 12000 X gで 10分間遠心し、 上清を回収して等量のクロ口ホルムを添加、 よ く攪拌し、 再び遠心することにより、 除タンパクを行った。 700 1の水層を回 収し、 これに 1/10量の 3 M酢酸ナトリゥムを添加し 6/10量の 2-propanolを加 え 20000 X gで遠心し、 DNAを沈殿させた。遠心後 70% エタノールにより DNA 沈殿を洗浄し、乾燥後 100 H iの TE bufferに溶かし、 これを抽出 DNAとした。 抽出した土壌 DNA溶液精製操作を施していないため、 腐植物質が相当量混入 していた。 このままでは吸光度などによって DNA量を測定することができなか つた。 そこで腐植物質の影響がない状態で DNAを定量するため、 抽出 DNAを 1% ァガロースゲルにて電気泳動し、 低分子のため移動距離の長い腐植物質を DNAから十分に分離したのち、 SYBR Green I (TAE緩衝液による 10000倍希 釈液） で染色し、 DNAのバンド部分の輝度を測定した。 DNAの輝度と DNA量の 相関を示す検量線を作成するため、 それぞれのゲルには、 λ Hind III Digestを標 準試料として 10 ng 〜 50 ngまで 10 ng間隔で 5点、 試料とともに泳動し検量 線を作成した。 なお、 2000 bp以上のサイズの DNAを定量対象とした。 測定機 器はフルォロ ·イメージアナライザ一 Fla-5000 (富士写真フィルム株式会社） を使用し、 輝度を数値化するための画像処理には ImageGauge 4.0 (富士写真フ イルム株式会社） を用いた。 ァガロースゲルにおける定量例を図 1に示す。  0.5 g of these soils were mixed with 2% SDS extract, 2% CTAB / 1.4 M NaCl extract, 8 M guanidine thiosinate extract, 2% SDS and benzyl chloride extract, and 2% TritonXlOO extract. 1.2 ml of each extract was added, and beads-beating treatment (intensity of 5 m / sec for 30 seconds) or heat treatment at 65 ° C for 1 hour was performed. Thereafter, the mixture was centrifuged at 12,000 X g for 10 minutes, the supernatant was recovered, an equal amount of black-mouthed form was added, the mixture was stirred well, and centrifuged again to remove proteins. A 7001 aqueous layer was collected, 1/10 volume of 3 M sodium acetate was added, 6/10 volume of 2-propanol was added, and the mixture was centrifuged at 20000 X g to precipitate DNA. After centrifugation, the DNA precipitate was washed with 70% ethanol, dried, dissolved in 100 Hi TE buffer, and used as extracted DNA. Because the extracted soil DNA solution was not purified, substantial amounts of humic substances were mixed. As it was, it was not possible to measure the amount of DNA by absorbance. Therefore, in order to quantify the DNA without the influence of humic substances, the extracted DNA was electrophoresed on a 1% agarose gel, and the humic substances that traveled a long distance due to small molecules were sufficiently separated from the DNA. (A 10000-fold dilution with TAE buffer), and the brightness of the DNA band was measured. To prepare a calibration curve showing the correlation between DNA brightness and DNA amount, each gel was run by running 5 points of λ Hind III Digest as a standard sample at 10 ng to 50 ng intervals at 10 ng intervals along with the sample. Created a line. In addition, DNA having a size of 2000 bp or more was subjected to quantification. The measurement equipment used was a Fluoro Image Analyzer I-Fla-5000 (Fuji Photo Film Co., Ltd.), and ImageGauge 4.0 (Fuji Photo Film Co., Ltd.) was used for image processing to quantify brightness. Fig. 1 shows an example of quantification on an agarose gel.
なお、 各界面活性剤の、 緩衝液組成、 および物理的諸条件を表 2に示す。 表 2 緩衝液 S成と界面活性剤の概要 Table 2 shows the buffer composition and physical conditions of each surfactant. Table 2 Overview of buffer solution S and surfactants
基本的な緩衝液組成  Basic buffer composition
lOOmM Tris-HCl lOOmM EDTA (pH8.3)  lOOmM Tris-HCl lOOmM EDTA (pH8.3)
界面活性剤組成  Surfactant composition
1. SDS 2% ドデシル硫酸ナトリウム（Wako)  1. SDS 2% sodium dodecyl sulfate (Wako)
2. CTAB 2% セチルトリメチルアンモニゥムブロミド (Sigma)  2. CTAB 2% cetyltrimethylammonium bromide (Sigma)
1.4M NaCl  1.4M NaCl
3. Guanidine 8M グァニジンチオシァネート  3. Guanidine 8M guanidine thiosinate
0.5% L-ラウロイルサルコシン  0.5% L-lauroyl sarcosine
4. Benzyl Chloride 2%SDSおよび塩化べンジル  4. Benzyl Chloride 2% SDS and Benzyl chloride
5. TritonXlOO 2% TritonXlOO  5. TritonXlOO 2% TritonXlOO
注 4. Benzyl Chloride法について  Note 4. About Benzyl Chloride method
2% SDS抽出液 1.0 mUこ Benzyl Chloride 500 jU l 添加  2% SDS extract 1.0 mU Add Benzyl Chloride 500 jUl
クロ口ホルムによる除タンパク作業を省略  Omission of protein removal work by black mouth holm
また、 本試験を含め後述する全ての実験で beads-beating処理は、 表 3に示す 条件にて行った。 表 3 Beads beating処理につしゝて The beads-beating treatment was performed under the conditions shown in Table 3 in all the experiments described below including this test. Table 3 Beads beating process
Beads Beating処理の条件  Beads Beating conditions
Beads組成 2 ml スクリユーキャップ付チューブを使用。  Beads composition Use 2 ml tube with screw cap.
Beads組成は 0.1 mm, 0.5 mmのシリカジルコニァビーズ  Beads composition of 0.1 mm and 0.5 mm silica zirconia beads
を 3 : 1で混合したもの lg および 5 mmのガラスビーズ 1個  3: 1 mixture of lg and 5 mm glass beads
Beads Beatin の条件  Beads Beatin conditions
FastPrep FP120 (Qbio社 USA)を使用  Use FastPrep FP120 (Qbio USA)
5m/secの強度で 30秒間処理  Treated at 5m / sec intensity for 30 seconds
この beads組成は Burgmann et aL (2001) の知見を参考に調製したものであ る。 なお実験はすべて 3連で行つた。 The beads composition was prepared by referring to the knowledge of Burgmann et aL (2001). All experiments were performed in triplicate.
(2) 結果と考察  (2) Results and discussion
土壌 DNA の抽出量についての結果を図 2 に示す。 加熱処理および beads-beating処理に関わらず SDSと Triton X100を使用した時が土壌 DNAの 抽出効率がよく、 また界面活性剤が原因と思われる DNAの低分子化はみられな かった。 beads-beating処理と SDSとの組み合わせは、 その他の組み合わせより 抽出量が多く、弥生圃場堆肥連用区土壌については、 l gあたりの土壌から 30 g近い DNAを抽出す'ることが可能であった。 Figure 2 shows the results for the amount of soil DNA extracted. Irrespective of the heat treatment and beads-beating treatment, the extraction efficiency of soil DNA was better when SDS and Triton X100 were used, and there was no DNA degrading that could be attributed to the surfactant. The combination of beads-beating treatment and SDS resulted in a higher extraction volume than the other combinations. It was possible to extract DNA close to g.
CTAB およびグアジニンチオシァネートでは、 土壌試料から十分な量の DNA を抽出することができなかったが、 抽出液はほぼ無色に近く、 土壌からの腐植物 質の抽出を妨げる効果が非常に強いことが明らかとなった。  CTAB and guanidin thiosinate failed to extract sufficient amounts of DNA from soil samples, but the extract was almost colorless and very effective in preventing humic substances from being extracted from soil. It became clear.
ベンジルクロライド法で抽出したものについては、 かなりの量の DNAが電気 泳動によって確認されたが、 抽出 DNAの低分子化が著しかった。 すなわち、 加 熱により抽出したものでは 8000 bp以下に、 beads-beatingしたものではその大 部分が 2000 bp以下のサイズになっており、 塩化ベンジルは DNAの断片化を容 易に引き起こすことが判明した。 なお、 図 2には 2000 bp以上のサイズの DNA のみについて定量した結果を示したため、 ベンジルクロライド法の収量は極めて 小さく表れている。  For the benzyl chloride extracted, a considerable amount of DNA was confirmed by electrophoresis, but the molecular weight of the extracted DNA was remarkable. In other words, those extracted by heating had a size of 8000 bp or less, and those of beads-beating had a size of 2000 bp or less, indicating that benzyl chloride easily caused DNA fragmentation. . Note that Figure 2 shows the results of quantification of only DNA having a size of 2000 bp or more, so that the yield of the benzyl chloride method is extremely small.
加熱処理と beads-beating処理を比較すると、 beads-beating処理は極めて短 時間で抽出を行うことができ、 また DNAの収量も加熱処理と比較して多かった。 また加熱処理は、 beads-beatin 処理よりも多くの腐植物質が抽出されてしまい、 この腐植物質の除去が困難であると推測された。 泳動ゲルから推定される DNA のサイズは、 beads-beating処理により得られたものは 20 kbp程度の大きさのも のを主体に 20 kbp〜7 kbpの範囲に若干低分子化していた。 一方、 加熱処理で得 られた DNAは 23 kbp以上のサイズで低分子化はほとんどみられなかった。  Comparing the heat treatment with the beads-beating treatment, the beads-beating treatment was able to perform extraction in a very short time, and the yield of DNA was higher than that of the heat treatment. Also, it was presumed that the heat treatment extracted more humic substances than the beads-beatin treatment, and it was difficult to remove this humic substance. The size of the DNA estimated from the electrophoresis gel, which was obtained by beads-beating, was slightly reduced to a molecular weight of 20 kbp to 7 kbp, mainly about 20 kbp. On the other hand, the DNA obtained by the heat treatment had a size of 23 kbp or more, and almost no low molecular weight was observed.
以上の結果より、 今後の実験では、 抽出効率が高い SDS を界面活性剤として 使用することとし、 細胞破壊の過程としては操作が短時間でかつ収量も高かった beads-beating処理を採用することとした。  Based on the above results, in future experiments, we will use SDS with high extraction efficiency as a surfactant, and use beads-beating treatment, which is a short-time operation and high yield, as the process of cell disruption. did.
〔実施例 2〕 SDSの最適濃度の決定 [Example 2] Determination of optimal concentration of SDS
実施例 2では、 実施例 1の結果を踏まえ、 土壌 DNAの抽出液に用いる SDSの 最適濃度を検討した。  In Example 2, based on the results of Example 1, the optimal concentration of SDS used in the extract of soil DNA was examined.
(1) 材料と方法 (1) Materials and methods
実験 1 Experiment 1
弥生圃場堆肥区土壌を対象に、 .SDS濃度に 0%から 2%までの段階を設け、 土 壌 DNA抽出に最適な SDS濃度の条件を検討した。 SDS濃度以外の実験条件は 抽出液が 100 mM Tris-HCl lOOmM EDTA (pH 8.6) とし、 それ以外は、 (beads-beatingの条件が）実施例 1と同様である。実験はすべて 3連で行った。 実験 2 For the soil in the Yayoi farmland compost area, the level of SDS concentration was set from 0% to 2%, The optimal SDS concentration conditions for the extraction of DNA were examined. The experimental conditions other than the SDS concentration were the same as in Example 1 except that the extract was 100 mM Tris-HCl lOOmM EDTA (pH 8.6). All experiments were performed in triplicate. Experiment 2
抽出液の組成を' 100 mM Tris-HCl 300 mM EDTA(pH8.6)とし、 それ以外は 実施例 1と同様の条件を用いて、 SDS濃度に 0%か.ら 2%までの段階を設け、 土 壌 DNA抽出に最適な SDS濃度を検討した。 EDTA濃度に関する詳細は後述する。 実験には表 4に示した 6種類の土壌を供試土壌とした。  The composition of the extract was' 100 mM Tris-HCl 300 mM EDTA (pH 8.6), and the same conditions as in Example 1 were used except that the SDS concentration was changed from 0% to 2%. Then, the optimal SDS concentration for soil DNA extraction was examined. Details regarding the EDTA concentration will be described later. In the experiment, six types of soils shown in Table 4 were used as test soils.
表 4 供試土壌の理化学性 ァロフェン (mg/g soil) Al 弥生圃場堆肥連用区土壌 ァロフェン質黒ポク土 35.73 栃木農試森林土壌 ァロフェン質黒ポク土 23.63 東北大学森林土壌 非ァロフェン質黒ポク ± 20.82 埼玉農試畑土壌 灰色低地土 1.22 大阪農試畑土壌 灰色低地土 0.59 兵庫農試畑土壌 灰色低地土
Figure imgf000052_0001
0.56 Table 4 Physicochemical properties of test soils Arophen (mg / g soil) Al Yayoi field compost continuous use soil Arofen black pork soil 35.73 Tochigi agricultural test forest soil Arofen black pok soil 23.63 Tohoku University forest soil Non-arofen black po H ± 20.82 Saitama agricultural test field soil Gray lowland soil 1.22 Osaka agricultural test field soil Gray lowland soil 0.59 Hyogo agricultural test field soil Gray lowland soil
Figure imgf000052_0001
0.56
これらのうち弥生圃場堆肥連用区土壌および栃木農試 森林土壌、 東北大学 森林土壌は火山灰土壌で、 大阪農試 畑土壌および兵庫農試 畑土壌、 埼玉農試 畑土壌は非火山灰土壌 （沖積土壌） である。 また実験は 3連で行った。 Of these, Yayoi Farm Compost Continuous Use Area and Tochigi Agricultural Test Forest Soil, Tohoku University Forest Soil is volcanic ash soil, Osaka Agricultural Test Field Soil and Hyogo Agricultural Test Field soil, Saitama Agricultural Test Field soil is non-volcanic ash soil (alluvial soil) It is. The experiment was performed in triplicate.
(2) 結果と考察  (2) Results and discussion
実験 1の結果を図 3に、 実験 2の結果を図 4に示す。 100 mM EDTAおよび 300 mM EDTAの場合も SDS濃度が 0 %から 0.5ずつ増加するのにしたがって土 壌 DNAの抽出量は増加し、 0.5° /。より高濃度の SDS濃度においても 0.5%の場合 と比較して抽出量に有意な差は認められなかった。 よって本試験条件下で最大量 の土壌 DNAを抽出するためには、 0.5%の SDS濃度が必要であることが示され た。 但し、 SDS濃度の増加に伴って腐植物質の抽出量も増加もみられ （抽出液の 着色程度の目視観察に基づく推定）、 さらに、 抽出液が泡立ち、 試料の扱いが困難 になるといった問題も生じるため、 SDS濃度 0. &〜 1 %にとどめておくのが良い。 〔実施例 3〕 抽出液の pHの検討 Figure 3 shows the results of Experiment 1 and Figure 4 shows the results of Experiment 2. In the case of 100 mM EDTA and 300 mM EDTA, as the SDS concentration increased from 0% in 0.5 increments, the amount of soil DNA extracted increased to 0.5 ° /. Even at higher SDS concentrations, there was no significant difference in the amount of extraction compared to 0.5%. Therefore, it was shown that an SDS concentration of 0.5% was required to extract the maximum amount of soil DNA under the test conditions. However, as the SDS concentration increases, the amount of humic substances extracted also increases (estimation based on visual observation of the degree of coloring of the extract), and further, there is a problem that the extract foams and handling of the sample becomes difficult. Therefore, it is better to keep the SDS concentration at 0. & ~ 1%. [Example 3] Examination of pH of extract
本実施例では、 抽出液の適切な pHおよび緩衝能を検討した。  In this example, the appropriate pH and buffer capacity of the extract were examined.
(1) 材料と方法 (1) Materials and methods
使用した土壌は弥生圃場堆肥連用区土壌および埼玉農試畑土壌の 2 点である。 理化学性は表 5に示した。 表 5 供試土壌の理化学性
Figure imgf000053_0001
抽出液は Tris-EDTA緩衝系の抽出液を用いた。抽出液の pHを 9.0、 8.0、 7.0、 6.0の 4段階に設定した 100 mM Tris-HCl/100 mM EDTA/1 % SDSから成る抽 出液 1.2 mlを土壌 0.5 gに添加し、 beads-beating処理により DNAを抽出し、 除タンパク後 DNAの沈殿回収および定量を行った。 Two soils were used: Yayoi field compost continuous use area soil and Saitama agricultural test field soil. The physicochemical properties are shown in Table 5. Table 5 Physical and chemical properties of test soil
Figure imgf000053_0001
The extract used was a Tris-EDTA buffer extract. 1.2 ml of an extract consisting of 100 mM Tris-HCl / 100 mM EDTA / 1% SDS with pH set to 9.0, 8.0, 7.0 and 6.0 was added to 0.5 g of soil, and beads-beating treatment was performed. The DNA was extracted by DNA extraction, and after the protein was removed, the DNA was recovered and quantified.
(2) 結果と考察  (2) Results and discussion
結果'を図 5に示す。 いずれの土壌からの DNA抽出についても、 試験した pH の範囲内ではアルカリ性の強い抽出液ほど高収量であった。 beads-beating処理 直後の粗抽出液の pHを測るといずれの土壌も土壌の酸度により pHが低下して いることが明らかになった。 抽出時の pHの低下は DNAの化学的安定性を損な うだけではなく、 土壌 DNAの収量が低下するためアルカリ性に維持される必要 があることがわかった。 このような理由で低濃度の緩衝系では土壌の酸度を考慮 した緩衝能を持つ抽出液を使用する必要があると考えられた。  The results are shown in FIG. For DNA extraction from all soils, the more alkaline extracts yielded higher yields within the pH range tested. Measuring the pH of the crude extract immediately after beads-beating treatment revealed that the pH of all soils was lowered due to the acidity of the soil. It was found that lowering the pH during extraction not only impaired the chemical stability of the DNA, but also needed to be maintained alkaline because the yield of soil DNA was reduced. For this reason, it was considered necessary to use an extract with a buffer capacity that takes into account the acidity of the soil in a low-concentration buffer system.
しかし、 pH 8.3の 1M Tris-HCl溶液および 0.5M EDTA溶液を混合して抽出 液を調整すると pHは 8.6に上昇し安定していること、 また、 後述の実施例 （実 施例 8 ) では高濃度の EDTAとリン酸緩衝液を使用することになつたので、 これ らがあわせもつ緩衝能により抽出後の溶液の pHはほとんど抽出前の抽出液と変 わらないことが確認できている。 従って高濃度の EDTA、 リン酸緩衝液を使用す る抽出液においては、 抽出前後の溶液の pHは 8.6でほぼ一定であり、 抽出時の pH変化は考慮しなくとも良いと考えた。 However, when the extract was adjusted by mixing a 1M Tris-HCl solution with a pH of 8.3 and a 0.5M EDTA solution to adjust the extract, the pH increased to 8.6 and was stable. Also, in the example described later (Example 8), Since the concentrations of EDTA and phosphate buffer were used, it was confirmed that the pH of the solution after extraction was almost the same as the extract before extraction due to the combined buffer capacity. Therefore, use high concentrations of EDTA and phosphate buffer. The pH of the solution before and after extraction was almost constant at 8.6, and it was thought that the pH change during extraction did not need to be considered.
〔実施例 4〕 土壌による DNA吸着の解消 [Example 4] Elimination of DNA adsorption by soil
本実施例では抽出液の EDTA濃度が DNA収量および金属イオン抽出量に与え る影響について検討した。  In this example, the effect of the EDTA concentration of the extract on DNA yield and metal ion extraction was examined.
(1) 材料と方法  (1) Materials and methods
土壌 DNA抽出の基本的な条件は実施例 1と同様とし、 抽出液の EDTA濃度の  The basic conditions for soil DNA extraction were the same as in Example 1, and the EDTA concentration of the extract was
Figure imgf000054_0001
土壌 DNAの抽出量および DNA粗抽出液 （beads-beating後遠心した上清） 中 に溶存していた Al、 Fe、 Ca、 Mgの濃度を分析した。 これらの金属元素は土壌中 において主要なものであり、 土壌による DNA吸着の原因となり得る元素である と考えられた。 土壌 DNAの抽出量は実施例 1と同様の方法で定量し、 金属元素 は適宜希釈後 ICP発光分析 （Seiko SPS-1200) により定量した。 実験は 3連で 行った。
Figure imgf000054_0001
The amount of soil DNA extracted and the concentration of Al, Fe, Ca, and Mg dissolved in the crude DNA extract (supernatant centrifuged after beads-beating) were analyzed. These metal elements were predominant in soil and were considered to be the elements that could cause DNA adsorption by soil. The amount of soil DNA extracted was quantified in the same manner as in Example 1, and the metal elements were appropriately diluted and quantified by ICP emission analysis (Seiko SPS-1200). The experiment was performed in triplicate.
(2) 結果と考察  (2) Results and discussion
EDTA濃度が土壌抽出 DNAに与える影響について、 図 6にァガロースゲル電 気泳動後染色した DNAの写真の一例を、 図 7に土壌 DNA抽出量を定量した結 果を示す。 また、 粗抽出 DNA液中の金属元素の定量結果を図 8 A〜C に示す。 EDTA によりキレートされ抽出されたと考えられる金属元素は EDTA-金属元素 複合体になっていると考えられる。 EDTAの総量に対する EDTA—金属元素複合 体形成率、 すなわち実際の EDTAが使われた割合を、 4種類の元素それぞれの複 合体形成率おょぴ 4種類の元素をあわせた EDTA金属元素複合体形成率について 求めた。 これを図 9 A〜Cに示す。 Figure 6 shows an example of the effect of EDTA concentration on soil-extracted DNA.Figure 6 shows an example of a photograph of DNA stained after agarose gel electrophoresis. The result is shown. Figures 8A to 8C show the results of quantification of metal elements in the crude extracted DNA solution. Metal elements that are considered to be chelated and extracted by EDTA are considered to be EDTA-metal element complexes. The ratio of EDTA-metal element complex formation relative to the total amount of EDTA, that is, the ratio of actual EDTA used, is calculated as the complex formation rate of each of the four elements. EDTA metal element complex formation combining the four elements The rate was determined. This is shown in FIGS.
(2-1) 火山灰土壌について  (2-1) Volcanic ash soil
火山灰土壌においては、 3種類の土壌すべてにおいて抽出液中の EDTA濃度の 上昇に伴い DNAの抽出量も増加した。特に栃木農試森林土壌においては、 EDTA 濃度が 100 mM以上でないと DNAが全く抽出されず、 さらに 300 mM以上とす ることで高分子の DNAがより多く得られた。 前述のように、 DNAが土壌に吸着 する主な原因であると推定されるリン酸基は、 DNA の構成単位であるヌクレオ チドごとに存在しており、 長い DNA分子のリン酸基の一部が土壌粒子に吸着さ れてしまうことで、 DNA が溶液中に放出されなくなると考えられる。 このよう に考えると、 高分子の DNAほどリン酸基を多く持つことになるので、 土壌粒子 に強固に吸着する能性が高くなることが推測される。 ァガロースゲルの電気泳動 結果より、弥生圃場堆肥連用土壌や栃木農試森林土壌において EDTA濃度が低い と低分子の DNAしか抽出できておらず、 高濃度の EDTAにより初めて高分子の DNAが抽出され始めている。火山灰土壌から高分子 DNAを抽出するためには高 濃度の EDTA溶液を使用する必要があることが明らかとなった。  In the volcanic ash soil, the amount of DNA extracted in all three types of soil increased as the EDTA concentration in the extract increased. Especially in the Tochigi agricultural test forest soil, no DNA was extracted unless the EDTA concentration was 100 mM or higher, and more high-molecular DNA was obtained by using 300 mM or higher. As described above, phosphate groups, which are presumed to be the main cause of DNA adsorption to soil, are present in each of the DNA constituent units, nucleotides, and are part of the phosphate groups of long DNA molecules. It is thought that DNA is not released into the solution due to the adsorption of the soil particles. In this way, it is assumed that the higher the molecular weight of DNA, the more phosphate groups it has, and the higher its ability to strongly adsorb to soil particles. Based on the results of agarose gel electrophoresis, only low-molecular-weight DNA could be extracted at low EDTA concentration in Yayoi field compost continuous soil and Tochigi agricultural test forest soil, and high-molecular-weight DNA began to be extracted for the first time with high-concentration EDTA. . It became clear that it was necessary to use a high-concentration EDTA solution to extract high molecular weight DNA from volcanic ash soil.
高濃度の EDTAは土壌から大量の金属元素を溶出した。弥生圃場堆肥連用区土 壌からは比較的低濃度の EDTAによっても DNAが抽出されている。 弥生圃場堆 肥連用区土壌から抽出された金属元素はほとんどが Caであり、 A1は少ない。 弥 生圃場は火山灰土壌でありながら長期の堆肥連用により Caが土壌に集積し、 土 壌中に含まれる金属元素組成が大きな影響を受けたことが窺い知れる。 栃木農試 の森林土壌や東北大学森林土壌のように大量の非晶質アルミニウムを含有してい る土壌では、 十分量の EDTAにより DNAを吸着ずる土壌の非晶質 A1が除去さ れなければ、 土壌 DNAは多くは抽出されない。 栃木農試森林土壌 よび東北大学森林土壌については、 A1以外の金属、 すなわ ち Fe、 Ca、 Mgは、 EDTA濃度が 50 mM以下の濃度において既に最大抽出量 が得られており、 50 mM以上に EDTA濃度を設定してもそれらの抽出量の増加 はほとんど見られなかった。しかし、 A1は EDTA濃度の影響を顕著に受け、 EDTA 濃度が 100 mMに達するまでは高い錯体形成率で、 EDTA濃度に対応して A1が 土壌から溶出している。'また 100 mM以上の濃度でも緩やかに A1の溶出量は増 加している。 東北大学森林土壌においては EDTA濃度が 50 mMで、 栃木農試森 林土壌においては EDTAが 100 ιηΜの濃度で土壌 DNAが抽出されはじめ、それ 以上では、 EDTA濃度に比例し DNA抽出量が増加している。 土壌による DNA の吸着は土壌の非晶質 A1が原因であり、 EDTAにより DNAを吸着する A1量が 除去されて初めて抽出できることが窺いしれる。 これらのことから、 従来の手法 で火山灰土壌から DNAが抽出できなかった一因は、 EDTA可溶の非晶質 A1によ る DNAの吸着であると考えられた。 High concentrations of EDTA eluted large amounts of metal elements from soil. DNA is also extracted from the Yayoi field compost continuous use soil with relatively low concentration of EDTA. Most of the metal elements extracted from the soil of the Yayoi Field Compost Zone are Ca, and A1 is low. Although the Yayoi field is a volcanic ash soil, Ca is accumulated in the soil due to long-term continuous use of compost, indicating that the metal element composition contained in the soil was greatly affected. In soils containing large amounts of amorphous aluminum, such as the forest soil of the Tochigi Agricultural Trial and the forest soil of Tohoku University, if amorphous A1 in the soil that adsorbs DNA with a sufficient amount of EDTA is not removed, Much of the soil DNA is not extracted. For Tochigi Agricultural Forest Soil and Tohoku University Forest Soil, metals other than A1, that is, Fe, Ca, and Mg, had already been extracted at the maximum EDTA concentration of 50 mM or less, and 50 mM or more. Even when the EDTA concentration was set in the above, almost no increase in the amount of those extracted was observed. However, A1 was significantly affected by the EDTA concentration, with a high complexation rate until the EDTA concentration reached 100 mM, and A1 was eluted from the soil corresponding to the EDTA concentration. 'Also, even at concentrations of 100 mM or more, the amount of A1 eluted gradually increased. In the Tohoku University forest soil, the EDTA concentration was 50 mM, and in the Tochigi Agricultural Forest soil, the soil DNA began to be extracted at an EDTA concentration of 100 ιηΜ. Above that, the DNA extraction amount increased in proportion to the EDTA concentration. ing. The adsorption of DNA by the soil is due to the amorphous A1 in the soil, suggesting that extraction can be performed only after the amount of A1 that adsorbs DNA is removed by EDTA. These results suggest that one of the reasons that DNA could not be extracted from volcanic ash soil by the conventional method was DNA adsorption by EDTA-soluble amorphous A1.
(2-2) 非火山灰土壌について  (2-2) Non-volcanic ash soil
非火山灰土壌においては、 EDTA濃度が 50 mM以下でも土壌 DNAが抽出さ れた。 3種類の土壌いずれについても EDTA濃度が 100〜200 mM程度で収量が 最大となっており、 それ以上の濃度では逆に DNA収量が低下した。 非火山灰土 壌からの金属イオンの抽出量を見るとその大部分が Caである。 これらの土壌に おいて、 EDTAで抽出されると考えられる Caが全量抽出できていない 50 mM以 下の低濃度の EDTAであっても、 すなわち Caが土壌から十分に除去できていな い状態でも DNAの収量が十分得られていることから （図 7、 図 8 A参照）、 土壌 中に含まれている Caは DNAを抽出に影響を及ぼす程度の吸着をしないことが 示される。 また埼玉農試は火山灰土壌が多少混入しており、 大阪農試畑土壌、 兵 庫農試畑土壌と比較すると A1 の溶出量は多いが、 火山灰土壌と比較すると、 非 火山灰土壌からの A1の溶出量は EDTA濃度を高くしても A1の溶出量にはほとん ど差が見られなかった。  In non-volcanic ash soil, soil DNA was extracted even when the EDTA concentration was less than 50 mM. In all three types of soil, the yield was maximum at EDTA concentrations of about 100 to 200 mM, and at higher concentrations, the DNA yield was conversely reduced. Looking at the amount of metal ions extracted from non-volcanic ash soil, most of it is Ca. In these soils, the amount of Ca that is considered to be extracted by EDTA cannot be fully extracted.Even in the case of low-concentration EDTA of 50 mM or less, that is, even when Ca is not sufficiently removed from the soil. Sufficient DNA yields were obtained (see Figures 7 and 8A), indicating that Ca contained in the soil did not adsorb enough to affect DNA extraction. In addition, Saitama Agricultural Trial contains a small amount of volcanic ash soil, and the amount of A1 eluted is larger than that of Osaka Agricultural Experimental Soil and Hyogo Agricultural Experimental Soil. There was almost no difference in the elution amount of A1 even when the EDTA concentration was increased.
(2-3) EDTA—金属元素複合体形成率について  (2-3) EDTA-metal element complex formation rate
図 8 Bより、 火山灰土壌、 非火山灰土壌ともに 10〜20 mM程度の低濃度の状 態では、 EDTAは、 のほとんどが今回分析した 4種類の金属元素と錯体を形成 していることが明らかとなった。 濃度が上昇するにつれ、 金属元素の種類によら ず錯体の形成率は減少し、 EDTA濃度が 300〜400 mMと高濃度になると、 土壌 の種類によらず EDTA-金属元素複合体形成率は約 20%でほぼ一定になっていた。 これは EDTAの金属元素とのキレート反応の平衡が EDTAの濃度および土壌の 金属元素組成によらず、 高濃度では一定の錯体形成率に達しているためと考えら れた。 つまり 10〜20 mM程度の低濃度の EDTAは 100%の割合で金属元素とキ レートし、 300〜400 mMと高濃度な状態では EDTAはその約 20%が金属元素を キレー卜すると考えられる。 このことより高濃度 EDTAを抽出の最初の段階から 使用しなくとも、 低濃度の EDTA溶液で抽出を繰り返す、 もしくは土壌に対し抽 出液の比を大きくすることにより、土壌 DNAを抽出できる可能性が考えられた。 From Fig. 8B, both volcanic ash soil and non-volcanic ash soil have low concentrations of about 10 to 20 mM. In this state, it was found that most of the EDTA complexed with the four metal elements analyzed in this study. As the concentration increases, the complex formation rate decreases irrespective of the type of metal element, and when the EDTA concentration is as high as 300 to 400 mM, the formation rate of the EDTA-metal element complex is about irrespective of the type of soil. It was almost constant at 20%. This is thought to be because the equilibrium of the chelating reaction of EDTA with the metal element reached a certain complex formation rate at high concentrations, irrespective of the concentration of EDTA and the composition of the metal element in the soil. In other words, it is considered that EDTA at a low concentration of about 10 to 20 mM chelates a metal element at a rate of 100%, and at a high concentration of 300 to 400 mM, about 20% of the EDTA chelate a metal element. Therefore, even if high-concentration EDTA is not used from the initial stage of extraction, it is possible to extract soil DNA by repeating extraction with low-concentration EDTA solution or increasing the ratio of extract to soil. Was thought.
(2-4) 高濃度 EDTAに関する留意点について  (2-4) Notes on high-concentration EDTA
EDTA使用時に留意すべき点として、 EDTAが 400 mM以上になると DNAの 低分子化が引き起こされたことがあげられる。 pHが 8.3程度の溶液では、 EDTA1 分子あたりカウンタ一イオンとしておよそ 3分子のナトリゥムイオンが存在して いる。 400 mMの EDTA溶液はィォン強度にして 1.2 M NaCl溶液に相当する高 濃度の塩溶液である。よってこの大量に含まれるナトリゥムイオンのために DNA が電気的に中和されてしまい、 この影響で beads-beating処理の際、 せん断され やすくなつているのではないかと考えられた。 また EDTA濃度が 300 mM以上 の場合、 高濃度の塩により常温では SDS が白い結晶として析出するため、 使用 時は 40 °C程度に溶液を加熱して溶解させる必要があった。 沖積土壌においては EDTA濃度が 200 mM以上の高濃度になると火山灰土壌の場合とは逆に DNAの 抽出量が低下した。 これは高濃度 EDTA溶液の高塩濃度により、 界面活性剤であ る SDS がミセル化、 もしくは析出してしまったために土壌微生物の溶菌率が下 がってしまったことに起因するものであると考えられた。  A point to keep in mind when using EDTA is that when EDTA exceeds 400 mM, the molecular weight of DNA was reduced. In a solution with a pH of about 8.3, about three sodium ions exist as one counter ion per EDTA molecule. The 400 mM EDTA solution is a high concentration salt solution corresponding to a 1.2 M NaCl solution in ion intensity. Therefore, it was thought that DNA was electrically neutralized due to the sodium ions contained in such a large amount, and this effect might have caused the beads to be easily sheared during beads-beating treatment. When the EDTA concentration is 300 mM or higher, SDS precipitates as white crystals at room temperature due to the high concentration of salt, so it was necessary to dissolve the solution by heating it to about 40 ° C before use. In alluvial soils, when the EDTA concentration was higher than 200 mM, the amount of DNA extracted was lower than in volcanic ash soils. This is due to the fact that the high salt concentration of the high-concentration EDTA solution resulted in micelles or precipitation of the surfactant SDS, which reduced the lysis rate of soil microorganisms. it was thought.
以上のことから beads-beating処理を行う際、 EDTAの最大使用濃度は 300 mM とし、 この濃度を越えると DNAは低分子化することを前提に実験をする必要が あると考えられた。 〔実施例 5〕 抽出回数が土壌 DNA抽出量に与える影響 From the above, it was considered that when beads-beating treatment was performed, the maximum use concentration of EDTA was set to 300 mM, and it was necessary to perform experiments on the premise that if this concentration was exceeded, DNA would be reduced in molecular weight. [Example 5] Effect of the number of extractions on the amount of soil DNA extracted
EDTAはほとんどの金属イオンに対して優れたキレ一ト安定定数を持つ化合物 である （株式会社同仁化学研究所カタログ 22nd Edition )。 これは極めて低濃度 であっても、大部分の EDTAが対象金属イオンと錯体を形成することを意味する。 今回検討した beads-beating処理を利用する DNA抽出においては、 土壌 0.5 g に対し抽出液量は 1.0 mlである。抽出を繰り返したり土壌に対する抽出液量の比 が大きくすることで、 低濃度の EDTA溶液であっても、 今回対象としている土壌 の非晶質 A1を除去できる可能性がある。 本実施例では EDTAの絶対量が重要な のか、 もしくは濃度が重要なのかを明らかにするため、 EDTA濃度を数段階設定 し、 同じ土壌から数回 DNAの抽出を繰り返し行った場合の DNAの合計収量を 検討した。  EDTA is a compound with an excellent chelate stability constant for most metal ions (Catalog 22nd Edition, Dojin Chemical Laboratory Co., Ltd.). This means that, even at very low concentrations, most EDTA will complex with the target metal ion. In the DNA extraction using beads-beating treatment studied in this study, the amount of extract was 1.0 ml for 0.5 g of soil. By repeating extraction and increasing the ratio of extract to soil, even if the concentration of EDTA solution is low, amorphous A1 in the target soil may be removed. In this example, in order to clarify whether the absolute amount or the concentration of EDTA is important, the EDTA concentration was set at several levels, and the total DNA obtained when DNA was repeatedly extracted several times from the same soil was determined. The yield was examined.
(1) 材料と方法  (1) Materials and methods
供試土壌は弥生圃場堆肥連用区土壌、 栃木農試森林土壌、 東北大学森林土壌の 3種類の土壌を用いた。 基本的な抽出液組成は前項と同じで Tris-HCl 100 mM、 SDS 1%を用い、 EDTA濃度を 0 mM、 50 mM、 100 mM、 200 mM、 300 mMと 5段階に設定した抽出液を用いた。 beads lgを含むスクリュ一キヤップ付チュー ブに土壌 0.5 gを分注し、 1.0 ml の抽出液を添加後 beads-beating処理を強度 5m/secで 30秒間行った。 その後 12000 X gで 5分間遠心後、 上清 500 μΐを回収 した。上清を回収した残りの土壌に再び 1回目の抽出と同じ濃度の EDTA抽出液 を 500 μΐ添加し、攪拌のために beads-beating処理を強度 4m/secで 5秒間行い、 再び遠心し、 上清を回収した。 こ'の作業を 5回繰り返した。 回収した上清 500 μ のうち 20 μΐを希釈後 ICP発光分析による金属イオンの定量に供し、残りの 480 μΐ にはクロ口ホルムを用いて除タンパク処理し、 最終的に 400 μΐを回収した。 これ に終濃度 0.3 Μになるように 5 Μ酢酸ナトリウム溶液（ρΗ 5.2)を添加し、 6/10 量の 2-pi'opanolを用いて DNAを回収した。 DNAの定量は 2_2_1と同様にして 行った。 実験は 3連で行った。  The test soil used was three types of soil: Yayoi field compost continuous use soil, Tochigi agricultural test forest soil, and Tohoku University forest soil. The basic extract composition is the same as in the previous section, using Tris-HCl 100 mM and SDS 1%, and using EDTA concentrations of 0 mM, 50 mM, 100 mM, 200 mM, 300 mM and 5 mM extract. Was. 0.5 g of soil was dispensed into a tube with a screw cap containing beads lg, and 1.0 ml of the extract was added, and beads-beating treatment was performed at a strength of 5 m / sec for 30 seconds. Then, after centrifugation at 12000 X g for 5 minutes, 500 µ 500 of the supernatant was recovered. To the remaining soil from which the supernatant was collected, add 500 μΐ of the same concentration of EDTA extract as the first extraction again, perform beads-beating treatment at 4 m / sec for 5 seconds for stirring, and centrifuge again. Qing was recovered. This process was repeated five times. After diluting 20 μΐ of 500 μ 回収 of the collected supernatant, it was subjected to quantification of metal ions by ICP emission spectrometry. The remaining 480 μΐ was subjected to protein removal using black-mouthed form, and finally 400 μΐ was recovered. To this was added a 5 （sodium acetate solution (ρΗ5.2) to a final concentration of 0.3Μ, and the DNA was recovered using 6/10 volume of 2-pi'opanol. DNA quantification was performed in the same manner as in 2_2_1. The experiment was performed in triplicate.
繰り返し抽出の段階ごとに再添加した抽出液により、 新たに得られたと考えら れる土壌 DNA量、 溶'出金属元素量は、 上記の操作にしたがって以下の計算式で 記述される。 ただし、 n回目の DNAまたは金属元素の抽出量を Xn (土壌 lgあ たりの DNAまたは金属元素の収量に換算した値、 単位は g/g soil) とする。 2回目の抽出により新たに得られた量 = X2 - X1 X 1/2 It is believed that the extract was re-added at each stage of the extraction, and that it was newly obtained. The amount of soil DNA and the amount of leached metal elements are described by the following formulas according to the above operation. However, the extraction amount of DNA or metal element at the nth time is Xn (value converted to the yield of DNA or metal element per lg of soil, the unit is g / g soil). New amount obtained from the second extraction = X2-X1 X 1/2
3回目の抽出により新たに得られた量 = X3 - X2X 1/2 - X1 X 1/4 New amount obtained by the third extraction = X3-X2X 1/2-X1 X 1/4
4回目の抽出により新たに得られた量 = X4 - X3 X 1/2 - X2 X 1/4 - XI X 1/8 New amount obtained from the fourth extraction = X4-X3 X 1/2-X2 X 1/4-XI X 1/8
5回目の抽出により新たに得られた量 = X5 - X4X 1/2 - X3 X 1/4 -X 2 X 1/8 - XIX5-X4X 1/2-X3 X 1/4 -X 2 X 1/8-XI
X 1/16 X 1/16
なお実験はすべて 3連で行った。 All experiments were performed in triplicate.
(2) 結果と考察  (2) Results and discussion
異なる濃度の E D TA抽出液を用いた繰り返し抽出によって得られた土壌 D N A の段階ごとの収量（lgあたりの土壌からの収量に換算した）および土壌から 5回 抽出した土壌 DNAの合計収量を図 10A〜Cに示す。 繰り返し抽出の際に、 追加 して添加した EDTAにより抽出されたと考えられる土壌 DNA量 （前述の計算に 従った値；計算例を図 11.にあげる）を図 12に示す。また追加して添加した EDTA のキレート形成により抽出されたと考えられる金属元素量について図 13A〜Bに 示す。  Figure 10A shows the yield of each step of the soil DNA (converted to the yield per lg of soil) and the total yield of soil DNA extracted five times from the soil obtained by repeated extraction using EDTA extracts at different concentrations. To C. Figure 12 shows the amount of soil DNA that was considered to have been extracted by the added EDTA during repeated extraction (values according to the above calculations; calculation examples are shown in Figure 11). Figures 13A and 13B show the amounts of metal elements considered to have been extracted by the chelation of the added EDTA.
5 回抽出し得られた土壌 DNA量は、 3 種類すベての土壌において 300 mM EDTAを含む抽出液によるものが最も多かった （図 10A〜C)。 最も DNA収量の 良かった 300 mMの EDTAを含む抽出液による繰り返し抽出では、 2回目以降の 土壌 DNAの抽出量はほぼ変わらなかった （図 12)。. 300 mMの EDTAを用いた 抽出では、 土壌 DNAは 1回目の抽出でほぼ全量抽出されたと思われ、 2回目以 降に EDTA溶液を新たに添加して抽出しても、 1回目の抽出時に回収しきれなか つた溶液 （遠心後の土壌およびビーズの間隙に含まれていた部分） を少しずつ回 収して抽出量が増えているだけであり、 土壌に吸着された DNAを剥離して回収 しているものではないと思われた。  The amount of soil DNA obtained by extracting five times was the highest in all three types of soils containing the extract containing 300 mM EDTA (Figs. 10A to 10C). Repeated extraction with the extract containing 300 mM EDTA, which had the highest DNA yield, showed almost no change in the amount of soil DNA extracted from the second time onward (Figure 12). In the extraction using 300 mM EDTA, it was considered that almost all of the soil DNA was extracted in the first extraction. The recovered solution (the part contained in the gap between the soil and beads after centrifugation) was recovered little by little and the amount of extraction increased, and the DNA adsorbed on the soil was separated and recovered. It didn't seem to be what I was doing.
栃木農試森林土壌からは 100 mM以下の EDTAを含む抽出液で繰り返し抽出 すると 2回目以降に 1回目に得られた土壌 DNA量の 2倍近い量の土壌 DNAが 得られた。 これについては 2回目に添加された EDTAが土壌から A1を除去する ことにより吸着されていた DNAが回収できたものと考えられる。 また東北大学 森林土壌においても 100 mM以下の濃度の抽出液で、 2回目に 1回目の抽出量の 約 3分の 1程度が新たに得られており、 これも 2回目に添加した EDTAにより A1が除去され DNAが回収できたものであると考えられた。 Repeated extraction from the Tochigi Agricultural Forest Soil with an extract containing 100 mM or less EDTA yields nearly twice the amount of soil DNA obtained from the first time after the second time. Obtained. It is probable that this was due to the fact that the adsorbed DNA could be recovered by removing A1 from the soil with EDTA added the second time. Also, in forest soil of Tohoku University, about one-third of the amount of the first extraction was newly obtained with the extract at a concentration of 100 mM or less. Was removed and the DNA was considered to be recovered.
土壌抽出に用いた EDTAの絶対量が同じ場合で比較すると、 例えば EDTAの 絶対量が土壌 0.5 gに対し 300 molとした場合、すなわち 300 mM EDTA抽出 液による 1回目の抽出量と 200 mMの EDTAの 1回目と 2回目の抽出合計量の 比較では、 弥生圃場堆肥連用区土壌および東北大学森林土壌においてはほぼ同じ 量の土壌 DNAが得られている。 これは EDTAの絶対 *が 200 nmol、 100 nmol の場合でも同様であった。しかし、栃木農試森林土壌においてはこの計算はあては まらず、 EDTAが絶対量で同じであっても抽出 DNA量には大きな差が生じた。 すなわち同じ EDTA量であっても 1回目に高濃度で抽出した方が抽出量は多く、 繰り返し抽出しても高濃度で 1度に抽出した DNA量に及ばなかった。  When the absolute amount of EDTA used for soil extraction is the same, for example, when the absolute amount of EDTA is 300 mol per 0.5 g of soil, that is, when the first extraction amount using 300 mM EDTA extract and 200 mM EDTA In the comparison between the first and second extractions, approximately the same amount of soil DNA was obtained in the Yayoi field compost continuous use soil and the Tohoku University forest soil. This was the same when the absolute * of EDTA was 200 nmol or 100 nmol. However, this calculation did not apply to the Tochigi Agricultural Forest Soil, and there was a large difference in the amount of extracted DNA even if the absolute amount of EDTA was the same. That is, even with the same amount of EDTA, the amount of DNA extracted at the first high concentration was larger than that of the first extraction, and even with repeated extraction, it did not reach the amount of DNA extracted at a high concentration at one time.
これらの結果より弥生圃場堆肥連用区土壌および東北大学森林土壌は DNAを 吸着する非晶質 Aiの作用が比較的弱く、 50 mM程度の比較的低濃度の EDTA溶 液で土壌 DNAの一定の量が抽出され、 それ以上の EDTAにより土壌へ吸着され ていた DNAを回収していて、 EDTA量に対応した土壌 DNAをほぼ安定して回 収できることが明らかとなった。 しかし、 栃木農試森林土壌においては非晶質 A1 の作用が強く、 DNAの抽出には 200 mM以上の EDTAが必要であり、 一度土壌 に吸着された DNAを再び解離させ回収するのは困難であると考えられた。  Based on these results, the Yayoi field compost continuous use soil and Tohoku University forest soil have a relatively weak effect of amorphous Ai that adsorbs DNA, and a certain amount of soil DNA is obtained with a relatively low concentration of about 50 mM EDTA solution. Was extracted, and the DNA adsorbed to the soil with more EDTA was recovered, and it became clear that soil DNA corresponding to the amount of EDTA could be recovered almost stably. However, the effect of amorphous A1 is strong in Tochigi agricultural test forest soil, and EDTA of 200 mM or more is required for DNA extraction, and it is difficult to dissociate and recover DNA once adsorbed on the soil. It was thought there was.
(3) 金属元素の抽出量  (3) Extraction amount of metal elements
弥生圃場堆肥区土壌からは、 他の 2種類の火山灰土壌と異なり、 吸着の原因と なる A1 がほとんど抽出されなかった。 弥生圃場堆肥連用区土壌は長年の堆肥連 用により Caが大量に土壌に集積し、 堆肥を連用していない対照区と比較すると 土壌中の A1量が少なく、 土壌が Caの影響を強く受けていると考えられた。 また この土壌のもう一つの特徴は非晶質の Siが多いことがあげられ、 非晶質 A1の大 部分がこの Siとアルミノシリケ一ト （またはケィ素比が高いァロフェン） を形成 している可能性が考えられた。 この土壌では Alは大部分が負電荷をもつ Siと複 合体を形成しているため、 土壌 DNAの吸着作用が小さいものと考えられた。 東北大学森林土壌からは A1 が多量に抽出された。 この土壌は、 非ァロフェン 質黒ポク土壌という火山灰土壌の中でも特殊な土壌であり、 ピロリン酸抽出 A1 (すなわち腐植 -A1 複合体の形態をとるもの） が多く、 ァロフェン含量が低いこ とがあげられる。 腐植 -A1複合体になっている A1が、 土壌中の非晶質 A1全体の 約半分の量に達しており、 腐植物質と錯体を形成している A1 が多い。 この東北 大学森林土壌は非晶質アルミ成分が非常に多いのにもかかわらず、 50 mMという 比較的低濃度の EDTAを含む抽出液でも DNAが抽出することが可能であった。 この土壌からは EDTAにより高効率で土壌から A1が除去されていることが示唆 される。 よって DNA抽出時の EDTAは、 ピロリン酸抽出の際のピロリン酸とほ ぼ同じ作用を土壌にもたらしていると考えられる。 Unlike the other two types of volcanic ash soil, A1 that caused adsorption was hardly extracted from the soil of the Yayoi compost plot. In the soil of the Yayoi Farm Compost Area, Ca was accumulated in the soil in large amounts due to long-term use of compost, and the amount of A1 in the soil was smaller than that of the control area where no compost was used, and the soil was strongly affected by Ca. Was thought to be. Another characteristic of this soil is that it is rich in amorphous Si, and most of the amorphous A1 forms aluminosilicate (or arophen with a high silicon ratio) with this Si. I thought it was possible. In this soil, Al mostly formed a complex with negatively charged Si, so it was considered that the adsorption of soil DNA was small. A1 was extracted in large amounts from Tohoku University forest soil. This soil is a special soil among the non-arofenous black pork soils of volcanic ash soil, which is rich in pyrophosphate-extracted A1 (that is, in the form of humus-A1 complex) and low in arofen content. Can be A1 in the humus-A1 complex accounts for about half of the total amount of amorphous A1 in soil, and many A1s are complexed with humic substances. In this Tohoku University forest soil, DNA was able to be extracted even with an extract containing a relatively low concentration of 50 mM EDTA, despite the extremely high content of amorphous aluminum. This suggests that A1 was removed from the soil with high efficiency by EDTA. Therefore, it is considered that EDTA at the time of DNA extraction has almost the same effect on the soil as pyrophosphate at the time of pyrophosphate extraction.
栃木森林土壌もピロリン酸抽出 A1 が多い土壌であるが、 ァロフェンも多く含 んでいる。 前述したように、 この土壌では繰り返し抽出を行っても DNA収量は 増加せず、 2回目以降に新しく添加した EDTAがほとんど DNAの吸着解消には 作用していない可能性が考えられる。しかし、東北大学森林土壌の場合と同じく、 A1 は 2 回目以降に添加された土壌より新たに EDTA により除去されている。 EDTAでさらに土壌から Alを除去しても DNA収量が上がらないという栃木農試 森林土壌で見られた現象により、土壌により DNAを吸着する非晶質 A1の主要な 形態が複数存在していること、 土壌によっては EDTAが除去する非晶質 A1の形 態がその土壌において DNAを吸着する形態の AIとは異なる可能性があることが 推測される。  Tochigi forest soil is also rich in pyrophosphate extract A1, but also rich in arofen. As mentioned above, repeated extraction in this soil did not increase the DNA yield, suggesting that the newly added EDTA from the second time onward may have little effect on the elimination of DNA adsorption. However, as in the case of Tohoku University forest soil, A1 was newly removed by EDTA from the soil added after the second time. Tochigi Agricultural Trial, in which the DNA yield does not increase even if Al is further removed from the soil by EDTA, due to the phenomenon seen in the forest soil, there are multiple main forms of amorphous A1 that adsorb DNA to the soil However, it is speculated that in some soils, the form of amorphous A1 removed by EDTA may be different from the form of AI that adsorbs DNA in the soil.
また 3種類の土壌ともに Caと Mgについては EDTA濃度によらず 1回目の抽 出でほぼ全量が溶液中に抽出されており、 2回目以降に新しく添加された EDTA により新たに抽出された Ca、 Mgはほとんどなかった。 これより、 Ca、 Mgは低 濃度の EDTAであっても容易に土壌から除去することが可能で、 土壌 DNAの吸 着にはあまり関与していないことが確認された。  In addition, in all three types of soil, almost all of Ca and Mg were extracted into the solution by the first extraction regardless of the EDTA concentration, and Ca and Mg newly extracted by the newly added EDTA after the second extraction There was almost no Mg. This confirms that Ca and Mg can be easily removed from soil even at a low concentration of EDTA, and does not significantly contribute to the adsorption of soil DNA.
以上のことから、 EDTAを 300 mMという高濃度での使用することが、 火山灰 土壌からの土壌 DNAの抽出には効果的であり、 土壌の種類によっては 1回目の 抽出に高濃度の EDTAを使用するか否かで、 DNA合計収量が左右されることが 明らかになった。 これは、 いったん吸着した DNAを放出しにくい土壌が存在す ることを意味する。 From the above, using EDTA at a concentration as high as 300 mM is an It was found to be effective in extracting soil DNA from soil, and that depending on the type of soil, the use of high concentrations of EDTA in the first extraction could determine the total DNA yield. This means that there are soils that are difficult to release the DNA once adsorbed.
また、 繰り返し抽出により吸着の原因となる A1 が除去されるにしたがって DNAが徐々に抽出され続ける土壌 （東北大学森林土壌） と、 Αίが除去されても DNA 抽出量が増えない土壌 （栃木農試森林土壌） が存在する。 東北大学森林土 壌は非ァロフェン質黒ポク土で、 非晶質 A1の大部分が腐植 -A1複合体であり、 ァ 口フェン質黒ポク度と比較するとァロフェン含量は少ない。 それゆえ土壌 DNA を吸着する A1の中には、 腐植 -A1複合体の A1の他に、 ァロフェン態の A1が存在 しており、 EDTAが主に DNAの吸着を解消するのは、 主に腐植 -ΑΙ複合体の A1 であることが推測された。  In addition, soil where DNA is gradually extracted as A1 that causes adsorption is removed by repeated extraction (Tohoku University forest soil), and soil where DNA extraction does not increase even if Αί is removed (Tochigi Agricultural Forest soil). The forest soil of Tohoku University is a non-arofenous black pork soil, in which most of the amorphous A1 is a humus-A1 complex, and the content of arophene is low as compared with the degree of apofenic black pork. Therefore, in A1 that adsorbs soil DNA, in addition to A1 in the humus-A1 complex, A1 in the arophen state exists, and EDTA mainly eliminates the adsorption of DNA by humus. -ΑΙ It was presumed to be A1 of the complex.
〔実施例 6〕 高濃度 EDTAでも DNAが抽出されない土壌からの DNA抽出 これまでの結果から、 従来の抽出法で火山灰土壌からの DNA抽出が困難であ つた最大の原因は、 土壌による DNAの吸着であったことが強く示唆された。 そこで、 DNAの吸着を解消する EDTAの最適濃度をさらに検討するため、 DNA の吸着力が強いと考えられる土壌を対象とし、 beads-beating後に高濃度 EDTA を再添加し、 高濃度 EDTAと加熱処理の併用により、 土壌に吸着された DNAを 回収する方法および条件を検討した。 [Example 6] DNA extraction from soil from which DNA is not extracted even with high-concentration EDTA From the results so far, the biggest cause of the difficulty in extracting DNA from volcanic ash soil by the conventional extraction method is the adsorption of DNA by soil. Was strongly suggested. Therefore, in order to further examine the optimal concentration of EDTA that eliminates DNA adsorption, soil that is considered to have strong DNA adsorption is targeted, and after beads-beating, high-concentration EDTA is added again, and heat treatment with high-concentration EDTA is performed. The method and conditions for recovering DNA adsorbed on soil by using a combination of these were investigated.
(1) 材料と方法 (1) Materials and methods
試験には弥生圃場対照区土壌を用いた。 検討した点は次の 3点である。 はじめ の抽出液の EDTA濃度（beads-beating時の抽出液）、 DNA回収液の EDTA濃度 (再添加する EDTA溶液）、 回収液添加後の処理 （攪拌もしくは加熱処理） であ る。 処理内容と抽出液及び回収液との組み合わせを表 7に示す。 表 7 The soil in the control section of the Yayoi field was used for the test. The following three points were considered. The EDTA concentration of the first extract (extract at the time of beads-beating), the EDTA concentration of the DNA recovery solution (the EDTA solution to be re-added), and the treatment after adding the recovery solution (stirring or heating). Table 7 shows the combinations of the treatment contents and the extracts and recovered liquids. Table 7
Beads beatin 回収液の Beads beatin
処理 時の EDTA濃度 EDTA濃度  EDTA concentration during processing EDTA concentration
200 600  200 600
200 800  200 800
200 1000  200 1000
60°C1時間加熱 3444344422233 33ooooooo oooooo ooo o oooooooo o o o o o 60 ° C for 1 hour 3444344422233 33ooooooo oooooo ooo o oooooooo o o o o o
100 mM Tris-HCl 1 1 % SDS (pH 8.3)および異なる濃度の EDTAから成る抽出 液 1.2 mlで beads-beatingを行い、 微生物細胞を破壌し、 その後いつたん遠心し て上清 600 μΐを回収した後に、 さらに濃度の高い EDTAを含む回収液 600 (ΐ \ を添加し攪拌のみを行う、 もしくは攪拌後 60 °Cで 1時間加熱処理を加えた。 そ の後遠心し、 先ほど回収した上清とあわせて除タンパク処理後 DNAを回収、 定 量した。 100 mM Tris-HCl 1 Beads-beating with 1.2 ml of extract containing 1% SDS (pH 8.3) and different concentrations of EDTA to break down microbial cells, and then centrifuging briefly to collect 600 μΐ of supernatant After that, a recovery solution 600 containing more concentrated EDTA was added (ΐ \ was added, and only stirring was performed, or heat treatment was performed at 60 ° C for 1 hour after the stirring. Then, centrifugation was performed, and the supernatant collected earlier was centrifuged. The DNA was recovered and quantified after deproteinization.
(2) 結果と考察  (2) Results and discussion
結果を図 14に示す。回収液添加後の操作によって DNA収量に顕著な差が生じ た。攪拌のみの処理と比較すると、加熱処理をしたものではより多くの土壌 DNA を抽出することが可能となった。 これは EDTAと土壌との反応、 すなわち DNA を吸着している非晶質 A1を EDTAがキレートして奪い取り、 DNAを解離させる 反応が温度により加速されることを示唆している。 また EDTA 濃度も高いほど DNA抽出効果が高いという結果が得られた。 今回の実験では、 使用した EDTA の最高濃度において土壌 DNAが最も多く得られているので、さらに多くの DNA が土壌中に吸着されている可能性もあり、 土壌中の DNAの総量を知ることはで きなかった。 しかし、 EDTAの溶解度は通常の条件では最高 1.5 M程度であるの で、 土壌に添加し、 着された DNAを回収する場合、 スモールスケールの抽出 では最終濃度で 1M以上の濃度で土壌を処理することは極めて難しいと考えられ、 EDTA濃度をあげるよりもむしろ反応時間を長くするほうが土壌 DNAの抽出効 率を上げることにとつては有効であるように思われる。 というのも EDTAは A1 と 1:1錯体を形成する。 1000 mMの EDTA溶液 1 mlは、 100%反応すると仮定 して計算すると、 l mniolすなわち 27 mgの A1をキレ一トすることができる。一 方、極めて非晶質成分の多い火山灰土壌 1 g (乾土）で非晶質の A1はおおよそ 100 mgである。 このことを考慮すると、 0.5 gの土壌 （湿潤土） には最高で 40 mg 程度の非晶質 A1が含まれることになり、 1 M EDTA溶液 1 mlを回収液として使 用すると、 ほとんど全ての非晶質 A1を溶解させられる EDTAを使用しているこ とになるからである。 The results are shown in FIG. The operation after the addition of the recovery solution caused a significant difference in DNA yield. Compared to the agitation only treatment, it was possible to extract more soil DNA with the heat treatment. This suggests that the reaction between EDTA and soil, that is, the reaction that dissociates DNA is accelerated by the chelation of EDTA by chelating and removing DNA-adsorbed amorphous A1. The results also showed that the higher the EDTA concentration, the higher the DNA extraction effect. In this experiment, since the highest amount of soil DNA was obtained at the highest concentration of EDTA used, it is possible that more DNA may be adsorbed in the soil, and it is not possible to know the total amount of DNA in the soil. could not. However, the solubility of EDTA is up to 1.5 M under normal conditions. Therefore, when recovering the DNA attached to the soil and recovering it, it is considered extremely difficult to treat the soil at a final concentration of 1M or more with small-scale extraction. It seems that increasing the length is effective in increasing the extraction efficiency of soil DNA. EDTA forms a 1: 1 complex with A1. Assuming that 1 ml of a 1000 mM EDTA solution reacts 100%, it is possible to clean lmniol, that is, 27 mg of A1. On the other hand, amorphous A1 is approximately 100 mg in 1 g (dry soil) of a volcanic ash soil with much amorphous components. Taking this into account, 0.5 g of soil (moist soil) contains up to about 40 mg of amorphous A1, and if 1 ml of 1 M EDTA solution is used as the recovery solution, almost all This is because EDTA that can dissolve amorphous A1 is used.
先にも述べたように、 300〜400 mMの濃度で EDTAを使用しても、 加熱をし ない状態ではその 15〜20%程度しか土壌中の金属元素と錯体を形成していない。 また抽出回数を増やして土壌の非晶質 A1の除去量を定量した実験により、 EDTA は火山灰土壌のおもに腐植 -A1複合体の A1を除去していることが推測できている。 高濃度の EDTA溶液を添加し加熱処理を加えることにより腐植 -A1複合体の A1 に加えァロフェンを初めとするその他の形態の非晶質 A1 とも錯体形成反応が進 み、 土壌からの A1の除去率が高くなり土壌 DNAが抽出できたと推測された。 しかし、反 Jfe時間を長くすると、短時間で抽出可能な beads-beating処理の利点 を生かすことができない。また、最初の抽出処理で遠心と上清回収を行った後に、 再び回収用緩衝液を加えてィンキュベーシヨンを行う作業は煩雑であること、 さ らに、溶液は SDSを含むためしばしば泡立ち、手作業を繰り返すことによって、 手や実験機具に付着した試料が他の試料に混入する危険性が高まる。 さらに、 高 濃度の EDTA添加および加熱処理は大量の腐植物質の溶出をも引き起こした。 腐植物質は DNAの土壌への吸着と同じ原理により、土壌中で非晶質 A1と錯体 を形成して安定化していると考えられる。 腐植をはじめとする土壌有機物を保持 していた非晶質 A1を EDTAが加熱処理中に徐々に取り除くため、団粒ゃ土壌粒子 内部に保持されていた腐植物質までが次第に抽出液中に溶出され、 DNA試料へ の腐植の混入も多く るという問題が考えられる。 As mentioned above, even when EDTA is used at a concentration of 300 to 400 mM, only about 15 to 20% of the complex forms with metal elements in soil without heating. In addition, experiments in which the number of extractions was increased and the amount of amorphous A1 removed from the soil were quantified, suggesting that EDTA removed A1 of the humus-A1 complex mainly in volcanic ash soil. Addition of high-concentration EDTA solution and heat treatment promotes complex formation with Arofen and other forms of amorphous A1 in addition to A1 in the humus-A1 complex, removing A1 from soil It was presumed that the soil DNA could be extracted at a higher rate. However, if the anti-Jfe time is increased, the advantage of beads-beating treatment, which can be extracted in a short time, cannot be used. In addition, after centrifugation and supernatant recovery in the first extraction process, the work of adding a recovery buffer and performing incubation again is complicated.Moreover, since the solution contains SDS, it is often necessary to perform the incubation. Repeated bubbling and manual work increases the risk of samples adhering to hands and laboratory equipment being mixed into other samples. In addition, the addition of high concentrations of EDTA and heat treatment also caused the elution of large amounts of humic substances. It is considered that humic substances are stabilized by forming a complex with amorphous A1 in soil by the same principle as the adsorption of DNA to soil. Since EDTA gradually removes amorphous A1 that has retained humus and other soil organic matter during the heat treatment, the aggregates and humic substances retained inside the soil particles are gradually eluted into the extract. To DNA samples There is a problem that humus is often mixed.
以上のことより、 腐植が少なくァロフェン態の A1 が多い火山灰土壌について は EDTAの錯形成反応を利用して A1を除去する方法は非効率的であると考えら れ、 ァロフェン態の AIによって吸着された土壌 DNAを抽出するためには、 その 他の方法を検討す'る必要があると考えられた （実施例 7 )。  Based on the above, it is considered that the method of removing A1 using the EDTA complexation reaction is inefficient in volcanic ash soils with little humus and a lot of Arofen-form A1, and it is adsorbed by Arophen-form AI. It was considered necessary to examine other methods in order to extract the soil DNA (Example 7).
〔実施例 7〕 土壌 DNA抽出液へのリン酸緩衝液の使用 [Example 7] Use of phosphate buffer in soil DNA extract
抽出液に様々な濃度でリン酸を加え、 土壌 DNAの抽出量を測定した。  Phosphoric acid was added to the extract at various concentrations, and the amount of soil DNA extracted was measured.
(1) 材料と方法 (1) Materials and methods
供試土壌には火山灰土壌として、 高濃 の EDTAを使用しても DNA抽出が困 難であった弥生圃場対照区土壌、 田無農場未耕地土壌、 栃木農試森林土壌の 3種 類のァロフェン質黒ポク土を、 また非火山灰土壌として、 埼玉農試畑土壌、 草地 試験場永年採草地土壌、 兵庫農試畑土壌の 3種類の沖積土壌を用いた （表 8 )。  Three types of alofenic materials were used as the volcanic ash soil: the Yayoi field control plot soil, the Tanashi farm uncultivated land soil, and the Tochigi agricultural test forest soil, for which DNA extraction was difficult even when highly concentrated EDTA was used as the test soil. Black alluvial soil and three types of alluvial soil were used as non-volcanic ash soil: Saitama Agricultural Experiment Station Soil, Grassland Experimental Station Permanent Grassland Soil, and Hyogo Agricultural Experiment Station Soil (Table 8).
表 8 供試土壌の理化学性  Table 8 Physicochemical properties of test soil
Figure imgf000065_0001
Figure imgf000065_0001
beads-beating の条件は実施例 1と同様に設定した。 抽出液組成は 100 mM Tris-HCl I 1% SDS (pH8.3) を基本として、 これに Κ2ΗΡθ4 (ρΗ 8.3) を 0、 50、 100、 250、 500、 750、 1000、 1250、 1500、 1750、 2000 mMになるよう添カロし た抽出緩衝液を調製し、 土壌 0.5 gに対してそれぞれ 1.2 mlずつ添加し、 土壌 DNAを抽出した。 粗抽出液は除タンパク後、 等量の 12 % PEG/ 1 M NaCl溶液 を添加し、 4 °Cにて 20000 X gで 20分間遠心することにより DNAを回収し、 ァガロース電気泳動により 2000 bp以上の DNAを定量した （これ以降の実験に は DNAの回収法に等量の 12 % PEG/ 1 M NaCl溶液を使用した）。 またリン酸と の比較のため、 EDTAについても 50、 100、 200、 300、 400、 500、 600、 800 mM に設定した抽出液を作成し、 同様に DNAを抽出し定量を行った。 全ての実験は 3連で行った。 . The conditions for beads-beating were set in the same manner as in Example 1. The extract composition 100 mM Tris-HCl I 1% SDS (pH8.3) as the base, to which Κ 2 ΗΡθ4 the (ρΗ 8.3) 0, 50, 100, 250, 500, 750, 1000, 1250, 1500, Extraction buffer solutions were prepared to which the concentration was 1750 and 2000 mM, and 1.2 ml each was added to 0.5 g of soil to extract soil DNA. After removing the protein from the crude extract, add an equal volume of 12% PEG / 1 M NaCl solution, and centrifuge at 20000 xg for 20 minutes at 4 ° C to recover DNA, and agarose gel electrophoresis for more than 2000 bp DNA was quantified (for subsequent experiments, an equal volume of 12% PEG / 1 M NaCl solution was used for DNA recovery). For comparison with phosphoric acid, 50, 100, 200, 300, 400, 500, 600, 800 mM EDTA was also used. The extraction liquid set in the above was prepared, and DNA was similarly extracted and quantified. All experiments were performed in triplicate. .
(2) 結果と考察 (2) Results and discussion
(2-1) 抽出液の EDTA濃度が土壌 DNA抽出量に及ぼす影響  (2-1) Effect of EDTA concentration of extract on soil DNA extraction
火山灰土壌においては、 前節で述べた通り、 十分に高濃度な EDTAを用いなけ れば土壌 DNAが十分に抽出できないことが再び示された （図 15A)。特に栃木農 試森林土壌と田無農場未耕地土壌においては、 EDTA濃度が最大濃度の時に DNA が最大収量となっており、 800 mMという高濃度な EDTA抽出液を用いても土壌 中に未抽出の DNAが吸着されている可能性が考えられた。  As described in the previous section, it was shown again that in the volcanic ash soil, the soil DNA could not be sufficiently extracted without using a sufficiently high concentration of EDTA (Fig. 15A). In particular, in the Tochigi agricultural test forest soil and Tanashi farm uncultivated land soil, the maximum yield of DNA was obtained when the EDTA concentration was at the maximum, and even when an EDTA extract with a high concentration of 800 mM was used, the DNA was not extracted into the soil. The possibility that DNA was adsorbed was considered.
非火山灰土壌においては、埼玉農試畑土壌と兵庫農試畑土壌では EDTA濃度が 高まるにつれて DNA収量の低下が観察された。 EDTA濃度の検討実験 （実施例 4参照） においても同様な現象が見られたが、 前述の通り SDS の析出が原因で あると考えられた。  In non-volcanic ash soils, DNA yield decline was observed in Saitama and Hyogo agricultural test soils as the EDTA concentration increased. Similar phenomena were observed in the examination of EDTA concentration (see Example 4), but as described above, it was considered that SDS was precipitated.
なお、火山灰土壌であっても、弥生圃場対照区土壌からの抽出 DNA量は、 EDTA 濃度が 500 mMの時に最大となり、 それ以上の EDTA濃度になると収量は低下 した。 火山灰土壌でも高濃度 EDTAによる SDSの析出および土壌微生物の溶菌 率の低下は起こっていると考えられるが、 吸着された DNAの回収率の上昇が著 しいため、 溶菌率低下による DNA収量の減少が現れにくくなつているだけであ り、 SDSによる土壌微生物の溶菌率が高まれば、 より多くの土壌 DNAが得られ ると考えられる。  Even in the case of volcanic ash soil, the amount of DNA extracted from the soil in the control plot of the Yayoi field was highest when the EDTA concentration was 500 mM, and the yield decreased when the EDTA concentration was higher. It is thought that the precipitation of SDS by high-concentration EDTA and a decrease in the bacteriolysis rate of soil microorganisms also occur in the volcanic ash soil, but the recovery rate of the adsorbed DNA is remarkably increased. It is only difficult to appear, and if the lysis rate of soil microorganisms by SDS increases, more soil DNA will be obtained.
草地試験場の永年採草地土壌は、 他の非火山灰土壌とは違い、 火山灰土壌型の 抽出傾向を示した （図 15A)。 草地試験場は那須火山帯に位置しており、 元来こ の地域の大部分の土壌はァロフェン質黒ポク土である。 しかし、 採集地である永 年採草地は、 河川の浸食により火山灰層が失われ、 火山灰に覆われていた元の地 層部分が地表に表れ、 現在の表土の母材となっている。 そのため、 火山帯に位置 していながら非火山灰土壌の性状を示している。 ところが、 酸性シユウ酸塩抽出 による A1や Feの量からも推定されるように、 実際には非晶質 A1が土壌中に残 存しており、 その非晶質 A1が土壌 DNAを吸着したと考えられた。 このように、 6 土壌中に存在する非晶質 A1 は、 火山灰土壌の混入というたとえ少量であっても DNAを強く吸着し、 DNA抽出を阻害する可能性が示唆された。 Unlike the other non-volcanic ash soils, the permanent grassland soil at the grassland test site showed a tendency to extract the volcanic ash soil type (Fig. 15A). The grassland test site is located in the Nasu volcanic belt, and most of the soil in this area is originally arofenic black pork soil. However, the perennial grassland, which is the collection site, loses its volcanic ash layer due to river erosion, and the original stratum that was covered by volcanic ash appears on the ground surface and is the base material of the current topsoil. Therefore, it shows the properties of non-volcanic ash soil while located in the volcanic zone. However, as can be inferred from the amounts of A1 and Fe from the acid oxalate extraction, amorphous A1 actually remains in the soil, and it was assumed that the amorphous A1 adsorbed soil DNA. it was thought. in this way, 6 It was suggested that amorphous A1 existing in soil could adsorb DNA strongly and inhibit DNA extraction, even in the case of a small amount of volcanic ash soil contamination.
(2-2) リン酸の濃度が土壌 DNA抽出に及ぼす影響  (2-2) Effect of phosphate concentration on soil DNA extraction
火山灰土壌からの DNA抽出量については、 リン酸緩衝液を用いることにより、 弥生圃場対照区土壌では 1 g土壌から 35 gの DNAを抽出することができ、 EDTA緩衝液を用いても収量の低かった田無農場未耕地では 1 g土壌から 15 g もの DNAを抽出することができた (図 15B)。また土壌 DNAの最大収量が得られ たリン酸濃度は、弥生圃場対照区土壌では 1500 mM、栃木農試森林土壌では 750 mM、 田無農場未耕地土壌では 1500 mMであった。 それ以上の濃度に設定した 場合でも、 抽出量はほぼ同じか多少減少する程度であったことから、 これらの濃 度のリン酸緩衝液で、 土壌に吸着したほとんど全ての土壌 DNAが回収できてい ることが示唆された。 また、 非火山灰土壌からの DNA抽出も良好であり、 火山 灰土壌が一部混入している草地試験場の土壌からも高い収量で土壌 DNAが抽出 された。  Regarding the amount of DNA extracted from volcanic ash soil, by using phosphate buffer, 35 g of DNA could be extracted from 1 g of soil in the Yayoi field control soil, and the yield was low even with EDTA buffer. In uncultivated land at Tanashi Farm, as much as 15 g of DNA could be extracted from 1 g of soil (Fig. 15B). The concentration of phosphoric acid that gave the maximum yield of soil DNA was 1500 mM in the Yayoi field control soil, 750 mM in the Tochigi agricultural test forest soil, and 1500 mM in the Tanashi farm uncultivated land. Even when the concentration was set higher than that, the amount of extraction was almost the same or slightly decreased.Therefore, almost all soil DNA adsorbed on soil could be recovered with phosphate buffer at these concentrations. It was suggested that DNA extraction from non-volcanic ash soil was also good, and soil DNA was extracted with high yield from grassland test site soil partially contaminated with volcanic ash soil.
EDTAとリン酸を同じモル濃度で比較すると、 栃木農試森林土壌ならびに草地 試験場永年草地土壌を除き、 EDTAの方が土壌 DNAの抽出効果は高かったが、 リン酸塩の溶解度は高く水に溶けやすく、 EDTAの溶解度が最大 1.5 M (pH 8.3) であるのに対しリン酸カリゥムの溶解度は 3 M程度と高いため、 EDTAよりもは るかに高い濃度で使用することが可能である。 リン酸イオンを高濃度で使用する と、 弥生圃場対照区土壌や田無農場未耕地土壌からは、 EDTAを用いて得られた DNAの最大収量の 2倍以上の DNAを抽出することができた。  Comparing EDTA and phosphoric acid at the same molar concentration, except for Tochigi Agricultural Forest and Grassland Experimental Grassland Grassland Soil, EDTA was more effective in extracting DNA from the soil, but it had higher phosphate solubility and was more soluble in water. The solubility of EDTA is 1.5 M (pH 8.3) at the maximum, whereas the solubility of potassium phosphate is as high as 3 M. Therefore, it can be used at a much higher concentration than EDTA. When phosphate ions were used at high concentrations, it was possible to extract more than twice the maximum yield of DNA obtained using EDTA from soil in the control plot of Yayoi field and uncultivated soil in Tanashi farm.
以上のように、 リン酸緩衝液は、 DNA収量においては優れていると言える。 但し、 以下の事項を考慮する必要がある。  As described above, it can be said that the phosphate buffer is excellent in DNA yield. However, the following items need to be considered.
すなわち、 火山灰土壌から土壌 DNA を抽出するための緩衝液は、 EDTA、 リ ン酸のいずれを使用しても、 DNAの最大収量が得られる条件 （EDTA、 リン酸、 SDSの濃度など）が土壌によって異なる。これは極めて重要なヒとを示している。 土壌 DNAの抽出の際、 土壌 DNAの回収率を上げようと高濃度 EDTA、 リン酸 緩衝液を使用すれば SDS による細胞破壊効率が下がってしまう、 細胞破壊効率 を上げようと低濃度緩衝液を使用すれば土壌への DNA吸着が多くなつてしまう。 今回の結果は、両者のバランスが土壌によって異なることを示している。従って、 すべての土壌で最大収量の DNA抽出を可能とする普遍的な抽出条件を確立する には、 さらに検討する必要があると思われた。 In other words, regardless of whether EDTA or phosphoric acid is used as the buffer for extracting soil DNA from volcanic ash soil, the conditions (maximum concentration of EDTA, phosphoric acid, SDS, etc.) at which the maximum yield of DNA is obtained are the same. Depends on This represents a very important event. If high concentration EDTA or phosphate buffer is used to increase the recovery rate of soil DNA when extracting soil DNA, the cell destruction efficiency by SDS will decrease. The use of low-concentration buffers will increase DNA adsorption to soil. The results show that the balance differs between soils. Therefore, further studies would be needed to establish universal extraction conditions that would allow for maximum DNA extraction in all soils.
分析対象とするすべての土壌試料から最大収量の土壌 DNAを抽出しようとす れば、 すべての土壌について理化学性を試験する必要が生じ、 その結果に応じて それぞれの土壌に最適な抽出緩衝液を個別に作成する必要があるといえる。  Attempts to extract the highest yields of soil DNA from all soil samples to be analyzed necessitate testing all of the soils for physicochemical properties. It can be said that it needs to be created individually.
〔実施例 8〕 高濃度 EDTA-リン酸混合液による土壌 DNAの抽出 [Example 8] Extraction of soil DNA with high concentration EDTA-phosphate mixture
本実施例では、 土壌 DNA抽出におよぼす EDTAとリン酸カリゥム緩衝液の相 乗効果を期待して、 両者の併用を試みた。  In this example, the combined use of EDTA and potassium phosphate buffer was attempted, expecting a synergistic effect on soil DNA extraction.
(1) 材料と方法 (1) Materials and methods
実験には、 実施例 7に示す 6種類の土壌を用いた。 抽出液は lOO mM Tris-HCl 1 1 % SDSに EDTAとリン酸を、表 9の 1番〜 16番に示す組み合わせの濃度で使 用した。 抽出条件、 DNAの定量条件は実施例 6と同様である。  In the experiment, six types of soils shown in Example 7 were used. For the extract, EDTA and phosphoric acid were used in a combination of lOO mM Tris-HCl 1 1% SDS and the combinations shown in Table 9 Nos. 1 to 16. The extraction conditions and DNA quantification conditions are the same as in Example 6.
表 9 抽出液に含まれる EDTAおよびリン酸の濃度  Table 9 Concentrations of EDTA and phosphoric acid in extract
EDTA (mM) K?HP04 (mM) EDTA (mM) K? HP0 4 (mM)
1 100 250  1 100 250
2 100 500  2 100 500
3 100 750  3 100 750
4 100 1000  4 100 1000
5 100 1250  5 100 1250
6 200 250  6 200 250
7 200 500  7 200 500
8 200 750  8 200 750
9 200 1000  9 200 1000
10 200 1 250  10 200 1 250
1 1 400 250  1 1 400 250
12 400 500  12 400 500
13 400 750  13 400 750
14 400 1000  14 400 1000
15 600 250  15 600 250
16 600 500 (2) 結果と考察 16 600 500 (2) Results and discussion
土壌 DNA抽出量の結果を図 16A〜Bに示す。 EDTAとリン酸をそれぞれ単独 で使用するより、混合液では低い濃度であっても、高い DNA抽出効果が得られ、 土壌 DNA抽出に関して EDTAとリン酸の補完効果が認められた。 また、 非火山 灰土壌ではこれまで、 EDTA濃度の増加に伴って DNA収量の低下が認められて いたが、 EDTAとリン酸緩衝液を組み合わせると、 EDTAを高濃度にしても収量 の低下が比較的低く抑えられた。 ·  Figures 16A-B show the results of soil DNA extraction. Compared to using EDTA and phosphate alone, a high DNA extraction effect was obtained even at a lower concentration in the mixed solution, and a complementary effect of EDTA and phosphate on soil DNA extraction was observed. In addition, in non-volcanic ash soil, a decrease in DNA yield was previously observed with an increase in EDTA concentration.However, when EDTA and phosphate buffer were combined, the decrease in yield was high even at high EDTA concentrations. Was kept very low. ·
これまでの結果から、 高濃度の EDTA でも抽出が難しかったァロフェン態の A1に富む弥生圃場対照区土壌や田無農場未耕地土壌からも、 リン酸緩衝液を用い ることにより高収量で DNAを抽出できることが明らかとなった。 他の 2種類の 火山灰土壌と比較して、ァロフェン態 A1が少ない栃木農試森林土壌に関しては、 EDTAでもリン酸でもほぼ同じ抽出量が得られている。そして EDTA-リン酸緩衝 液の混合液を使用することにより、 EDTA単独およびリン酸単独で得られる土壌 DNAの合計量にほぼ近い量の土壌 DNAが得られた。 これまでの結果も考慮する と、 EDTAは主に腐植 -A1複合体の A1を除去し、 リン酸はァロフェン態 A1のマ スキングを行う効果があるものと考えられる。  From the results so far, high-yield DNA was extracted from phosphate-buffered soil in the Aroen-rich A1-rich Yayoi field control plot soil and the uncultivated field in Tanashi farm, which were difficult to extract even with high concentrations of EDTA. It became clear what we could do. Compared to the other two types of volcanic ash soils, almost the same amount of extraction was obtained with EDTA and phosphoric acid in the Tochigi Agricultural Forest Soil, which has less arophenic A1. By using a mixed solution of EDTA-phosphate buffer, an amount of soil DNA almost close to the total amount of soil DNA obtained with EDTA alone and phosphate alone was obtained. Considering the results to date, EDTA mainly removes A1 in the humus-A1 complex, and phosphoric acid is thought to have the effect of masking the arofen-form A1.
EDTA-リン酸緩衝液併用の利点は、 1つには同じ DNA収量を得たい場合それ ぞれ単独で抽出する際の濃度よりも、 いずれもの濃度を低く抑えられることであ る。 2つめは DNAの最大収量が得られる濃度は、 やはり土壌によって若干異な るが、 400 mMの EDTAに 750 mMのリン酸カリウムを組み合わせると、 ほと んどの土壌から DNAの最大収量を得られることが明らかとなった（図 16A〜B)。 これは、 土壌の種類を問わない、 ユニバーサルな DNA抽出液の組成を突き止め たことを意味する。 またこの濃度では、 その後の DNAの精製操作および沈殿操 作時に塩濃度をさげるための希釈操作が不要であったことからも、 今後は上記の 組成を土壌 DNA抽出用緩衝液に適用することとした。  The advantage of the combined use of EDTA-phosphate buffer is that, in part, if one wants to obtain the same DNA yield, the concentration of each can be kept lower than the concentration when extracting alone. Second, the concentration at which the maximum yield of DNA is obtained also varies slightly depending on the soil, but combining 400 mM EDTA with 750 mM potassium phosphate will yield the maximum yield of DNA from most soils. (Figs. 16A-B). This means that the composition of the universal DNA extract was determined regardless of the type of soil. At this concentration, it was not necessary to perform a dilution operation to reduce the salt concentration during subsequent DNA purification and precipitation operations.Therefore, the above composition should be applied to the soil DNA extraction buffer in the future. did.
このユニバーサルな緩衝液組成は、'供試した土壌のほぼすベてから最大収量の This universal buffer composition provides the maximum yield from almost all of the soil tested.
DNAを得ることができる。 しかしながら、 土壌によっては土壌 DNAの低分子化 (20〜7kbp)が起きることが判明した。多数の試料を対象にすることが多い土壌 微生物群集構造解析 は実験操作の簡便さが要求され、 また一般に比較的短い DNA領域 （200〜： 1500 bp) しか解析対象としないことなどから、 多少 DNAが 低分子化されても十分に目的は達成できると考えられた。 よって、 微生物群集構 造解析をする上では、 本実施例の条件により土壌 DNAを抽出して問題はないと 5 考えられた。 但し、' クロ一ニングなど断片化した DNAでは不都合な操作や、 群 集構造解析であってもより正確に解析したい場合には（高度に低分子化した DNA を鐃型として PCR増幅を行うとキメラ配列が発生しやすくなる）、 より高分子の DNAが得られる抽出手法が必要であると考えられた。 DNA can be obtained. However, it was found that depending on the soil, the molecular weight of the soil DNA was reduced (20-7 kbp). Soil often used for many samples Microbial community structure analysis requires simple experimental procedures, and generally involves analysis of only relatively short DNA regions (200 to 1500 bp). It was thought that it could be achieved. Therefore, in analyzing the microbial community structure, it was considered that there was no problem in extracting soil DNA under the conditions of this example. However, if fragmented DNA such as cloning is used for inconvenient operations, or when it is desired to perform more accurate analysis even in the case of community structure analysis, it is necessary to perform PCR amplification using highly degraded DNA as a cycling type. Chimera sequences are more likely to be generated), and it was considered necessary to use an extraction technique to obtain higher molecular weight DNA.
10 〔実施例 9〕 「2ステップ法」 の検討 10 [Example 9] Examination of "two-step method"
実施例 8で示したように、 高濃度の EDTA-リン酸による抽出は高収量で DNA を抽出できるが、 DNA の低分子化の可能性がある。 そこで、 最も強く物理的衝 撃が DNAに加わる beads-beating時に低濃度の EDTA-リン酸溶液を用い、 次に 土壌に吸着された DNAを高濃度 EDTA-リン酸溶液で抽出することにより、 DNA 15 を効率良く抽出しつつ低分子化を回避できるかどうか検討した。  As shown in Example 8, extraction with high concentration of EDTA-phosphate can extract DNA in high yield, but may reduce the molecular weight of DNA. Therefore, by using a low-concentration EDTA-phosphate solution during beads-beating, when the strongest physical impact is applied to the DNA, and then extracting the DNA adsorbed on the soil with a high-concentration EDTA-phosphate solution, We examined whether it is possible to avoid low molecular weight while extracting 15 efficiently.
(1) 材料と方法  (1) Materials and methods
供試土壌として表 10に示すものを用いた。  The soil shown in Table 10 was used as the test soil.
表 1 0 供試土壌の理化学性 酸性シユウ酸塩抽出 ピロリン酸抽出 ァロフェン Table 10 0 Physicochemical properties of test soil Acid oxalate extraction Pyrophosphate extraction
Ο Γ. pH pH(KCI)全窒素 全炭素 （mg/g soil) (mg/g soil) (mg/g soil)PH Γ . PH pH (KCI) Total nitrogen Total carbon (mg / g soil) (mg / g soil) (mg / g soil)
2 (J (%) (%) Si Fe Al Fe Al Al 弥生圃場対照区土壌 ァロフェン質黒ポク土 6.98 2.38 45.14 田無農場未耕地 ァロフェン質黒ポク土 5.18 6.55 50.52 栃木農試森林土壌 ァロフェン質黒ポク土 5.28
Figure imgf000070_0001
14.72 23.63 2 (J (%) (%) Si Fe Al Fe Al Al Yayoi field control plot soil Arofen black pork soil 6.98 2.38 45.14 Tanashi farm uncultivated land Arofen black black soil 5.18 6.55 50.52 Tochigi agricultural test forest soil Alofen black Soil 5.28
Figure imgf000070_0001
14.72 23.63
100 mM Tris-HCl I l % SDS (pH8.3) 400 lを土壌 0.5 gの入ったスクリュー 25 キャップチューブに加え、 beads-beating後、 数秒の高速遠心で土壌溶液をチュ ーブ下部に集め、 キャップをあけて高濃度 EDTA 溶液、 リン酸溶液もしくは EDTA-リン酸混合緩衝液を 800 \添加した。 その後 vortexにより土壌と溶液 をよく攪拌して直ちに遠心、 上清を回収して除タンパクし、 DNA を沈殿回収し た。 beads-beating後にチューブに添加した溶液の組成、 および添加後の抽出液 の終濃度を表 11に示す。 Add 400 l of 100 mM Tris-HCl I l% SDS (pH 8.3) to a screw 25 cap tube containing 0.5 g of soil, and after beads-beating, collect the soil solution at the bottom of the tube by high-speed centrifugation for several seconds. The cap was opened, and a high-concentration EDTA solution, a phosphate solution or an EDTA-phosphate mixed buffer solution was added for 800 yen. Then, mix the soil and solution with vortex well, centrifuge immediately, collect the supernatant, remove protein, precipitate and recover DNA. It was. Table 11 shows the composition of the solution added to the tube after beads-beating and the final concentration of the extract after addition.
表 11  Table 11
EDTA EDTA-リン酸  EDTA EDTA-phosphate
回収液濃度 (mM) 終濃度 (mM) 回収液濃度 (mM) 終濃度 (mM)  Recovered liquid concentration (mM) Final concentration (mM) Recovered liquid concentration (mM) Final concentration (mM)
EDTA K2HP04 EDTA K。HP04 EDTA K 2 HP0 4 EDTA K. HP0 4
1 75 50 1 75 50
2 150 100 375 250 2 150 100 375 250
3 300 200 巧 0 、5003 300 200 Skill 0, 500
4 450 300 125 7504 450 300 125 750
5 600 400 500 10005 600 400 500 1000
6 750 500 875 12506 750 500 875 1250
7 900 600 575 2507 900 600 575 250
8 1200 800 750 500 8 1200 800 750 500
125 750 125 750
500 1000 K2HP04_ 875 1250 回収液濃度 (mM) 終濃度 (mM) 575 250500 1000 K 2 HP0 4 _ 875 1250 Concentration of recovered solution (mM) Final concentration (mM) 575 250
00 oooooo55500 o 55oo oooooo ooooooo o !50 500 00 oooooo55500 o 55oo oooooo ooooooo o! 50 500
1 75 50 125 7501 75 50 125 750
2 150 100 500 10002 150 100 500 1000
3 375 250 $75 2503 375 250 $ 75 250
4 750 500 0 5004 750 500 0 500
5 1125 750 5 1125 750
6 1500 1000  6 1500 1000
7 1875 1250  7 1875 1250
8 2250 1500  8 2250 1500
9 2625 1750 ooooooo o ooooooo o  9 2625 1750 ooooooo o ooooooo o
10 3000 2000 ooooooo o ooooooo o  10 3000 2000 ooooooo o ooooooo o
(2) 結果と考察 (2) Results and discussion
結果を図 17に示す。 beads-beating時に EDTAやリン酸を含まない Tris'HCl 緩衝液を用い、その後に高濃度の EDTAおよびリン酸、 EDTA-リン酸緩衝液を用 いても、 弥生圃場対照区土壌および田無未耕地土壌からは、 beads-beating 時に 高濃度緩衝液を用いて抽出した時 （実施例 6参照） とほぼ同程度の量の DNAを 得ることができた。 しかし、 栃木農試森林土壌からは、 最も高い収量が得られた 濃度で比較すると約半分の DNA抽出量しか得られなかった。 初めから高濃度の EDTA, リン酸緩衝液を含む抽出液で抽出した場合と同じく、 3 種類全ての土壌 において EDTA 400mM、 リン酸 750 mMの濃度の抽出液がもっとも DNAの回 収量が多かった。 これにより EDTA 400 mM、 リン酸 750 mMの混合溶液は、 溶 菌した微生物から放出された DNAが土壌に吸着するのを防ぐとともに、 土壌に 吸着した DNAを回収'する効果も、 検討した組み合わせの範囲においては最も高 いと考えられた。 The results are shown in FIG. Even if a Tris'HCl buffer solution that does not contain EDTA or phosphate is used during beads-beating, and then high concentrations of EDTA, phosphate, and EDTA-phosphate buffer are used, the soil in the Yayoi field control plot and the land without cultivation From, DNA was obtained in approximately the same amount as when extraction was performed using a high-concentration buffer during beads-beating (see Example 6). However, from Tochigi Agricultural Forest soil, only about half the amount of DNA extracted was obtained when compared with the concentration that yielded the highest yield. In all three types of soil, the extract with a concentration of 400 mM EDTA and 750 mM phosphate gave the highest DNA recovery, as was the case with the extract containing high concentrations of EDTA and phosphate buffer from the beginning. As a result, a mixed solution of 400 mM EDTA and 750 mM phosphoric acid prevents DNA released from the lysed microorganisms from adsorbing on the soil, The effect of recovering the adsorbed DNA was also considered to be the highest in the range of the combinations examined.
DNAの低分子化は多少減少したものの、一部の試料では DNAの低分子化が依 然として認められた。 これは、 遠心操作によりペレット化した土壌を再懸濁する ため高濃度緩衝液を添加後に激しく vortex撹拌する必要があったこと、 および、 はじめに 400 i lという少量の溶液中で beads-beatingしたため、より大きな物理 的破壊力が加わったことが原因と考えられる。 少ない液量で beads-beatingを行 うと B rgmann et al. (2001) の報告にあるように DNAは低分子にせん断され やすくなる。 また土壌の種類によっては、 DNA が不可逆的に土壌に吸着し、 そ の剥離が困難な土壌も存在すると考えられた。  Degradation of DNA was somewhat reduced, but in some samples DNA degradation was still observed. This is due to the need to vigorously vortex after adding a high concentration buffer to resuspend the soil pelletized by centrifugation, and beads-beating in a small solution of 400 il first. This is probably due to the addition of a large physical destructive force. If beads-beating is performed with a small volume, as described in Burgmann et al. (2001), DNA is likely to be sheared into small molecules. In addition, depending on the type of soil, it was considered that DNA was irreversibly adsorbed on the soil, and there were some soils that were difficult to exfoliate.
そこで追加検討条件として、 beads-beating時の抽出液に DNAの低分子化を引 き起こさない上限の濃度の緩衝液の使用について検討した。 すなわち、 DNA抽 出液の終濃度は 400 mM EDTA-750 mMリン酸で固定し、 beads-beatingを表 12 に示す濃度の 800 1の抽出液により、 フラッシュ遠心後にキヤップをあけ表 12 に示す濃度の 400 1の回収液を追加し、 攪拌して DNAを土壌より剥離させ、 回 収する改良法を検討した。  Therefore, as an additional study condition, the use of a buffer solution with an upper limit concentration that does not cause the lowering of DNA in the extract during beads-beating was examined. That is, the final concentration of the DNA extract was fixed with 400 mM EDTA-750 mM phosphoric acid, beads-beating was removed by flash centrifugation with 8001 extract at the concentration shown in Table 12, and the concentration shown in Table 12 was removed. 4001 of the recovered solution was added and stirred to separate the DNA from the soil, and an improved method for recovery was studied.
表 12  Table 12
800 μ \ 400 μ \ 800 μ \ 400 μ \
beads - beating時の抽出液 DNA回収液  beads-extracted liquid during beating DNA recovery liquid
EDTA K2HPO EDTA K2HPO, EDTA K 2 HPO EDTA K 2 HPO,
1 400 750 400 750  1 400 750 400 750
2 100 100 1000 2050  2 100 100 1000 2050
3 100 250 1000 1750  3 100 250 1000 1750
4 100 500 1000 1250  4 100 500 1000 1250
5 100 750 1000 750  5 100 750 1000 750
6 200 100 800 2050  6 200 100 800 2050
7 200 250 800 1750  7 200 250 800 1750
8 200 500 800 1250  8 200 500 800 1250
9 200 750 800 750  9 200 750 800 750
10 300 100 600 2050  10 300 100 600 2050
1 1 300 250 600 1750  1 1 300 250 600 1750
12 300 500 600 1250  12 300 500 600 1250
13 300 750 600 750  13 300 750 600 750
14 400 100 400 2050  14 400 100 400 2050
15 400 250 400 1750  15 400 250 400 1750
16 400 500 400 1250 改良法による結果を図 18Aおよび B (DNA抽出量） およぴ図 19 (ァガロース 電気泳動写真） に示す。 この 3種類の土壌に関しては beads-beating時に DNA の低分子化を引き起こさない限界の濃度は EDTAが 400 mM、 リン酸が 250 mM 程度であると考えられた。 両者の混合系では 400 mM EDTA、 100 ιηΜリン酸の 混合液が低分子化を引き起こさない限界濃度であると考えられた。 16 400 500 400 1250 The results of the improved method are shown in Figs. 18A and 18B (DNA extraction amount) and Fig. 19 (agarose electrophoresis photograph). For these three types of soil, it was thought that the limit concentrations that did not cause DNA depolymerization during beads-beating were about 400 mM for EDTA and about 250 mM for phosphoric acid. It was considered that the mixed solution of 400 mM EDTA and 100 ιηΜ phosphoric acid had the limit concentration that did not cause lower molecular weight in the mixed system of both.
ここまでの結果から、 2ステップ法として、 DNAの低分子化を抑制しつつ収量 が最も高い抽出液、 すなわち 300 mM EDTAと 100 mMリン酸を含む緩衝液を beads-beating 処理時の抽出液として用い （第 1 ステップ）、 その後高濃度の EDTA-リン酸緩衝液を添加して攪拌し、 土壌に吸着した DNAを回収する （第 2 ステツプ） 方法を効率的な土壌 DNA抽出法として提案する。  From the results so far, as a two-step method, the extract with the highest yield while suppressing DNA depolymerization, that is, a buffer containing 300 mM EDTA and 100 mM phosphoric acid, was used as the extract during beads-beating treatment. Use (1st step), then add a high concentration of EDTA-phosphate buffer, stir and recover the DNA adsorbed on the soil (2nd step). We propose a method for efficient soil DNA extraction.
〔実施例 10〕 土壌 DNAの沈殿方法の選択 [Example 10] Selection of precipitation method for soil DNA
本実施例では、 2-プロパノール、 エタノール、 PEGの 3種類を用いた DNAの 沈殿方法について、 DNAの回収率および DNAとともに沈殿する腐植物質の量を 測定した。  In this example, the recovery rate of DNA and the amount of humic substances precipitated together with DNA were measured for the method of DNA precipitation using three types of 2-propanol, ethanol and PEG.
(1) 材料と方法 (1) Materials and methods
栃木農試森林土壌、 東北大学森林土壌、 埼玉農試畑土壌を供試した。 それらの 理化学性を表 13に示す。  The Tochigi agricultural test forest soil, Tohoku University forest soil, and the Saitama agricultural test field soil were tested. Table 13 shows their physicochemical properties.
表 1 3 供試土壌の理化学性  Table 13 Physical and chemical properties of test soil
酸性シユウ酸塩抽出 ピロリン酸抽出 ァロフェン pH pH(KCI)全窒素 全炭素 （mg/g soil) (mg/g soil) (mg/g soil)  Acid oxalate extraction Pyrophosphate extraction Alofen pH pH (KCI) Total nitrogen Total carbon (mg / g soil) (mg / g soil) (mg / g soil)
(%) (%) Si Fe Al Fe Al Al 東北大学森林土壌 非ァロフェン質黒ポク土 20.82 栃木農試森林土壌 ァロフェン質黒ポク土 23.63 埼玉農試畑土壌 灰色低地土
Figure imgf000073_0001
1.22 栃木農試森林土壌および東北大学森林土壌は、 全炭素量が 9%におよび、 腐植 物質の集積量が多い土壌である。 30種類の土壌より土壌 DNAを抽出した際、 他 の黒ポク土の抽出液が茶色に着色していた程度であつたのに対し、 これらの土壌 の抽出液は黒色に着色していたことからも、 腐植物質の混入量の多さが推測され た。 (%) (%) Si Fe Al Fe Al Al Tohoku University forest soil Non-arofen black pork soil 20.82 Tochigi agricultural test forest soil Alofen black pork soil 23.63 Saitama agricultural test field soil Gray lowland soil
Figure imgf000073_0001
1.22 Tochigi Agricultural Forest Soil and Tohoku University Forest Soil have a total carbon content of 9% and a large accumulation of humic substances. When soil DNA was extracted from 30 types of soil, the extracts of these black soils were colored brown, whereas the extracts of these black soils were colored brown. Suggests that the amount of humic substances mixed is high. It was.
土壌 DNAの抽出には 100 mM Tris-HCl I 300 mM EDTA I 1 % SDSの抽出液 を用い、 土壌 0.5 gに抽出液 1 mlを添加後、 bead-beateing処理を行い、 遠心後 に等量のクロ口ホルムによる除タンパク処理を行った。 この抽出液 500 l に 1/10量の 5 M 酢酸ナトリウム（pH 5.2)を添加し、 2·プロパノールは 7/10等量、 エタノ一ルは 2等量、 20%PEGは 6/10等量を加え、 4 °Cにて 20000 X gで 20 分間遠心し、 沈殿を得た。 沈殿は TE 緩衝液 に溶解させ、 土壌 DNAの 回収量を定量した。 また腐植物質の一つの特徴として、 400 nm以上の可視光吸 収を有することがあげられる。 腐植の分析法 （山本 1997) では波長 400 nmお よび 600 nmにおける吸光度を土壌の腐植量として定義していることから、 土壌 DNA 溶液への腐植物質の混入量を推定するため、 これら 2波長における吸光度 を測定した。測定は UV/VIS Spectrophotometer V-550 (日本分光）を使用した。 (2) 結果と考察  For extraction of soil DNA, use 100 mM Tris-HCl I 300 mM EDTA I 1% SDS extract, add 1 ml of extract to 0.5 g of soil, perform bead-beateing treatment, and centrifuge. A protein removal treatment was carried out using a black mouth form. To 500 l of this extract, add 1/10 volume of 5 M sodium acetate (pH 5.2), 7/10 volume for 2-propanol, 2 volume for ethanol, 6/10 volume for 20% PEG Was added and centrifuged at 20000 X g for 20 minutes at 4 ° C to obtain a precipitate. The precipitate was dissolved in TE buffer and the amount of soil DNA recovered was determined. One of the characteristics of humic substances is that they have a visible light absorption of 400 nm or more. The humus analysis method (Yamamoto 1997) defines the absorbance at wavelengths of 400 nm and 600 nm as the amount of humus in the soil. Therefore, in order to estimate the amount of humic substances mixed into the soil DNA solution, these two wavelengths were used. The absorbance was measured. UV / VIS Spectrophotometer V-550 (JASCO) was used for the measurement. (2) Results and discussion
土壌 DNAの回収量を図 20Aに、 土壌 DNA溶液の 400 nmおよび 600 nmに おける吸光度を図 20Bに示した。  The recovered amount of soil DNA is shown in FIG. 20A, and the absorbance of the soil DNA solution at 400 nm and 600 nm is shown in FIG. 20B.
2-プロパノールおよび PEGによる土壌 DNAの回収率が高かった。エタノール では土壌 DNAの回収量が少なく、 DNAの大部分が沈殿していないと考えられた。 また PEGによる沈殿法では、 土壌 DNAに混入している大部分の RNAを除去で きていた。 土壌 DNA溶液の 400 nmおよび 600 nmにおける吸光度から、 PEG による沈殿操作により得られた DNAは、 最も腐植物質の混入が少ないことが示 された。 PEG を用いた沈殿操作は 2-プロパノールを用いた場合と比較して腐植 物質の混入が半分以下であり、 回収した DNAの純度が高いことがわかった。 ま たァガロース電気泳動の結果から 2-Propanolでは回収されていた rRNAが PEG による沈殿操作では除去されていたことがわかった。このように、 PEGには DNA の回収率が高い、腐植物質の混入が少ない、 RNAを除去できるといった優れた点 があるので、この方法を土壌 DNAの沈殿法として採用することにした。しかし、 この方法で得られた土壌 DNAを錡型として 16S I'RNA遺伝子のほぼ全長を対象 に PCR反応を行ったところ、 全く増幅がなく、 混入している腐植物質の更なる 除去が必要であると えられた The recovery of soil DNA by 2-propanol and PEG was high. It was considered that the recovery of soil DNA was low with ethanol, and most of the DNA did not precipitate. In addition, the PEG precipitation method was able to remove most of the RNA contaminating the soil DNA. The absorbance at 400 nm and 600 nm of the soil DNA solution indicated that the DNA obtained by the PEG precipitation procedure had the least contamination with humic substances. Precipitation using PEG contained less than half the amount of humic substances compared to using 2-propanol, indicating that the purity of the recovered DNA was high. The results of agarose electrophoresis also showed that rRNA that had been recovered with 2-Propanol was removed by precipitation with PEG. As described above, PEG has the advantages of high DNA recovery rate, low humic substance contamination, and the ability to remove RNA. Therefore, this method was adopted as a method for precipitating soil DNA. However, when the PCR reaction was performed on the 16S I 'RNA gene using the soil DNA obtained by this method as type I, almost no amplification was observed, and further contamination of the humic substances was observed. Needed to be removed
〔実施例 11〕 PEGによる沈殿操作の条件検討 [Example 11] Examination of conditions for precipitation operation using PEG
本実施例では、 PEGの濃度による DNAの回収量を検討した。  In this example, the amount of DNA recovered according to the PEG concentration was examined.
(1) 材料と方法 ' (1) Materials and methods ''
弥生圃場対照区土壌および栃木農試森林土壌より 400 mM EDTA / 750 mMリ ン酸 / 1 % SDSを用いて、 beads-beating処理により土壌 DNAを抽出した。 除 タンパク処理後、 等量の 10、 11、 12、 13、 14、 および 15%PEG溶液を用いて土 壌 DNAの沈殿回収を行い、 回収量と PEG濃度の関係を検討した。 DNA沈殿時 の溶液中の PEG濃度はそれぞれ 5、 5.5、 6、 6.5、 7、 7.5%である。  Soil DNA was extracted from the Yayoi field control plot soil and the Tochigi agricultural test forest soil by beads-beating treatment using 400 mM EDTA / 750 mM phosphoric acid / 1% SDS. After protein removal, soil DNA was precipitated and recovered using equal amounts of 10, 11, 12, 13, 14, and 15% PEG solutions, and the relationship between the recovered amount and the PEG concentration was examined. The PEG concentrations in the solution during DNA precipitation were 5, 5.5, 6, 6.5, 7, and 7.5%, respectively.
(2) 結果と考察 (2) Results and discussion
結果を図 21に示す。 検討濃度範囲では加える PEG濃度が 12%の場合に DNA 回収量が最も多かった。 腐植物質の混入量には大差がなかったため、 DNA の回 収量のみを考慮して、 今後の DNA沈殿回収方法としては、 抽出液に対し等量の 12% PEG溶液を添加して行うこととした。  The results are shown in FIG. Within the range of concentrations studied, the highest amount of DNA was recovered when the added PEG concentration was 12%. Since there was no significant difference in the amount of humic substances mixed, the future DNA precipitation recovery method was to add an equal volume of 12% PEG solution to the extract, taking into account only the DNA recovery. .
〔実施例 12〕 土壌 DNAの精製方法についての検討 [Example 12] Study on purification method of soil DNA
前述の通り（実施例 1参照）、 DNA抽出に最適な界面活性剤の検討を行った際、 CTABでは土壌 DNAを抽出することができなかったが、 CTABによる抽出後の 溶液は非常に透明で腐植物質の除去に極めて大きな効果があることが示された。 また Zhou et al. (1996) 、 Porteous et al. (1997)、 Wilstrom et al. (1996) の報告 では、 腐植物質の除去には CTAB が効果的であるとされている。 そこで CTAB を利用した土壌 DNAの簡易精製と、前項の PEGによる沈殿を組み合わせること により、 PCR可能な土壌 DNAを調製することを試みた。  As described above (see Example 1), when examining the best surfactant for DNA extraction, CTAB was unable to extract soil DNA, but the solution after extraction with CTAB was very transparent. It was shown that the removal of humic substances was extremely effective. Zhou et al. (1996), Porteous et al. (1997), and Wilstrom et al. (1996) report that CTAB is effective in removing humic substances. Therefore, we attempted to prepare PCR DNA soil DNA by combining the simple purification of soil DNA with CTAB and the precipitation with PEG as described in the previous section.
(1) 材料と方法 (1) Materials and methods
供試土壌としては実施例 10 と同じく栃木農試森林土壌、 東北大学森林土壌、 埼玉農試畑土壌の 3種類の土壌を用いた。土壌 DNAの抽出には 100 mM Tris-HCl I 300mM EDTA I 1 % SDSの抽出液を用い、 土壌 0.5 gに抽出液 1 mlを添加後、 bead-beateing処理を'行い、 遠心して粗抽出液を得た。 この粗抽出液 300 1に対 し、 10% CTAB溶液および 5 M NaClを用いて、 終濃度で CTABの濃度が 0%、 1%、 2%、 3%の 4段階と、 NaClの濃度が 0.7 M、 1.4 M、 2.1 Mの 3段階のいず れかの組み合わせとなるように添加した。 溶液をよく混合し 60°Cで 10分間加熱 処理を行った後、 抽出液をよく混合し、 等量のクロ口ホルムを添加して除タンパ ク操作を行い、水層を回収し 20%の PEGを 6/10等量加え、土壌 DNAを回収し、 TE 緩衝液に溶解させた。 As the test soil, three types of soil were used as in Example 10, namely, Tochigi Agricultural Forest Soil, Tohoku University Forest Soil, and Saitama Agricultural Test Field Soil. For extraction of soil DNA, 100 mM Tris-HCl I 300 mM EDTA I 1% SDS extract was used.After adding 1 ml of extract to 0.5 g of soil, A bead-beateing treatment was performed and centrifuged to obtain a crude extract. Using the 10% CTAB solution and 5 M NaCl, the crude extract 3001 was used in four stages of final CTAB concentrations of 0%, 1%, 2%, and 3%, and a NaCl concentration of 0.7%. M, 1.4 M, and 2.1 M were added in one of three combinations. After the solution was mixed well and heat-treated at 60 ° C for 10 minutes, the extract was mixed well, an equal amount of black-mouthed form was added, and the protein was removed.The aqueous layer was recovered and the 20% 6/10 equivalents of PEG were added, and soil DNA was recovered and dissolved in TE buffer.
この場合 CTABと結合した腐植物質はクロ口ホルムによる除タンパク時に変性 層に集まり、 除去される。  In this case, the humic substances combined with CTAB are collected and removed in the denatured layer during protein removal by black-mouthed form.
土壌 DNAの回収量を定量し、また腐植物質の混入について 2-4-2と同様に 400 nmにおける吸光度を測定した。  The amount of soil DNA recovered was quantified, and the absorbance at 400 nm was measured for humic substances in the same manner as in 2-4-2.
(2) 結果と考察 (2) Results and discussion
土壌 DNAの回収量を図 22Aに示す。 また、 精製操作後の土壌 DNAの 400mn における吸光度を図 22B〜Cに示す。  Figure 22A shows the amount of soil DNA recovered. Figures 22B to 22C show the absorbance at 400 mn of the soil DNA after the purification operation.
CTABによる精製は、 土壌 DNAの損失がほとんどないことが示された。 なか でも 1.4 M以上の NaCl存在下で処理を行ったものが最も土壌 DNAの回収量が 多かった。 また CTABの使用により、 どの土壌試料についても腐植物質が除去さ れ、 特に CTAB を 2%以上使用したときに腐植物質の大部分が除去されていた。 通常精製に使用される CTAB濃度は 1 %であるが、 1 % では腐植物質の除去率が 悪く、 2 % 以上の使用が効果的であった。 CTABは陽イオン界面活性剤であり、 陰イオン界面活性剤である SDS とは親水基同士で速やかに結合し、 疎水基が外 側に向いたミセルもしくは塩を形成し沈殿する。 よって CTABを 1%で使用する と、 その大部分が溶液中に残存している SDS との反応で消費されてしまい腐植 物質の除去に働ける CTABが減少し、その結果として吸光度が高くなつていると 考えられた。 2%以上の CTABの使用は残存する SDSがすべて CTABにより除去 された後、 余剰の CTABが十分量存在し、 腐植物質の除去を行えるために腐植物 質が除去され、 吸光度が低くなつていると考えられた。 Zhou et al. (1996)ほ、既に抽出液に CTABを添加することを考案しているが、 CTAB 1 %に対し 2%の SDSを使用している。これでは SDSにより CTABが機能 を失ってしまっており、 十分な腐植除去ができていないと推測される。 Purification by CTAB showed little loss of soil DNA. Among them, those treated in the presence of 1.4 M or more NaCl showed the largest recovery of soil DNA. The use of CTAB also removed humic substances from all soil samples, especially when more than 2% of CTAB was used. The concentration of CTAB usually used for purification is 1%, but at 1%, the humic substances removal rate was poor, and the use of 2% or more was effective. CTAB is a cationic surfactant. It binds quickly with hydrophilic groups to SDS, which is an anionic surfactant, and forms micelles or salts with hydrophobic groups facing outward and precipitates. Therefore, when CTAB is used at 1%, most of it is consumed in the reaction with SDS remaining in the solution, and the amount of CTAB used to remove humic substances is reduced, resulting in higher absorbance. It was considered. Use of more than 2% of CTAB means that after the remaining SDS has been removed by CTAB, there is a sufficient amount of surplus CTAB, and humic substances are removed to remove humic substances, resulting in low absorbance. It was considered. Zhou et al. (1996) have already considered adding CTAB to the extract, but use 2% SDS for 1% CTAB. It is presumed that CTDS has lost its function due to SDS, and that humus has not been sufficiently removed.
以上のことより、 1% SDSを含む抽出液で土壌 DNAを抽出する場合は、 2%以 上の CTAB、 1.4 'Μ 以上の NaClを添加し、 60°Cで 10分間のインキュべ一ショ ンを行い、 クロ口ホルムによる除タンパク後、 PEGにより土壌 DNAを沈殿回収 することにより、 ほとんどの腐植物質が除去された土壌 DNAを得ることができ ることが明らかとなった。  From the above, when extracting soil DNA with an extract containing 1% SDS, add 2% or more of CTAB and 1.4'Μ of NaCl and incubate at 60 ° C for 10 minutes. It was clarified that the soil DNA from which most humic substances were removed could be obtained by precipitating and recovering the soil DNA using PEG after protein removal using black-mouthed form.
土壌 DNAの沈殿法について求められるのは、  What is required for the soil DNA precipitation method is
(i) DNAの回収率が高いこと、  (i) high DNA recovery;
(ii) DNAのみを選択的に沈殿させ、 腐植物質を初めとする夾雑物を沈殿させな いこと  (ii) Selective precipitation of DNA only and no precipitation of humic substances or other contaminants
である。  It is.
また、 DNAの精製法に求められるのは、  In addition, what is required for DNA purification methods is
(i) 夾雑物を取り除き、 DNAの純度を上げること、  (i) removing contaminants and increasing DNA purity;
(ii) 精製時に DNAが失われないこと、  (ii) no loss of DNA during purification;
(iii) 簡便でかつコストがかからないこと、  (iii) simple and inexpensive;
(iv) 特殊な技術、 装置を必要としないこと  (iv) Does not require special technology or equipment
である。 It is.
CTABによる簡易精製および PEGによる DNAの回収の組み合わせは、上記の 条件を全て満たしていると考えられ、 この精製、 沈殿法と抽出法を組み合わせる ことにより、 本発明におけるオリジナルの抽出法として使用することができる。  The combination of simple purification by CTAB and recovery of DNA by PEG is considered to satisfy all of the above conditions.By combining this purification, precipitation and extraction, it can be used as the original extraction method in the present invention. Can be.
〔実施例 13〕 本発明の方法と既往の手法による土壌 DNA収量の比較 本発明の方法、 ならびに既往の方法を用いて様々な土壌から DNAを抽出し、 土壌 DNAの収量の比較を行つた。 [Example 13] Comparison of soil DNA yield between the method of the present invention and the existing method DNA was extracted from various soils using the method of the present invention and the existing method, and the yield of soil DNA was compared.
(1) 材料と方法 (1) Materials and methods
現在までの実験において様々な理化学性を示す 12種類の土壌を供試土壌とし た。 その理化学性を表 14に示す。 Twelve soils exhibiting various physicochemical properties in the experiments up to now It was. Table 14 shows their physicochemical properties.
表 14 供試土壌の理化学性 Table 14 Physicochemical properties of test soil
Figure imgf000078_0001
本発明において開発した手法として、 抽出操作条件が異なる以下の 4種類の手 法を比較検討した
Figure imgf000078_0001
The following four methods with different extraction operation conditions were compared and studied as the methods developed in the present invention.
1. 0.5 gの土壌から、 100 mMTris-HCl/400 mM EDTA/ 750mM リン酸力 リウム / 1°/。SDS (pH 8.6) からなる抽出液 1200 により DNAを抽出し、 2% CTAB / 1 M NaClによる簡易精製を経て、 12% PEGにより土壌 DNAを沈殿回 収する方法。 この方法を、 以下 「オリジナル 1 Step法」 と記す。  1. From 0.5 g of soil, 100 mM Tris-HCl / 400 mM EDTA / 750 mM potassium phosphate / 1 ° /. DNA is extracted using SDS (pH 8.6) extract 1200, and after simple purification using 2% CTAB / 1M NaCl, soil DNA is recovered using 12% PEG. This method is hereinafter referred to as “original one-step method”.
2. 0.5 gの土壌に、 100 mMTris-HCl/ 300 mM EDTA/ 100 mM リン酸カリ ゥム I 1 % SDS (pH 8.6)からなる抽出液 800^1を添加して beads-beating処理 を行い、 フラッシュ遠心後 400 lの 600 mMEDTA， 2050 mM リン酸カリウム 緩衝液 （pH8.6) を加え、 攪拌後に遠心して上清を回収し、 2% CTAB / 1 M NaCl による簡易精製を経て、等量の 12% PEGにより土壌 DNAを沈殿回収する方法。 この方法を、 以下 「オリジナル 2Step法」 と記す。  2.To 0.5 g of soil, add 800 mM extract solution consisting of 100 mM Tris-HCl / 300 mM EDTA / 100 mM potassium phosphate I 1% SDS (pH 8.6) and perform beads-beating treatment. After flash centrifugation, 400 l of 600 mM EDTA, 2050 mM potassium phosphate buffer (pH 8.6) was added, and the mixture was centrifuged. After centrifugation, the supernatant was recovered, subjected to simple purification with 2% CTAB / 1 M NaCl, and Precipitation and recovery of soil DNA using 12% PEG. This method is hereinafter referred to as “original 2Step method”.
3. 0.5 gの土壌に beads-beating処理まで 2と同様の操作を加えた後、 400 l の 600mMEDTA/2050 mM リン酸カリウム緩衝液 （pH8.6) を添加し、 60。C で 1時間加熱処理を行ってから、 遠心、 簡易精製、 DNAの沈殿回収を 2と同様 の操作を行った方法。 この方法を、 以下 「オリジナル 2Step加熱法」 と記す。  3. After adding the same operation as in 2 to beads-beating treatment to 0.5 g of soil, 400 l of 600 mM EDTA / 2050 mM potassium phosphate buffer (pH 8.6) was added. A method in which heat treatment is performed for 1 hour at C, followed by centrifugation, simple purification, and DNA precipitation and collection. This method is hereinafter referred to as “original 2Step heating method”.
4. 0.5 gの土壌に、 100 mM Tris-HCl / 300 mM EDTA / 100 mM リン酸カリ ゥム / 1 % SDSからなる抽出液 1200 にて beads-beating処理を行い、 遠心し て上清を回収し、 2 °/。 CTAB/ l MMaClによる簡易精製を経て、等量の 12% PEG により土壌 DNA 沈殿回収する方法。 この方法を、 以下 「オリジナル LowConcBuffer (LCB) 法」 と記す。 4.Beads-beating treatment is performed on 0.5 g of soil with an extract 1200 consisting of 100 mM Tris-HCl / 300 mM EDTA / 100 mM potassium phosphate / 1% SDS, and the supernatant is collected by centrifugation. And 2 ° /. After simple purification with CTAB / l MMaCl, an equal amount of 12% PEG Method for recovering soil DNA by precipitation. This method is hereinafter referred to as “original LowConcBuffer (LCB) method”.
既往の方法として、 Zhou et al. (1996) の方法および Cullen & Hirsch (1998) の 2種類の方法、さらに Bio 101 Fast DNA spin kit (Qbio, USA) , UltraClean™ Soil DNA kit (MoBio、 USA) の 2種類のキットを用いる方法により土壌 DNA を抽出した。 土壌 DNA 抽出はそれぞれのプロトコールに基づいて行ったが、 Cullen & Hirsch (1998)の方法および UltraClean Soil DNA kit (MoBio USA) については、 beadsによる破砕に特別な装置が必要とされたため、 この部分につ いては両者とも Fast Prep FP101 (Qbio、 USA)を代用し、 強度 5 m / sec、 30sec の処理で代替し、 抽出を行った。  Previous methods include the method of Zhou et al. (1996) and two methods of Cullen & Hirsch (1998), as well as Bio101 Fast DNA spin kit (Qbio, USA) and UltraClean ™ Soil DNA kit (MoBio, USA) Soil DNA was extracted by the method using two kinds of kits. Soil DNA extraction was performed according to each protocol.However, Cullen & Hirsch (1998) and UltraClean Soil DNA kit (MoBio USA) required special equipment for crushing with beads. For both, extraction was performed by substituting Fast Prep FP101 (Qbio, USA) with a treatment at an intensity of 5 m / sec for 30 sec.
また、 本実施例では土壌からの DNA抽出効率の評価を目的としたが、 ァガロ —ス電気泳動後の切りだしにより DNAを精製する Zhou et al.(1996)の方法、 お よびゲルろ過により DNAを精製する Cullen & Hirsch (1998)の方法では、 研究 者の技術や操作方法、 用いる装置などにより、 抽出量にかなりの誤差が生じると 考えられたため、 精製前の DNA溶液による収量の比較を行った。  Although the purpose of this example was to evaluate the efficiency of DNA extraction from soil, the method of Zhou et al. (1996), which purifies DNA by excision after agarose electrophoresis, and the method of gel filtration, The method of Cullen & Hirsch (1998), which purifies DNA, was considered to cause considerable errors in the amount of extraction due to the technique, operation method, and equipment used of the researchers. Was.
土壌 DNA の抽出の際、 土壌と抽出液の懸濁液を遠心して土壌 （あるいは beads-beating処理を利用する方法では土壌と beadsの混合物） を沈殿させ、 抽 出液を回収することになるが、 この際、 土壌や beadsの間隙に回収しきれない抽 出液が残る。'精製操作などの違いにより、 この抽出液の回収量がそれぞれの方法 で異なる。 土壌からの抽出効率を計算する際、 遠心により回収した上清と、 土壌 や beadsの間隙に含まれる溶液中には等しい濃度で DNAが含まれているものと 仮定し、 土壌 DNAの抽出量を、 添加した抽出液全量に相当する量に換算して算 出した。  In the extraction of soil DNA, the suspension of the soil and the extract is centrifuged to precipitate the soil (or a mixture of soil and beads if beads-beating is used), and the extract is collected. At this time, unextracted extract remains in the soil and in the gaps between beads. 'The amount of this extract recovered differs depending on the method of purification. When calculating the extraction efficiency from soil, it is assumed that the supernatant contained by centrifugation and the solution contained in the space between the soil and beads contain the same concentration of DNA. The amount was calculated by converting to the amount corresponding to the total amount of the added extract.
(計算例）  (Example of calculation)
土壌 0.5 gに抽出液 1200 lを添加し、 beads-beating処理後、 そこから 750 lを回収し、 その後の精製操作でその溶液が 1250 1になり、 最終的にそのう ち 1000 1の溶液から DNAを沈殿回収した場合、  1200 l of extract was added to 0.5 g of soil, and after bead-beating treatment, 750 l of the extract was collected, and the resulting solution became 1250 1 in the subsequent purification operation. If DNA is collected by precipitation,
土壌 1 g 当たりの DNA抽出量 = 沈殿回収された DNA 量 X 1250/1000 X 1200/750 X 2 Amount of DNA extracted per gram of soil = Amount of precipitated and recovered DNA X 1250/1000 X 1200/750 X 2
(2) 結果と考察  (2) Results and discussion
本発明において開発した方法の一例である上記 4種類のオリジナル法によって、 12種類の土壌から DNAを抽出した。  DNA was extracted from 12 kinds of soils by the above four kinds of original methods, which are examples of the method developed in the present invention.
結果を図 23に示す。 また 4種類の方法による抽出 DNAのサイズについて図 24に示す。 オリジナル IStep法、 2Step加熱法、 および既往の代表的な方法によ る土壌 DNAの抽出量について図 25に示す。  The results are shown in FIG. Figure 24 shows the size of DNA extracted by the four methods. Figure 25 shows the amount of soil DNA extracted by the original IStep method, the 2-step heating method, and the typical methods already used.
(2-1) 4種類のオリジナル法の土壌 DNA収量比較  (2-1) Comparison of soil DNA yields of the four original methods
火山灰土壌である弥生圃場対照区土壌、栃木農試森林土壌、東北大学森林土壌、 およびすベての非火山灰土壌から、 オリジナル IStep法およびオリジナル 2Step 加熱法により、 ほぼ同じ量の土壌 DNAを抽出することができた。 しかし、 千葉 農試森林土壌、 茨城農試畑土壌、 田無農牧草地土壌、 群馬畜試牧草地土壌につい ては、 オリジナル LCB法では土壌 DNAがほとんど抽出できていない。 またオリ ジナル 2Step法でも、 土壌 DNAの抽出量は、 オリジナル 1 Step法のそれと比較 して 1/3から 1/5以下となっている。 これらの土壌では、 土壌に吸着した DNA を溶出させる作用が強いォリジナル 2Step加熱法でも十分な量の土壌 DNAを回 収することができなかった。 この 4種類の土壌は、 いずれもァロフェン態 A1が 50 mg / g soilと火山灰土壌の中でも特に多い土壌であり、 DNAの土壌への吸着 が強く、 いったん土壌に吸着されてしまうと、 DNA を再抽出し回収することは 困難と思われた。 非火山灰土壌からは、 加熱を伴わない 2Step法でも、 極めて高 収量で土壌 DNAを回収することが可能であり、 土壌による DNA吸着の解消が 容易になされた。  Approximately the same amount of soil DNA is extracted from the volcanic ash soils of the Yayoi field control plot soil, Tochigi agricultural test forest soil, Tohoku University forest soil, and all non-volcanic ash soils by the original IStep method and the original 2Step heating method I was able to. However, for the Chiba Agricultural Forest Soil, Ibaraki Agricultural Farmland Soil, Tanashi Agricultural Pasture Soil, and Gunma Livestock Agricultural Pasture Soil, almost no soil DNA was extracted by the original LCB method. Also in the original two-step method, the amount of soil DNA extracted is one-third to one-fifth that of the original one-step method. In these soils, a sufficient amount of soil DNA could not be recovered by the original 2-step heating method, which has a strong effect of eluting the DNA adsorbed on the soil. All of these four types of soil have 50 mg / g soil arofen-A1 and are particularly high in volcanic ash soils.The DNA is strongly adsorbed on the soil. Extraction and recovery seemed difficult. From non-volcanic ash soil, even with the two-step method without heating, it was possible to recover soil DNA with extremely high yield, and DNA adsorption by soil was easily eliminated.
またオリジナル I Step法により得られた土壌 DNAが、 20〜7 kbpに低分子化 していたのに対し、 オリジナル 2Step法およびオリジナル 2Step加熱法、 オリジ ナル LCB法により得られた土壌 DNAは、 20 kbp以上の高分子に保たれ、 低分 子化は抑えられていた。  Soil DNA obtained by the original I Step method had a molecular weight of 20 to 7 kbp, whereas soil DNA obtained by the original 2 Step method, the original 2 Step heating method, and the original LCB method had a molecular weight of 20 kbp. It was kept at a high molecular weight of kbp or more, and underpolymerization was suppressed.
最終的な DNA溶液への腐植物質の混入はオリジナル 2Step法およびオリジナ ル LCB 法ではほとんどみられず、 溶液はほぼ無色であった。 しかし、 オリジナ ル IStep法およびォ ύジナル 2Step加熱法については、栃木農試森林土壌および 東北大学森林土壌から抽出した DNA溶液は茶色に着色しており腐植物質を完全 に除去することだできていなかった。 この 2種類の土壌は腐植の集積量が多く、 抽出時に多量の腐植物質が抽出されてしまっていた。 しかし、 後述するが踌型 DNA量を少なくすると PCR反応が成功する範囲の腐植物質の混入であった。 (2-2) 既往の方法との収量比較 Almost no humic substances were mixed into the final DNA solution by the original 2Step method and the original LCB method, and the solution was almost colorless. But the original In the IStep method and the original 2Step heating method, DNA solutions extracted from Tochigi agricultural test forest soil and Tohoku University forest soil were colored brown and could not completely remove humic substances. These two types of soil had a large accumulation of humus, and a large amount of humic substances had been extracted during extraction. However, as will be described later, if the amount of type II DNA was reduced, humic substances were present in a range where the PCR reaction was successful. (2-2) Yield comparison with existing methods
いずれの土壌においても、本発明のオリジナル IStep法および 2Step加熱法は、 既往の手法よりも多くの土壌 DNAを抽出することが可能であった。 特にァロフ ェン質黒ポク土および草地試験場永年採草地土壌からは、 既往の方法では Zhou et al. (1996) の方法でしか土壌 DNAが得られず、 またその場合でも DNAの収 量は低いものであった。  In any of the soils, the original IStep method and the 2Step heating method of the present invention were able to extract more soil DNA than the existing methods. In particular, soil DNA can be obtained only from the arophenic black pork soil and the grassland of the grassland test site by the method of Zhou et al. (1996), and even in that case, the yield of DNA is low. It was low.
非火山灰土壌および東北大学森林土壌からは、 UltraCleanTM Soil DNA kitを 除く既往の方法でも土壌 DNAが得られた。既往の方法では、 Bio 101 FastSpinKit による抽出量が多く、 Cullen & Hirscli (1998) 方法でも土壌 DNAは抽出され た。 しかし、 Zliou et al. (1996) の方法による抽出量は、 非火山灰土壌において も少なく、 この収量の差は、加熱処理および bead-beating処理の有無によって生 じたと考えられた。 しかし、 Zhou et al. (1996) の方法では、 23kbp以上の高分 子土壌 DNAが得られていた。 Non-volcanic ash soils and Tohoku University forest soils were also obtained by conventional methods except for the UltraClean TM Soil DNA kit. In the previous method, the amount of extraction by Bio 101 FastSpinKit was large, and soil DNA was also extracted by the method of Cullen & Hirscli (1998). However, the amount extracted by the method of Zliou et al. (1996) was small even in non-volcanic ash soil, and this difference in yield was considered to be caused by the presence or absence of heat treatment and bead-beating treatment. However, according to the method of Zhou et al. (1996), polymer soil DNA of 23 kbp or more was obtained.
この比較検討の結果より、 本発明のオリジナルの方法は、 さまざまな土壌から 高収量で土壌 DNAを抽出することが可能であり、またァロフェン態 A1に富む土 壌からは、 オリジナルの方法を用いないと土壌 DNAが十分に、 あるいは全く得 られないことが明らかになった。  Based on the results of this comparative study, the original method of the present invention can extract soil DNA from various soils with high yield, and does not use the original method from soil rich in arophenic form A1. And found that sufficient or no soil DNA was obtained.
〔実施例 14〕 DNA純度の比較 [Example 14] Comparison of DNA purity
土壌 DNAの純度は、 PCR反応にどれぐらいの濃度の DNAを鐯型として使用 できるかを指標として検討を行った。 濃度が高いほど夾雑物も多くなるため、 純 度の高い DNA試料でなければ、高濃度で使用したときに PCR反応が阻害される からである。 (1) 材料と方法 The purity of soil DNA was examined using as an index how much DNA could be used as type III in the PCR reaction. This is because the higher the concentration, the more contaminants are present, and unless a highly pure DNA sample is used, the PCR reaction is inhibited when used at a high concentration. (1) Materials and methods
DNAの抽出効率の差により、 最終的な DNA溶液量を一定にしても土壌 DNA 含量は全く異なる。 そこで、 定量した土壌 DNA濃度をもとに、 土壌 DNAを 100 ng、 50 ng、 10 ngの 3段階で铸型として用い、 PCR反応の成否を試験すること により、 土壌 DNAの純度を検定した。  Due to differences in DNA extraction efficiencies, the soil DNA content is completely different, even if the final DNA solution volume is constant. Therefore, based on the quantified soil DNA concentration, the purity of the soil DNA was tested by testing the success or failure of the PCR reaction by using the soil DNA in three stages of 100 ng, 50 ng, and 10 ng.
検討した土壌 DNAはオリジナル 1 Step法およびオリジナル 2Step加熱法によ り得られたものである。 この 2種類の方法は、 bead-beating後の粗抽出液が腐植 物質の混入により茶色もしくは黒色に着色しており、 他の 2種類の方法よりも腐 植物質の混入量は多いと考えられ、 精製による腐植物質の除去効率を検討するの に適していると考えられたからである。  The soil DNA studied was obtained by the original one-step method and the original two-step heating method. In these two methods, the crude extract after bead-beating is colored brown or black due to the incorporation of humic substances, and it is considered that the amount of humic substances mixed is greater than in the other two methods. This is because it was considered suitable for examining the efficiency of humic substances removal by purification.
PCRは 3連で行い、その反応条件および使用したプライマーについては以下の通 りである。  PCR was performed in triplicate, and the reaction conditions and primers used were as follows.
PCR反応の条件 PCR reaction conditions
プライマ一セット 27F-1494R  Primer set 27F-1494R
27Fmix： AGAGTTTGATCMTGGCTCAG (配列番号 1 )  27Fmix: AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 1)
1494R： GGTTACCTTGTTACGACTT (配列番号 2 )  1494R: GGTTACCTTGTTACGACTT (SEQ ID NO: 2)
上記プライマーセットにより、 16Si'RNA遺伝子のほぼ全長を増幅することが できる。  With the above-mentioned primer set, almost the full length of the 16Si ′ RNA gene can be amplified.
反応液組成は以下の通りである。  The composition of the reaction solution is as follows.
反応液： 50 1  Reaction solution: 50 1
プライマ一： 0.5 mM  Primer: 0.5 mM
Taq DNA Polymerase vsigma)： 2.5 unit  Taq DNA Polymerase vsigma): 2.5 unit
BSAを終濃度 400ng/ lで含む。  Contains BSA at a final concentration of 400 ng / l.
反応条件は、まず 94°Cで 2.5分反応させた後、次に 94 30秒の変性、 50°C 30 秒のアニーリング及び 72 2分の伸長反応を 1サイクルとしてこれを 30サイ クル行い、 最後に、 72でで 10分反応させた。  The reaction conditions were as follows: a reaction was first performed at 94 ° C for 2.5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, and elongation at 722 minutes as one cycle. The reaction was carried out at 72 for 10 minutes.
(2) 結果と考察  (2) Results and discussion
PCRによる土壌 DNAの純度検定の結果について図 26A〜： Bに示す。 腐植物質の集積量および混入量が多い 2種類の土壌以外、 すなわち栃木農試森 林土壌および東北大学森林土壌以外では、 オリジナル IStep法により得られた土 壌 DNAは、 50 lの PCR反応液中に 50ngを使用しても、： PCR反応が成功した。 オリジナル 2Step加熱法により得られた土壌 DNAでも同様の結果が得られた。 オリジナル 1 Step'法により得られた栃木農試森林土壌および東北大学森林土壌 は、 10 ng土壌 DNAを鐃型にすると PCRが成功した。 26A to 26B show the results of the soil DNA purity test by PCR. Except for the two types of soils with high accumulation and contamination of humic substances, that is, except for Tochigi Agricultural Forest and Tohoku University forest soils, the soil DNA obtained by the original IStep method was used in a 50 l PCR reaction solution. Using 50 ng: PCR reaction was successful. Similar results were obtained with the soil DNA obtained by the original 2Step heating method. In Tochigi agricultural test forest soil and Tohoku University forest soil obtained by the original 1 Step 'method, PCR was successful when 10 ng soil DNA was changed to a cylin type.
これに対し、 2Step加熱法により得られた栃木農試森林土壌の DNAでは、 い ずれの DNA量の場合でも PCR反応が成功しなかった。東北大学森林土壌よりォ リジナル 2Step加熱法により得られた土壌 DNAは、 さらに希釈して铸型として の使用量を 1 ng程度にしないと、 PCRは成功しなかった。 このことから、 腐植 の集積量が多い土壌から、 高濃度 EDTA-リン酸混合液を使用し、さらに加熱処理 を施して抽出された DNAは、 CTAB精製および PEG沈殿を行つても十分に腐植 物質を除去しきれないと考えられた。  On the other hand, in the DNA of Tochigi agricultural test forest soil obtained by the 2-step heating method, the PCR reaction was not successful at any amount of DNA. PCR was not successful unless soil DNA obtained from Tohoku University forest soil by the original two-step heating method was further diluted to reduce the amount used as type I to about 1 ng. Therefore, DNA extracted from soil with high accumulation of humus using a high-concentration EDTA-phosphoric acid mixed solution and heat treatment can be sufficiently humic substances even after CTAB purification and PEG precipitation. Was not completely removed.
しかし、 この 2種類の土壌を除く大部分の土壌については、 50 1の PCR反応 に 50 ngの土壌 DNAを铸型として使用しても PCRが成功したことから、オリジ ナル法によって高純度な土壌 DNAが得られたと考えられる。 オリジナル法で得 られる土壌 DNAは PCR反応の錶型として多量に用いることができるため、 PCR 反応サイクル数を少なく設定することが可能であり、 また、 土壌中のマイナーな 微生物群も効率よく検出できることが示された。  However, for most soils except for these two types of soil, PCR was successful even when 50 ng of soil DNA was used as type I in the 501 PCR reaction. It is considered that DNA was obtained. Since the soil DNA obtained by the original method can be used in large quantities as a type II PCR reaction, the number of PCR reaction cycles can be reduced, and minor microorganisms in soil can be detected efficiently. It has been shown.
〔実施例 15〕 土壌 DNA組成の比較 [Example 15] Comparison of soil DNA composition
本実施例では、 本発明の各種手法により得られた土壌 DNAの組成について、 PCR-DGGE法により比較検討した。  In this example, the composition of soil DNA obtained by various methods of the present invention was compared and examined by PCR-DGGE method.
(1) 材料と方法  (1) Materials and methods
抽出された土壌 DNA 1 lを錶型として、 16S rRNA遺伝子の V3領域を PCR により増幅し、 これを DGGEにより解析した。 PCR反応の条件および使用した プライマ一、 DGGEの条件を以下に示す。  Using 1 l of the extracted soil DNA as type II, the V3 region of the 16S rRNA gene was amplified by PCR and analyzed by DGGE. The conditions of the PCR reaction and the primer and DGGE conditions used are shown below.
(1-1) PCR プライマ一セット 341FGC-534R (1-1) PCR Primer set 341FGC-534R
16SrRNA遺伝子の V3領域を増幅  Amplifies V3 region of 16S rRNA gene
341FGC： 341FGC:
CGCCCGCCGC GCCCCGCGCC CGGCCCGCCG CCCCCGCCCC CCTACGGGAG GCAGCAG (配列番号 3 )  CGCCCGCCGC GCCCCGCGCC CGGCCCGCCG CCCCCGCCCC CCTACGGGAG GCAGCAG (SEQ ID NO: 3)
534R： 534R:
ATTACCGCGGCTGCTGGCAC (配列番号 4 )  ATTACCGCGGCTGCTGGCAC (SEQ ID NO: 4)
反応条件は、まず 94°Cで 2.5分反応させた後、次に 94°C 30秒の変性、 55 30 秒のアニーリング及び 72°C 1 分の伸長反応を 1サイクルとしてこれを 24又は 30サイクル行い、 最後に、 72°Cで 10分反応させた。 反応液組成は以下の通りで ある。  The reaction conditions are as follows: First, the reaction is performed at 94 ° C for 2.5 minutes, and then the denaturation at 94 ° C for 30 seconds, annealing for 30 seconds at 55 ° C, and the elongation reaction at 72 ° C for 1 minute are performed for 24 or 30 cycles. The reaction was finally performed at 72 ° C for 10 minutes. The composition of the reaction solution is as follows.
BSAを終濃度 400ng/ で含む。  Contains BSA at a final concentration of 400 ng /.
Taq DNA Polymerase (sigma) 2.5unit  Taq DNA Polymerase (sigma) 2.5unit
反応液 50 1  Reaction liquid 50 1
(1-2) DGGE  (1-2) DGGE
ゲル濃度 8 %  Gel concentration 8%
変性剤濃度勾配 35 %〜65 %  Denaturant gradient 35% to 65%
Buffer温度 60  Buffer temperature 60
電圧 100V  Voltage 100V
泳動時間 10時間  Running time 10 hours
オリジナルの 4種類の方法および Zhou et al. (1996) の方法により得られた土 壌 DNA溶液はおおよそ 5〜50 ng / lである。 この 10〜20 gを铸型として、 24サイクルの PCR反応を行った。 またその他の既往の方法では、 火山灰土壌か ら十分量の土壌 DNAが得られなかったが、 条件を揃えるため、 それぞれ 1 1ず っを錶型として 30サイクルの PCR反応を行った。既往の方法で非火山灰土壌か ら得られた土壌 DNAについては、 同じく l lを铸型として 24サイクルの PCR 反応を行った。  The soil DNA solution obtained by the four original methods and the method of Zhou et al. (1996) is approximately 5-50 ng / l. Using this 10 to 20 g as type III, 24 cycles of PCR reaction were performed. In the other existing methods, sufficient amounts of soil DNA could not be obtained from the volcanic ash soil. However, in order to adjust the conditions, 30 cycles of PCR were performed using 11-types for each type. Soil DNA obtained from non-volcanic ash soil by the previous method was subjected to 24 cycles of PCR using the same type of lll.
(2) 結果と考察 各方法で得られた土壌 DNAの DGGE解析結果を図 27A〜Eに示す。 (2) Results and discussion FIGS. 27A to 27E show the results of DGGE analysis of soil DNA obtained by each method.
弥生圃場対照区土壌、 千葉農試森林土壌、 茨城農試畑土壌、 田無農場牧草地土 壌、 群馬畜試牧草地土壌、 栃木農試森林土壌の 6種類の土壌については、 オリジ ナルによる方法では十分な増幅産物が得られたが、 既往の方法で抽出した土壌 DNAを錶型として行った PCR反応では、 Zhou et al. (1996) 方法以外では全く 増幅産物を得ることができなかった。 非火山灰土壌においてはオリジナル法およ び既往の方法でも十分な増幅産物が得られた。 これらの PCR反応により得られ た増幅産物を DGGEにより分析し、その土壌 DNAの由来生物組成について検討 した結果、 一部抽出の過程において土壌以外から混入したと思われる DGGE バ ンドがまれに生じることもあったが、 全ての抽出法により同様の DGGE プロフ アイルが得られた。すなわち、 少なくとも本実験で対象にした 16S rRNA を持つ 細菌については、 DNAが抽出される細菌群および各細菌群間における DNA抽出 率の比がいずれの抽出法でも同等であることが示された。  For the six types of soil in the Yayoi field control plot soil, Chiba agricultural trial forest soil, Ibaraki agricultural trial field soil, Tanashi farm pasture soil, Gunma animal trial pasture soil, and Tochigi agricultural trial forest soil, the original method was used. Although sufficient amplification products were obtained, amplification products could not be obtained at all by PCR other than the method of Zhou et al. (1996) using a type I soil DNA extracted by the conventional method. In non-volcanic ash soil, sufficient amplification products were obtained by the original method and the existing method. The amplification products obtained by these PCR reactions were analyzed by DGGE, and the composition of the biological origin of the soil DNA was examined.As a result, rare DGGE bands that appeared to have been contaminated from sources other than the soil during the extraction process were found to be rare. However, all extraction methods yielded similar DGGE profiles. That is, at least for the bacteria having 16S rRNA targeted in this experiment, it was shown that the ratio of the DNA extraction ratio between the bacterial group from which DNA was extracted and each bacterial group was the same in any of the extraction methods.
以上をまとめると次のことが示された。  Summarizing the above, the following was shown.
1. オリジナル IStep法、 オリジナル 2Step加熱法は、 既往の方法よりも高収 量で土壌 DNAを抽出することができた。  1. The original IStep method and the original 2Step heating method were able to extract soil DNA with higher yield than the previous methods.
2. オリジナル IStep法、 オリジナル 2Step加熱法は、 既往の方法では抽出が 困難であつたァロフェン質 A1に富む火山灰土壌からも土壌 DNAを抽出すること ができた。  2. With the original IStep method and the original 2Step heating method, it was possible to extract soil DNA from volcanic ash soil rich in arofenic A1, which was difficult to extract using the existing methods.
3. オリジナル IStep法、 オリジナル 2Step加熱法は、 ほとんどの土壌より得 られた土壌 DNAの 50 ngをも铸型として PCRに供試でき、 高純度な土壌 DNA が得られていることが示された。  3. In the original IStep method and the original 2Step heating method, 50 ng of soil DNA obtained from most soils can be used for PCR as type III, indicating that high-purity soil DNA was obtained. .
PCR-DGGEの結果より、各方法により得られた土壌 DNAに含まれる細菌由来 の DNA組成は、 ほぼ同じであることが推測された。  From the results of PCR-DGGE, it was estimated that the bacterial DNA composition contained in the soil DNA obtained by each method was almost the same.
〔実施例 16〕 CTABによる精製時に pHを低下させることによる精製率の向 上 （CH3COONaを単 で使用した場合) [Example 16] Improvement of purification rate by lowering pH during purification by CTAB (when CH 3 COONa is used alone)
土壌からアルカリ条件下で抽出される腐植物質の大部分は腐植酸である。 腐植 酸は土壌化学的にはプルカリで土壌から抽出され、 pH 2の酸性条件により電荷を 失い、 沈殿する物であるとの定義がなされている。 The majority of humic substances extracted from soil under alkaline conditions are humic acids. Humus Acid is chemically defined as a substance that is extracted from the soil with pulchali, loses its charge under acidic conditions of pH 2, and precipitates.
土壌から DNAを抽出する際に混入する夾雑物質もほとんどがこの腐植酸であ ると考えられる。  It is considered that most of the contaminants contaminated when extracting DNA from soil are humic acids.
通常、 CTABによる DNA溶液の精製は NaCl存在下で行われているが、 本実 施例ではこの NaClを酸性側に p|iを調製した CH3COONaに変更し、 CTABに よる精製時に pHを低下させることにより腐植除去効率の向上を試みた。 Usually, purification of DNA solution by CTAB is carried out in the presence NaCl, in this real施例the NaCl p acidified side | Change in CH 3 COONa prepared a i, the pH during purification by CTAB We tried to improve the humus removal efficiency by lowering it.
(1) 材料と方法 (1) Materials and methods
東北大学森林土壌 10 gに対し、次の 3種類の抽出液を用いて土壌からの DNA 抽出液を得た。  A DNA extract from soil was obtained using the following three types of extract with respect to 10 g of Tohoku University forest soil.
(i) 1% SDS I 100 mM Tris-HCl I 100 mM EDTA I 100 mM Na2HP04 (pH 8.6) , (ii) 1% SDS I 200 mM EDTA/ 375 mM Na2HP04 (pH 8.6) ， (i) 1% SDS I 100 mM Tris-HCl I 100 mM EDTA I 100 mM Na 2 HP0 4 (pH 8.6), (ii) 1% SDS I 200 mM EDTA / 375 mM Na 2 HP0 4 (pH 8.6),
(iii) 1% SDS I 400 mM EDTA I 750 mM Na2HP04 (pH 8.6) 上記抽出液を土壌に対し 24 ml添加し、 65°Cにおいて 30分加熱処理を加え、 土壌から DNA の加熱抽出を行った。 土壌を抽出液で長時間加熱抽出を行うと BeadsBeating処理の場合よりも多くの腐植物質が DNAと共に抽出される。差を より明確にするためこの実験では土壌 DNAを加熱抽出により抽出し、 得られた 土壌 DNA溶液を、 腐植物質が多量に混入した汚染サンプルとして精製実験を行 つ Tこ。 (iii) 1% SDS I 400 mM EDTA I 750 mM Na 2 HP0 4 (pH 8.6) The above extracts were added 24 ml to soil, a 30-minute heat treatment at 65 ° C was added, heating extracted from soil DNA Was done. Long-term heat extraction of the soil with the extract will extract more humic substances along with DNA than in the BeadsBeating treatment. To clarify the difference, in this experiment soil DNA was extracted by heat extraction, and a purification experiment was performed using the obtained soil DNA solution as a contaminated sample containing a large amount of humic substances.
その後 6000 X g 10分間の遠心により上清を得た。 この上清は BeadsBeating 処理による抽出操作よりもはるかに多くの腐植物質が混入していることが確認で きた。  Thereafter, supernatant was obtained by centrifugation at 6000 × g for 10 minutes. It was confirmed that this supernatant contained much more humic substances than the extraction operation by BeadsBeating treatment.
この溶液を土壌 DNA抽出液とし、 以下の精製実験に供試した。  This solution was used as a soil DNA extract and subjected to the following purification experiments.
3種類の土壌 DNA抽出液 750 lに 10% CTAB溶液 250 lおよび 5 M NaCl 溶液もしくは pHを 5.2、 5.4、 5.6、 5.8、 6.0、 6.2、 6.5に調製した 5 M CH3COONa 溶液を 250 1添加した。 すなわち土壌 DNA抽出液にこれらの溶液を添加する ことにより、 2% CTAB 1 1M NaClもしくは 1 M CHaCOONa (pHが 7条件） に 調製し、 クロ口ホルムをこれらの溶液に等量添加し、 vortex後 12000 X g 25°C で 10分間遠心を行い、 水相を精製処理後の DNA抽出液として回収した。 この CTAB による精製時の塩に酸性緩衝能を付加することにより、 pH を低下させ DNA溶液の純度がどのような影響を与えるかを明らかにするため、 この上清を 適時希釈して 400 nmにおける吸光度を測定した。 250 l of 10% CTAB solution and 250 l of 5 M NaCl solution or 5 M CH 3 COONa solution adjusted to pH 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.5 were added to 750 l of three types of soil DNA extracts did. That is, add these solutions to the soil DNA extract Prepare 2% CTAB 1 1M NaCl or 1M CHaCOONa (pH 7 conditions), add equal volume of form to these solutions, vortex, and centrifuge at 12000 X g at 25 ° C for 10 minutes The aqueous phase was recovered as a DNA extract after the purification treatment. The supernatant was diluted appropriately at 400 nm by adding an acidic buffer to the salt used for purification by CTAB to lower the pH and clarify the effect of the purity of the DNA solution. The absorbance was measured.
結果を図 28に示す。  The results are shown in FIG.
また、この上清 750 1に対し等量の 12% PEG溶液を添加し、 20000 X g 4°C 20 分間の遠心により土壌 DNAを沈殿させた。上清を除去、 70%エタノールで洗浄、 乾燥後、この DNAの沈殿を 200 lの TE buffer(pH 8.0)に溶解させて DNA溶液 とした。  Further, an equal amount of a 12% PEG solution was added to the supernatant 7501, and soil DNA was precipitated by centrifugation at 20000 × g at 4 ° C. for 20 minutes. After removing the supernatant, washing with 70% ethanol and drying, this DNA precipitate was dissolved in 200 l of TE buffer (pH 8.0) to obtain a DNA solution.
この DNA溶液を適時希釈して 400 nmにおける吸光度を測定した結果を図 29 に示す。 また最終的に得られた DNA溶液の DNAの濃度を測定し、 土壌 DNAの 回収率を 1% SDS I 200 mM EDTA/ 375 mM Na2HP04 (pH 8.6) による DNA抽 出液について定量した結果を図 30に示す。 FIG. 29 shows the results obtained by appropriately diluting the DNA solution and measuring the absorbance at 400 nm. Results also for measuring the concentration of DNA in the finally obtained DNA solution was quantified for DNA extraction exudates by a recovery of soil DNA in 1% SDS I 200 mM EDTA / 375 mM Na 2 HP0 4 (pH 8.6) Is shown in FIG.
(2) 結果と考察 (2) Results and discussion
図 28 より、 CTAB による精製時に塩溶液に酸性緩衝能を持った CH3COONa 溶液を使用することで通常の NaCl溶液を使用した場合と比較して、 DNA抽出液 からクロ口ホルム添加後の遠心時に 30%~60°/0の腐植物質が除去できていること が示された。 またこの場合の CH3COONa溶液の pHによっては差が生じなかつ た。 From Fig. 28, the centrifugation of the DNA extract after the addition of a black hole form was achieved by using a CH 3 COONa solution with acidic buffering capacity for the salt solution during purification by CTAB, as compared to using a normal NaCl solution. It was shown that 30% to 60 ° / 0 humic substances were sometimes removed. In this case, there was no difference depending on the pH of the CH 3 COONa solution.
図 29より、最終的に 12% PEG溶液により回収した DNAの純度において.3種 類のいずれの抽出液で得た土壌 DNA抽出液の精製に対しても、 酸性緩衝能を持 つた CH3COONaの使用により NaClの場合よりも純度の高い土壌 DNAを得る ことが可能であることが示された。 From Fig. 29, the purity of the DNA finally recovered with the 12% PEG solution was determined.CH 3 COONa with acidic buffer capacity was used for the purification of the soil DNA extract obtained from all three extracts. It was shown that soil DNA with higher purity than that obtained with NaCl could be obtained by the use of NaCl.
また 1% SDS I 100 mM Tris-HCl I 100 mM EDTA I 100 mM Na2HP04 (pH 8.6)で抽出した DNA抽出液については酸性緩衝能を持った CHaCOONaの効 果は高く pHが 5.2'の場合に最も純度が高かった。 しかし、 その他 2種類の抽 出液では pHによる効果はあまりみられなかった。 The 1% SDS I 100 mM Tris- HCl I 100 mM EDTA I 100 mM Na 2 HP0 4 virtue of CHaCOONa for extracted DNA extract was (pH 8.6) is having an acidic buffering capacity The fruits were high and the purity was highest when the pH was 5.2 '. However, the other two extracts did not show much effect of pH.
しかし、図 30からもわかるように CH3COONaの pHは DNAの回収率に大き な影響を与えており、 CH3COONaを使用した場合は pHが高いものほど DNAの 回収率が悪くなつていることが明らかになった。 However, as can be seen from Fig. 30, the pH of CH 3 COONa has a large effect on the DNA recovery rate, and when CH 3 COONa is used, the higher the pH, the lower the DNA recovery. It became clear.
以上の結果より、酸性緩衝能を持つた CH3COONaの使用は CTABによる精製 時の腐植除去に極めて効果的であり、 その pHは DNAの回収率がよい pH5.2の 使用がよいと考えられた。 〔実施例 17〕 CTABによる精製時に pHを低下させることによる精製率の向 上 （NaClと CH3COONaの混合液を使用した場合） Based on the above results, it is considered that the use of CH 3 COONa, which has acidic buffer capacity, is extremely effective in removing humus during purification by CTAB, and its pH should be pH 5.2, which has a good DNA recovery rate. Was. [Example 17] Improvement of purification rate by lowering pH during purification by CTAB (when a mixed solution of NaCl and CH 3 COONa is used)
本実施例では、 CTABによる精製時の塩溶液として NaClと CH3COONaの混 合液を使用することを試みた。 In this example, an attempt was made to use a mixed solution of NaCl and CH 3 COONa as a salt solution during purification by CTAB.
(1) 材料と方法 (1) Materials and methods
実験操作は実施例 16の (1)と同様であり、 CTAB精製時に添加する塩に NaCl と CH3COONaの混合液を使用した。 DNA抽出液 750 に 10% CTAB溶液 250 lと以下の塩の混合液を 250 1添加した。 The experimental procedure was the same as (1) in Example 16, and a mixed solution of NaCl and CH 3 COONa was used as the salt to be added during the purification of CTAB. 250 l of a 10% CTAB solution and 250 1 of the following salt mixture were added to 750 of the DNA extract.
2.5 M CH3COONa I 2.5 M NaCl (pH 5.2) , 2.5 M CH 3 COONa I 2.5 M NaCl (pH 5.2),
1.67 Μ CHsCOONa I 3.33 Μ NaCl (pH 5.2)，  1.67 Μ CHsCOONa I 3.33 Μ NaCl (pH 5.2),
1.25 Μ CHsCOONa I 3.75 M NaCl (pH 5.2)，  1.25 Μ CHsCOONa I 3.75 M NaCl (pH 5.2),
3.33 M CHsCOONa 1 1.67M NaCl (pH 5.2),  3.33 M CHsCOONa 1 1.67M NaCl (pH 5.2),
3.75 M CHsCOONa I 1.25 M NaCl (pH5.2)，  3.75 M CHsCOONa I 1.25 M NaCl (pH5.2),
5 M CHsCOONa I 1.25 M NaCl (pH 5.2),  5 M CHsCOONa I 1.25 M NaCl (pH 5.2),
5 M CH3COONa I 2.5 M NaCl (pH 5.2), 5 M CH 3 COONa I 2.5 M NaCl (pH 5.2),
3.75 M CHsCOONa I 2.5 M NaCl (pH 5.2) ,  3.75 M CHsCOONa I 2.5 M NaCl (pH 5.2),
3.75 M CHsCOONa I 3.75 M NaCl (pH 5.2)  3.75 M CHsCOONa I 3.75 M NaCl (pH 5.2)
添加後の塩の組成はそれぞれ CH3COONa I NaCl で 0.5 M / 0.5 M , 0.33 M/ .67 M， 0.25 M I 0.75 M， 0.67 M I 0.33 M， 0.75 M /0.25 M， 1 M / 0.25 M , 1 M / 0.5 M , 0.75 M I 0.5'M， 0.75 M I 0.75 Mとなる。 The composition of the salt after addition was 0.5 M / 0.5 M, 0.33 M / .67 M, 0.25 MI 0.75 M, 0.67 MI 0.33 M, 0.75 M / 0.25 M, 1 M / 0.25 M, 1 M in CH 3 COONa I NaCl, respectively. M / 0.5 M, 0.75 MI 0.5'M, 0.75 MI 0.75 M
これらの溶液を添加してクロ口ホルムを等量添加、 vortex、 遠心後の水相を採 取し、 適宜希釈後 400nmにおける吸光度を測った結果を図 31に示す。 また最終 的に 12% PEG溶液により回収して、 TE Bufferに溶解させた DNA溶液の 400nm における吸光度を図 32に示した。  Fig. 31 shows the results obtained by adding these solutions, adding an equal amount of black form, collecting the aqueous phase after vortex and centrifugation, and after appropriate dilution, measuring the absorbance at 400 nm. FIG. 32 shows the absorbance at 400 nm of the DNA solution finally recovered with a 12% PEG solution and dissolved in TE Buffer.
また土壌 DNAの回収量を図 33に示した。  Figure 33 shows the amount of soil DNA recovered.
(2) 結果と考察 (2) Results and discussion
図 31より塩溶液に NaClおよび CH3COONaの混合液を使用した場合でも、添 加後の塩の組成が CH3COONa I NaClで 0.67 M I 0.33 M , 0.75 M /0.25 M， 1 M I 0.25 M , 1 M / 0.5 M , 0.75 M / 0.5 M , 0.75 M I 0.75 Mの場合は、 1M CHsCOONa とほぼ同様もしくはそれ以上の腐植除去率を得られることが明らか となった。 また図 32に示されるように、 PEG溶液により回収した土壌 DNAの 純度も、 同様に従来用いられている NaClを CTABによる精製時に使用したもの よりも、高い純度が得られていた。図 33に示されるように、 CH3COONaと NaCl の混合液を使用すると従来用いられている NaCl単独の場合よりも DNAの回収 率が高くなつていた。 検討した条件においては CTAB による精製時の塩組成が CHsCOONa I NaCl で 0.25 M I 0.75 M , 0.67 M I 0.33 M の場合の効果が DNA の純度、 回収率の両方において最も高いと考えられた。 Even when using a mixture of NaCl and CH 3 COONa in salt solution than 31, added salt composition after pressure is CH 3 COONa I NaCl in 0.67 MI 0.33 M, 0.75 M /0.25 M, 1 MI 0.25 M, In the case of 1 M / 0.5 M, 0.75 M / 0.5 M, 0.75 MI 0.75 M, it was clarified that the humus removal rate was almost the same as or higher than that of 1M CHsCOONa. In addition, as shown in FIG. 32, the purity of the soil DNA recovered by the PEG solution was also higher than that of the conventionally used NaCl used for purification by CTAB. As shown in FIG. 33, the recovery of DNA was higher when a mixed solution of CH 3 COONa and NaCl was used than when NaCl alone was used conventionally. Under the conditions studied, it was considered that the effect when the salt composition during purification by CTAB was 0.25 MI 0.75 M and 0.67 MI 0.33 M with CHsCOONa I NaCl was the highest in both DNA purity and recovery.
〔実施例 18〕 アル力リ緩衝能を持つ PEG溶液の DNAの沈殿回収への使用 土壌からの DNA抽出にともなう夾雑物はほとんどが腐植酸であると考えられ るが、 腐植酸は先に述べたように酸性で沈殿し、 アルカリ性では再びイオン化し て水溶液に溶解する性質を持つ。 [Example 18] Use of PEG solution with buffer capacity for DNA precipitation recovery of DNA DNA from soil is thought to be mostly humic acid. As described above, it has the property of precipitating under acidic conditions and ionizing again under alkaline conditions and dissolving in an aqueous solution.
CTABによる精製時に酸性緩衝能をもった塩溶液を添加することにより、 腐植 物質の除去率を高めることができた。 しかし、その後の PEGによる DNAの沈殿 回収の際には除去しきれなかった腐植物質が DNAとともに沈殿してしまってお り、 この除去が必要である。 そこで CTABによ ¾精製時に低下させた pHを、 PEGによる DNAの沈殿回収 時に再び上昇させることにより、 腐植物質を再びイオン化させ、 腐植物質がより 沈殿しにくい条件に調製して、 DNA のみを選択的に沈殿させることが可能であ るか否かを検討した。 pH を上昇させるためには、 アルカリ性の緩衝能を持った 溶液が必要である。 PEG溶液は水溶液であるので、 PEG とアルカリ緩衝能を持 つ試薬を溶解し、 一つの溶液として作成して使用することを試みた。 The removal rate of humic substances could be increased by adding a salt solution having an acidic buffering capacity during purification by CTAB. However, in the subsequent precipitation of DNA by PEG, humic substances that could not be completely removed have precipitated together with the DNA, and this must be removed. Therefore, the pH that was reduced during purification by CTAB was increased again during the precipitation and recovery of DNA by PEG, so that the humic substances were ionized again and adjusted to conditions under which humic substances were less likely to precipitate, and only DNA was selected. It was examined whether it was possible to precipitate sedimentarily. To raise the pH, a solution with an alkaline buffering capacity is required. Since the PEG solution is an aqueous solution, an attempt was made to dissolve the PEG and a reagent having an alkaline buffering capacity, and to prepare and use the solution as a single solution.
アルカリ緩衝能を持つ試薬としては Tris (トリスヒドロキシァミノメタン） や CAPS、 CAPSO、 CHES、 TAPS, Bicine などが挙げられるが、 今回の実験では 最もよく使用されている Trisを使用した。 Tris (trishydroxyaminoaminomethane), CAPS, CAPSO, CHES, TAPS, and Bicine are examples of reagents with alkaline buffering ability. In this experiment, the most commonly used Tris was used.
(1) 材料と方法 (1) Materials and methods
実施例 16と同様に土壌 DNAを抽出し、 CTABによる精製処理をおこなった。 この精製処理の時には NaClに加えて、 酸性緩衝能を持った CH3COONaおよび NaClと CH3COONaの混合液を塩溶液として実施例 17と同様の条件で使用した。 Soil DNA was extracted and purified by CTAB in the same manner as in Example 16. At the time of this purification treatment, in addition to NaCl, CH 3 COONa having an acidic buffer capacity and a mixed solution of NaCl and CH 3 COONa were used as a salt solution under the same conditions as in Example 17.
CTABにより精製し、 クロ口ホルムを等量添加、 vortex, 遠心後の水相を採取 し、この溶液 750 /A lに対し 12% PEG溶液もしくはアル力リ緩衝能を持った PEG 溶液として 12% PEG / 3M Tris-HCl (pH 8.6)を等量添加し、 20000 X g 4 20分 間遠心を行い得られた土壌 DNAの沈殿を 70% エタノールで洗浄および乾燥後、 200 lの TE buffer (pH 8.0)に溶解させ、 土壌 DNA溶液とした。  Purify by CTAB, add an equal volume of black-mouthed form, vortex, collect the aqueous phase after centrifugation, and add 12% PEG solution or PEG solution with buffer capacity to 750 / Al. Add an equal volume of PEG / 3M Tris-HCl (pH 8.6), centrifuge at 20,000 X g for 20 minutes, wash and dry the resulting sediment of soil DNA with 70% ethanol, and add 200 l of TE buffer (pH 8.0) to give a soil DNA solution.
こうして獰られた土壌 DNA溶液を適宜希釈して、 400 ηπιにおける吸光度を測 定した結果を図 34に示す。 またこれらのアルカリ緩衝能を持つ PEG溶液の使用 が土壌 DNAの回収率に与える影響を図 35に示す。  FIG. 34 shows the results obtained by appropriately diluting the fermented soil DNA solution and measuring the absorbance at 400 ηπι. Figure 35 shows the effect of using these PEG solutions with alkaline buffering capacity on soil DNA recovery.
またアル力リ緩衝能を持つ PEG溶液のより詳細な条件検討として、 1% SDS I 200 mM EDTA/ 375 mM Na2HP04 (pH 8.6) で抽出した土壌 DNA抽出液につ いて上記の実験と同様の操作を行い、 PEG溶液として 12 % PEG , 12% PEG / 3 M Tris-HCl (pH 8.6)， 12% PEG I 2 M Tris-HCl (pH 8.6) , 12% PEG I 1.5 M Tris-HCl (pH 8.6)を使用した場合に得られる土壌 DNA溶液の純度についての結 果を図 36に示す。 また回収率について図 37に示す。 (2) 結果と考察 As a more detailed examination of conditions PEG solution with Al force re buffer capacity, 1% SDS I 200 mM EDTA / 375 mM Na 2 HP0 4 (pH 8.6) extracted by had soil DNA extract Nitsu in the above experiment and Perform the same operation as PEG solution, 12% PEG, 12% PEG / 3 M Tris-HCl (pH 8.6), 12% PEG I 2 M Tris-HCl (pH 8.6), 12% PEG I 1.5 M Tris-HCl Figure 36 shows the results for the purity of the soil DNA solution obtained when (pH 8.6) was used. Figure 37 shows the recovery rate. (2) Results and discussion
図 34よりアル力リ緩衝能をもつた 12% PEG I 3M Tris-HCl (pH 8.6)の土壌 DNAの沈殿回収への使用により CTABによる精製時のいずれの条件においても 従来用いられている方法に比較して、 DNA の純度が高くなつていることが明ら かとなつた。 '  From Fig. 34, the use of 12% PEG I 3M Tris-HCl (pH 8.6) with buffer capacity for the precipitation and recovery of soil DNA allows the conventional method to be used under any conditions during purification by CTAB. In comparison, it became clear that the purity of the DNA was higher. '
図 35 よりアルカリ緩衝能を持った PEG溶液は精製後の. DNA抽出液からの DNA回収に使用した場合、 通常の PEG溶液と回収率は全く変わらないことが明 らかとなつた。  From Fig. 35, it was clarified that when the PEG solution with alkaline buffering ability was used for DNA recovery from the purified DNA extract, the recovery rate was not different from that of the normal PEG solution at all.
以上のことからアル力リ緩衝能を持った PEG溶液は通常の PEG溶液と DNA を沈殿回収させる能力は全く同様であるが、 腐植物質をほとんど沈殿させないこ とを明らかにすることができた。  From the above, it was clarified that the PEG solution having the buffer capacity was almost the same as a normal PEG solution in the ability to precipitate and recover DNA, but hardly precipitated humic substances.
CTAB処理時に酸性緩衝能を持つ塩溶液を使用することによる pHを低下させ る操作と組み合わせることで、 純度が最も高い DNAを得ることができた。  The highest purity DNA could be obtained by combining it with the operation of lowering the pH by using a salt solution having acidic buffer capacity during CTAB treatment.
またアルカリ緩衝能を持った PEG溶液については、 図 36より、 アルカリ緩衝 能を持たない PEG溶液と比較して、純度の高い DNAが得られた。 CTABによる 精製時の塩条件として採用した 0.67 M CH3COONa / 0.33 M NaClで CTABによ る精製処理を行った土壌 DNA抽出液に対しては、 12% PEG / 3 M Tris-HCl (pH 8.6) , 12% PEG I 2 M Tris-HCl (pH 8.6) , 12% PEG 1 1.5 M Tris-HCl (pH 8.6)の いずれの条件であっても純度の高い DNAが得られた。 In addition, as shown in FIG. 36, the PEG solution having the alkaline buffering ability showed higher purity than the PEG solution having no alkaline buffering ability. 12% PEG / 3 M Tris-HCl (pH 8.6) was used for the soil DNA extract purified by CTAB with 0.67 M CH 3 COONa / 0.33 M NaCl, which was used as the salt condition for purification by CTAB. ), 12% PEG I 2 M Tris-HCl (pH 8.6), and 12% PEG 1 1.5 M Tris-HCl (pH 8.6).
図 37より、 CTABによる精製時の塩条件として採用した 0.67 M CH3COONa / 0.33 M NaClで CTABによる精製処理を行つた土壌 DNA抽出液に対しては、 12% PEG I 3 M Tris-HCl (pH 8.6) , 12% PEG I 2 M Tris-HCl (pH 8.6) , 12% PEG I 1.5 M Tris-HCl (pH 8.6)のいずれであっても DNAの回収率はほぼ変わらないこ とが明らかとなった。 From Fig. 37, the soil DNA extract purified by CTAB with 0.67 M CH 3 COONa / 0.33 M NaCl, which was adopted as the salt condition for purification by CTAB, was 12% PEG I 3 M Tris-HCl ( pH 8.6), 12% PEG I 2 M Tris-HCl (pH 8.6), and 12% PEG I 1.5 M Tris-HCl (pH 8.6). became.
〔実施例 19〕 抽出法の追加条件 · [Example 19] Additional conditions for extraction method
高分子の DNAは、 DNAの損傷が少なく、 クローニングやより長い塩基配列を 解析の対象にする研究に必要である。 高分子 DNAを得るためには物理的なせん 断を避けるため BeadsBeating法ではなく、 加熱処理という緩やかな条件で土壌 を処理することが望ましい。 High-molecular-weight DNA requires less DNA damage and is needed for cloning and studies that target longer nucleotide sequences. No physical way to get high molecular weight DNA It is desirable to treat the soil under mild conditions such as heat treatment instead of the BeadsBeating method to avoid cutting.
土壌に抽出液を添加し、 長時間加熱処理を行って抽出した土壌 DNA抽出液に は腐植物質が大量に混入するため腐植物質の除去が困難であつたが、 前述の新た に改良した DNA'の精製法によりほぼ完全な腐植物質の除去を達成でき、 加熱処 理法による土壌 DNA抽出を行うことが可能となった。  It was difficult to remove humic substances because the humic substances were mixed in a large amount in the soil DNA extract extracted by adding the extract to the soil and performing heat treatment for a long period of time. Almost complete removal of humic substances was achieved by the purification method, and soil DNA extraction by the heat treatment method became possible.
そこで本実施例では、加熱抽出により DNA抽出を行うための条件を検討した。 (1) 材料と方法  Therefore, in this example, conditions for performing DNA extraction by heat extraction were examined. (1) Materials and methods
土壌として東京大学農学部 弥生圃場対照区土壌、 東京大学附属農場 田無牧 草地土壌、 栃木農試 森林土壌、 埼玉農試 畑土壌、 兵庫農試 畑土壌を対象に 加熱処理による土壌 DNA抽出実験を行った。 抽出液としては弥生圃場対照区土 壌からの抽出には以下の組成の抽出液を使用した。  Soil DNA extraction experiments by heat treatment were performed on soil at the Yayoi Farm Control Area, Faculty of Agriculture at the University of Tokyo, Tanashi Maki Grassland Soil, Tochigi Agricultural Forest Soil, Saitama Agricultural Experimental Soil, and Hyogo Agricultural Experimental Soil. . As the extract, an extract having the following composition was used for extraction from the soil of the control plot in the Yayoi field.
1% SDS I 200 mM EDTA (ρΗ8.6) ,  1% SDS I 200 mM EDTA (ρΗ8.6),
1% SDS I 400 mM EDTA (pH8.6)，  1% SDS I 400 mM EDTA (pH8.6),
1% SDS I 250 mM Na2HP04 (pH8.6) , 1% SDS I 250 mM Na 2 HP0 4 (pH8.6),
1% SDS I 375 mM Na2HP04 (pH8.6), 1% SDS I 375 mM Na 2 HP0 4 (pH8.6),
1% SDS I 500 mM Na2HP04 (pH8.6) , 1% SDS I 500 mM Na 2 HP0 4 (pH8.6),
1% SDS 1 100 mM Tris-HCl I 100 mM EDTA / 100 mM Na2HP04 (pH8.6) ,1% SDS 1 100 mM Tris- HCl I 100 mM EDTA / 100 mM Na 2 HP0 4 (pH8.6),
1% SDS / 200 mM EDTA / 250 mM Na2HP04 (pH8.6), 1% SDS / 200 mM EDTA / 250 mM Na 2 HP0 4 (pH8.6),
1% SDS I 200 mM EDTA I 375 mM Na2HP04 (pH8.6) , 1% SDS I 200 mM EDTA I 375 mM Na 2 HP0 4 (pH8.6),
1% SDS I 200 mM EDTA / 500 mM Na2HP04 (pH8.6), 1% SDS I 200 mM EDTA / 500 mM Na 2 HP0 4 (pH8.6),
1% SDS I 400 mM EDTA / 250 mM Na2HP04 (pH8.6) , 1% SDS I 400 mM EDTA / 250 mM Na 2 HP0 4 (pH8.6),
1% SDS I 400 mM EDTA I 375 mM Na2HP04 (pH8.6), 1% SDS I 400 mM EDTA I 375 mM Na 2 HP0 4 (pH8.6),
1% SDS I 400 mM EDTA / 500 mM Na2HP04 (pH8.6)， 1% SDS I 400 mM EDTA / 500 mM Na 2 HP0 4 (pH8.6),
1% SDS I 400 mM EDTA I 750 mM Na2HP04 (pH8.6) その他の土壌については、 以下の組成の抽出液を使用した。 The 1% SDS I 400 mM EDTA I 750 mM Na 2 HP0 4 (pH8.6) other soil was used an extract of the following composition.
1% SDS 1 100 mM Tris-HCl /100 mM EDTA / 100 mM Na2HPO4 (pH8.6) , 1% SDS I 200 mM EDTA / 250 mM Na2HP04 (pH8.6), 1% SDS 1 100 mM Tris-HCl / 100 mM EDTA / 100 mM Na 2 HPO 4 (pH8.6), 1% SDS I 200 mM EDTA / 250 mM Na 2 HP0 4 (pH8.6),
1% SDS I 200 mM EDTA / 375 mM Na2HP04 (pH8.6)， 1% SDS I 200 mM EDTA / 375 mM Na 2 HP0 4 (pH8.6),
1% SDS I 200 mM EDTA / 500 mM Na2HP04 (pH8.6), 1% SDS I 200 mM EDTA / 500 mM Na 2 HP0 4 (pH8.6),
1% SDS I 400 mM EDTA / 250 mM Na2HP0 (pH8.6) , 1% SDS I 400 mM EDTA / 250 mM Na 2 HP0 (pH8.6),
1% SDS I 400 mM EDTA I 375 mM Na2HP04 (pH8.6), 1% SDS I 400 mM EDTA I 375 mM Na 2 HP0 4 (pH8.6),
1% SDS I 400 mM EDTA / 500 mM Na2HP04 (pH8.6) , 1% SDS I 400 mM EDTA / 500 mM Na 2 HP0 4 (pH8.6),
1% SDS I 400 mM EDTA I 750 mM Na2HP04 (pH8.6) 土壌 0.5 gに対し、 抽出液 1.2 mlを添加し、 適宜攪拌しながら 65°Cにおいて 1 時間加熱処理を行った。その後 12000 X g 25t 5分間の遠心により上清を得た。 この上清 750 1に対し 10% CTAB溶液 250 1および 3.33 M CH3COONa / 1.67 M NaCl溶液を 250 1添カ卩し、 vortex後等量のクロ口ホルムを添加し、 vortex 後 12000 X g 25°C 20分間の遠心を行った。 水相を回収し等量の 12 % PEG / 1.5 M Tris-HCl (pH 8.6)を添加し、 vortex後、 20000 X g 4°C 20分間遠心を行い、 土壌 DNAの沈殿を回収し、 70% エタノールで洗浄および乾燥後、 200 lの TE buffer (pH 8.0)に溶解させ、 土壌 DNA溶液とした。 To 1% SDS I 400 mM EDTA I 750 mM Na 2 HP0 4 (pH8.6) soil 0.5 g, extract was added 1.2 ml, was carried out for 1 hour heat treatment at 65 ° C with stirring as appropriate. Thereafter, a supernatant was obtained by centrifugation at 12000 × g for 25 minutes at 25 t. Add 10% CTAB solution 250 1 and 3.33 M CH 3 COONa / 1.67 M NaCl solution to 250 1 of this supernatant 750, add vortex, add an equal amount of clonal form, and after vortex 12000 X g 25 Centrifugation was performed at ° C for 20 minutes. Collect the aqueous phase, add an equal volume of 12% PEG / 1.5 M Tris-HCl (pH 8.6), vortex, centrifuge at 20000 X g at 4 ° C for 20 minutes to collect the soil DNA precipitate, After washing with ethanol and drying, it was dissolved in 200 l of TE buffer (pH 8.0) to obtain a soil DNA solution.
弥生圃場対照区土壌を用いて様々な組成の抽出液を用いて抽出した DNA抽出 量について図 38 に示す。 絞り込んだ条件について弥生圃場対照区土壌、 田無牧 草地土壌、 栃木農試 森林土壌、 埼玉農試 .畑土壌、 兵庫農試 畑土壌から抽出 した DNA抽出量についての結果を図 39に示す。  Figure 38 shows the amount of DNA extracted using extracts of various compositions using soil from the Yayoi field control plot. Fig. 39 shows the results of the extracted conditions for the DNA extraction amounts extracted from the Yayoi field control plot soil, the Tanashi pasture soil, the Tochigi agricultural test forest soil, the Saitama agricultural test field soil, and the Hyogo agricultural test field soil.
(2) 結果と考察 (2) Results and discussion
図 38より、 以下の抽出液において、 抽出量が高いことが明らかとなった。 . 1% SDS I 250 mM Na2HPO4 (pH8.6)， From Fig. 38, it was clarified that the amount of extraction was higher in the following extracts. 1% SDS I 250 mM Na 2 HPO 4 (pH8.6),
1% SDS I 375 mM Na2HP04 (pH8.6) , 1% SDS I 375 mM Na 2 HP0 4 (pH8.6),
1% SDS 1 100 mM Tris-HCl I 100 mM EDTA / 100 mM Na2HP04 (pH8.6) , 1% SDS /200 mM EDTA / 250 mM Na2HP04 (pH8.6) また図 39より 1% SDS I 200 mM EDTA / 250 mM Na2HP04 (pH8.6)の組成が どの土壌においても高い収量が得られることが明らかとなった。 1% SDS 1 100 mM Tris- HCl I 100 mM EDTA / 100 mM Na 2 HP0 4 (pH8.6), 1% SDS / 200 mM EDTA / 250 mM Na 2 HP0 4 (pH8.6) Also that a higher yield in any soil composition of 39 than 1% SDS I 200 mM EDTA / 250 mM Na 2 HP0 4 (pH8.6) obtained revealed.
〔実施例 20〕 土壌以外の環境サンプルからの DNA [Example 20] DNA from environmental samples other than soil
土壌以外の微生物を含む環境サンプルとしては、 人や家畜の糞便、 堆肥、 活性 汚泥、 湖底など水系の堆積物などが挙げられる。 '  Environmental samples containing microorganisms other than soil include feces of humans and livestock, compost, activated sludge, and water-based sediments such as lake bottoms. '
本実施例では、 土壌をサンプルとして開発した DNA抽出法によりその他の環 境サンプルからも DNAが抽出できるかを検討した。 (1) 糞便からの DNA抽出  In this example, it was examined whether DNA could be extracted from other environmental samples by the DNA extraction method developed using soil as a sample. (1) DNA extraction from feces
成人男子 2名より、 3 日間サンプリングを行った糞便サンプルから DNA抽出 を行つた。  DNA extraction was performed on stool samples sampled for three days from two adult males.
サンプルは 6種類: R-l、 R-2、 R-3、 N-l、 N_2、 Ν·3である。  There are six kinds of samples: R-l, R-2, R-3, N-l, N_2, Ν · 3.
まずサンプル R- 1を用いて抽出液の組成を検討した。抽出液は以下の組成のも のを用い、 BeadsBeatingおよび加熱抽出による抽出法の両方を検討した。  First, the composition of the extract was examined using sample R-1. The extract used had the following composition and both BeadsBeating and extraction by heating were examined.
1% SDS I 100 mM Tris-HCl I 200 mM EDTA I 500 mM Na2HPO4 (pH 8.6) , 1% SDS 1 100 mM Tris-HCl I 200 mM EDTA I 250 mM Na2HPO4 (pH 8.6) , 1% SDS I 100 mM Tris-HCl I 100 mM EDTA / 100 mM Na2HP04 (pH 8.6)， 1% SDS I 100 mM Tris-HCl I 200 mM EDTA (pH 8.6) , 1% SDS I 100 mM Tris-HCl I 200 mM EDTA I 500 mM Na 2 HPO 4 (pH 8.6), 1% SDS 1 100 mM Tris-HCl I 200 mM EDTA I 250 mM Na 2 HPO 4 (pH 8.6), 1% SDS I 100 mM Tris- HCl I 100 mM EDTA / 100 mM Na 2 HP0 4 (pH 8.6), 1% SDS I 100 mM Tris-HCl I 200 mM EDTA (pH 8.6),
1% SDS I 100 mM Tris-HCl I 100 mM EDTA (pH 8.6) ,  1% SDS I 100 mM Tris-HCl I 100 mM EDTA (pH 8.6),
1% SDS I 100 mM Tris-HCl I 50 inM EDTA (pH 8.6) ,  1% SDS I 100 mM Tris-HCl I 50 inM EDTA (pH 8.6),
1% SDS I 100 mM Tris-HCl 1 10 mM EDTA (pH 8.6)  1% SDS I 100 mM Tris-HCl 1 10 mM EDTA (pH 8.6)
糞便サンプル 0.5 gに抽出液 1.2mlを添加し、 BeadsBeatingによる方法は 4m I secで 30秒間の BeadsBeating処理を行い、その後 12000 X g 25で 5分間の遠 心により上清を得た。この上清 750 lに対し 10% CTAB溶液 250 1および 3.33 M CH3COONa 1 1.67 M NaCl溶液を 250 1添カ卩し、 vortex後等量のクロ口ホル ムを添加し、 vortex後 12000 X g 25 20分間の遠心を行った。 水相を回収し 等量の 12 % PEG / 1.5 M Tris-HCl (pH 8.6)を添加し、 vortex後、 20000 X g 4で 20分間遠心を行い、 DNAの沈殿を回収し、 70% ェタノ一ルで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、 糞便 DNA溶液とした。 To the stool sample (0.5 g) was added 1.2 ml of the extract, BeadsBeating was performed at 4 ml sec for 30 seconds, and the supernatant was obtained by centrifugation at 12000 X g25 for 5 minutes. To 750 l of this supernatant, add 250% of 10% CTAB solution 250 1 and 3.33 M CH 3 COONa 1 1.67 M NaCl solution, add vortex and add an equal amount of cloak form, and add 12000 X g after vortex Centrifugation was performed for 25 to 20 minutes. Collect the aqueous phase, add an equal volume of 12% PEG / 1.5 M Tris-HCl (pH 8.6), vortex, and The precipitate of DNA was collected by centrifugation for 20 minutes, washed with 70% ethanol, dried, and dissolved in TE buffer (pH 8.0) to obtain a fecal DNA solution.
加熱処理法による抽出は 65 において 1時間ィンキュベート処理を行い、その 後 12000 Xg25°C 5分間の遠心により上清を得た。この上清 750 lに対し 10% CTAB溶液 2501 lぉょび3.33MCH3COONa/1.67MNaCl溶液を 250 l添加 し、 vortex後等量のクロ口ホルムを添加し、 vortex後 12000Xg 25°C 20分間 の遠心を行った。 水相を回収し等量の 12 % PEG / 1.5 M Tris-HCl (pH 8.6)を添 力！]し、 vortex後、 20000 Xg4°C 20分間遠心を行い、 DNAの沈殿を回収し、 70% エタノールで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、 糞便 DNA溶液 とした。 Extraction by a heat treatment method was performed by incubating at 65 ° C. for 1 hour, and then centrifuged at 12000 × g at 25 ° C. for 5 minutes to obtain a supernatant. The supernatant 750 l to 10% CTAB solution 250 1 l Oyobi 3.33MCH 3 COONa / 1.67MNaCl solution was added 250 l, was added an equal amount of black hole Holm After vortex, vortex after 12000Xg 25 ° C 20 Centrifugation was performed for minutes. Collect the aqueous phase and add an equal volume of 12% PEG / 1.5 M Tris-HCl (pH 8.6)! After vortexing, centrifugation was performed for 20 minutes at 20000 Xg at 4 ° C. The DNA precipitate was collected, washed and dried with 70% ethanol, dissolved in TE buffer (pH 8.0), and used as a fecal DNA solution.
それぞれの方法で得られた DNAの抽出量について図 40に示す。  Figure 40 shows the amount of DNA extracted by each method.
また BeadsBeating法、加熱抽出法ともに 1。/。 SDS 1100 mM Tris-HCl I 50 mM 1 for both BeadsBeating method and heat extraction method. /. SDS 1100 mM Tris-HCl I 50 mM
EDTA (pH 8.6)を用いて上記の同様の操作でその他のサンプルからも抽出を行つ た。 Extraction was performed from other samples using EDTA (pH 8.6) in the same manner as described above.
得られた糞便 DNAについての電気泳動の結果を図 41に示す。  FIG. 41 shows the results of electrophoresis of the obtained fecal DNA.
また従来法と純度、 収量の点で比較を行った。 従来法は 1% SDS I 100 mM Tris-HCl / 50 mM EDTA (pH 8.6) の抽出液で 65°Cにおける 1時間インキュベー ト処理で加熱抽出を行い、 その後 12000Xg 25°C 5分間の遠心により上清を得 た。 この上清に対し、 等量のクロ口ホルムを添加し、 vortex後 12000 Xg 25°C 20分間の遠心を行った。水相を回収し 0.6倍量の 2-プロパノ一ルを添加し、 vortex 後、 20000Xg4°C 20分間遠心を行い、 DNAの沈殿を回収し、 70% エタノール で洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、 糞便 DNA溶液とした。  In addition, a comparison was made with the conventional method in terms of purity and yield. In the conventional method, 1% SDS I 100 mM Tris-HCl / 50 mM EDTA (pH 8.6) extract was used to perform heat extraction by incubating at 65 ° C for 1 hour, and then centrifuged at 12000Xg at 25 ° C for 5 minutes. I got Qing. To this supernatant, an equal volume of black-mouthed form was added, and after vortexing, centrifugation was performed at 12000 Xg at 25 ° C for 20 minutes. Collect the aqueous phase, add 0.6 volumes of 2-propanol, vortex, centrifuge at 20000Xg4 ° C for 20 minutes, collect the DNA precipitate, wash and dry with 70% ethanol, and add TE buffer (pH 8.0) to give a fecal DNA solution.
BeadsBeating法、加熱抽出法、従来法により得られた糞便 DNAの収量につい ての結果を図 42、 純度についての結果を図 43に示す。  Figure 42 shows the results for the yield of fecal DNA obtained by the BeadsBeating method, the heat extraction method, and the conventional method, and Figure 43 shows the results for the purity.
(2) 結果と考察 (2) Results and discussion
図 40より、 BeadsBeating法、 加熱抽出法ともに 1% SDS 1100 mM Tris-HCl / 50 mM EDTA (pH 8.6)による抽出量が最も多かった。 図 42より、 収量め面では従来法よりも今回開発した BeadsBeating法、 加熱 抽出法による DNAの抽出量は低いことが明らかとなった。 しかし、 今回開発し た BeadsBeating法、 加熱抽出法は精製操作を一連の操作として行っており、 そ の操作の過程での一部 DNAの損失がある。また今回開発した方法では PEG溶液 により DNAを回収しているため RNAは沈殿しないのに対し、 従来法では DNA の沈殿に 2-プロパノールを使用しているため、 RNA も沈殿させてしまう。 従つ て、見かけ上は、 この RNAが DNAの定量結果に影響を及ぼし収量に差が生じた ものと考えられる。 According to FIG. 40, the extraction amount with 1% SDS 1100 mM Tris-HCl / 50 mM EDTA (pH 8.6) was the largest in both the BeadsBeating method and the heat extraction method. From Fig. 42, it became clear that the amount of DNA extracted by the newly developed BeadsBeating method and the heat extraction method was lower than that of the conventional method in terms of yield. However, in the BeadsBeating method and the heat extraction method developed this time, the purification operation is performed as a series of operations, and some DNA is lost during the operation. In the method developed this time, RNA is not precipitated because the DNA is recovered using the PEG solution, whereas in the conventional method, RNA is also precipitated because 2-propanol is used for DNA precipitation. Therefore, apparently, this RNA affected the results of DNA quantification, resulting in a difference in yield.
図 43より、 従来法に比較して、 今回開発した BeadsBeating法、 加熱抽出法 で抽出した糞便 DNAの純度ははるかに高いことが明らかとなった。  Figure 43 shows that the purity of fecal DNA extracted by the BeadsBeating method and the heat extraction method developed this time is much higher than that of the conventional method.
これにより、今回開発した DNAの精製、沈殿方法は糞便サンプルから得た DNA 抽出液から選択的に DNAのみを取り出すことに極めて有効であることが示され た。 〔実施例 21〕 堆肥および活性汚泥からの DNA抽出  This demonstrates that the newly developed DNA purification and precipitation methods are extremely effective in selectively extracting only DNA from DNA extracts obtained from stool samples. [Example 21] DNA extraction from compost and activated sludge
本実施例では、 堆肥および活性汚泥サンプルから DNA抽出を行った。  In this example, DNA was extracted from compost and activated sludge samples.
サンプルは堆肥が 5種類 落ち葉堆肥、 牛糞バラ堆肥、 醱酵牛糞堆肥、 鶏糞堆 肥、腐葉土、活性汚泥サンプルが 3種類 Kanagawa， Ochiail , Ochiai2である。 まず落ち棄堆肥を用いて抽出液の組成を検討した。抽出液は以下の通りである。 1% SDS I 100 mM Tris-HCl I 200 mM EDTA I 500 mM Na2HP04 (pH 8.6) , 1% SDS I 100 mM Tris-HCl I 200 mM EDTA / 250 mM Na2HPO4 (pH 8.6)， 1% SDS I 100 mM Tris-HCl I 100 mM EDTA / 100 mM Na2HP04 (pH 8.6)， 1% SDS I 100 mM Tris-HCl I 200 mM EDTA (pH 8.6) , There are five types of compost: fallen leaf compost, cow dung rose compost, fermented cow dung compost, chicken dung compost, humus, and activated sludge. Three types of samples are Kanagawa, Ochiail, and Ochiai2. First, the composition of the extract was examined using abandoned compost. The extract is as follows. 1% SDS I 100 mM Tris- HCl I 200 mM EDTA I 500 mM Na 2 HP0 4 (pH 8.6), 1% SDS I 100 mM Tris-HCl I 200 mM EDTA / 250 mM Na 2 HPO 4 (pH 8.6), 1% SDS I 100 mM Tris- HCl I 100 mM EDTA / 100 mM Na 2 HP0 4 (pH 8.6), 1% SDS I 100 mM Tris-HCl I 200 mM EDTA (pH 8.6),
1% SDS I 100 mM Tris-HCl 1 100 mM EDTA (pH 8.6) ,  1% SDS I 100 mM Tris-HCl 1 100 mM EDTA (pH 8.6),
1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6)，  1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6),
1% SDS 1 100 mM Tris-HCl 1 10 mM EDTA (pH 8.6) 上記抽出液を用い、 BeadsBeating による抽出法と加熱抽出による抽出法の両 方を検討した。 1% SDS 1 100 mM Tris-HCl 1 10 mM EDTA (pH 8.6) Using the above extract, both extraction by BeadsBeating and extraction by heat Was considered.
堆肥サンプル 0.5 gに抽出液 1.2mlを添加し、 BeadsBeatingによる方法は 4m I secで 30秒間の BeadsBeating処理を行い、その後 12000 X g 25°C 5分間の遠 心により上清を得た。この上清 750 1に対し 10% CTAB溶液 250 1および 3.33 MCH3COONa/ϊ.67MNaCl溶液を 250 l添加し、 vortex後等量のクロ口ホル ムを添加し、 vortex後 12000 Xg 25°C 20分間の遠心を行った。 水相を回収し 等量の 12 % PEG/ 1.5 M Tris-HCl (pH 8.6)を添加し、 vortex後、 20000Xg4。C 20分間遠心を行い、 DNAの沈殿を回収し、 70% ェ夕ノ一ルで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、 堆肥 DNA溶液とした。 To a 0.5 g compost sample, 1.2 ml of the extract was added, and BeadsBeating was performed at 4 ml sec for 30 seconds, followed by centrifugation at 12000 xg at 25 ° C for 5 minutes to obtain a supernatant. Add 250 l of 10% CTAB solution 250 1 and 3.33 MCH 3 COONa / ϊ.67 M NaCl solution to 750 1 of this supernatant, add an equal amount of cloche form after vortex, and 12000 Xg 25 ° C 20 after vortex Centrifugation was performed for minutes. The aqueous phase was collected, an equal volume of 12% PEG / 1.5 M Tris-HCl (pH 8.6) was added, and after vortexing, 20000Xg4. C The DNA precipitate was collected by centrifugation for 20 minutes, washed and dried with 70% ethanol, dissolved in TE buffer (pH 8.0), and used as a compost DNA solution.
加熱処理法による抽出は 65°Cにおいて 1時間ィンキュベート処理を行い、その 後 12000Xg25°C 5分間の遠心により上清を得た。この上清 750 lに対し 10% CTAB溶液 250 /X 1および 3.33 M CH3COONa 11.67 M NaCl溶液を 250 1添加 し、 vortex後等量のクロ口ホルムを添加し、 vortex後 12000Xg 25°C 20分間 の遠心を行った。 水相を回収し等量の 12 % PEG I 1.5 M Tris-HCl (pH 8.6)を添 加し、 vortex後、 20000 4 20分間遠心を行い、 DNAの沈殿を回収し、 70% ェ夕ノールで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、 堆肥 DNA溶液 とした。 . Extraction by the heat treatment method was performed by incubating at 65 ° C for 1 hour, and then centrifuged at 12000Xg at 25 ° C for 5 minutes to obtain a supernatant. The supernatant 750 l of 10% CTAB solution 250 / X 1 and 3.33 M CH 3 COONa 11.67 M NaCl solution 250 1 was added to, was added an equal amount of black hole Holm After vortex, vortex after 12000Xg 25 ° C 20 Centrifugation was performed for minutes. Collect the aqueous phase, add an equal volume of 12% PEG I 1.5 M Tris-HCl (pH 8.6), vortex, centrifuge at 20000 for 20 minutes, collect the DNA precipitate and collect with 70% ethanol. After washing and drying, it was dissolved in TE buffer (pH 8.0) to obtain a compost DNA solution. .
それぞれの方法で得られた DNAの抽出量について図 44に示す。  FIG. 44 shows the amount of DNA extracted by each method.
また BeadsBeating法、加熱抽出法ともに.1% SDS 1100 mM Tris-HCl/ 50 mM EDTA (pH 8.6)を用いて上記の同様の操作でその他のサンプルからも抽出を行つ た。  In addition, in both BeadsBeating method and heat extraction method, extraction was performed from other samples by the same operation as above using 0.1% SDS 1100 mM Tris-HCl / 50 mM EDTA (pH 8.6).
この際活性汚泥サンプルは懸濁状態であつたので 500 1 をサンプルとし、 BeadsBeating処理、 加熱抽出時に 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6)となるように、 500^1の 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6)を添加し、 BeadsBeating処理、 加熱処理を行って DNAを抽出した。 以 後の操作は堆肥サンプルと同様におこなった。  At this time, since the activated sludge sample was in a suspended state, 5001 was used as the sample, and 500 ^ 1 was used so that it became 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6) during BeadsBeating treatment and heat extraction. Of 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6) was added, and the DNA was extracted by performing BeadsBeating treatment and heating treatment. Subsequent operations were performed in the same manner as the compost sample.
得られた堆肥 DNA、活性汚泥 DNAについての電気泳動の結果を図 45に示す。 また従来法と純度、 収量の点で比較を行った。 従来法は 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6) の抽出液で 65°Cにおける 1時間インキュベート処理で加熱抽出操作を行い、 その後 12000 X g 25°C 5分間の遠心により上清を得た。 （活性汚泥サンプルについては上記のよ うに 2 % SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6)を等量添加して、 抽 出操作を行った。）'この上清に対し、等量のクロ口ホルムを添加し、 vortex後 12000 X g 25°C 20分間の遠心を行った。 水相を回収し 0.6倍量の 2-プロパノールを 添加し、 vortex後、 20000 X g 4°C 20分間遠心を行い、 DNAの沈殿を回収し、 70 % エタノールで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、堆肥 DNA 溶液、 活性汚泥 DNA溶液とした。 The results of electrophoresis of the obtained compost DNA and activated sludge DNA are shown in FIG. In addition, comparison was made with the conventional method in terms of purity and yield. In the conventional method, 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6) was used for extraction by heating at 65 ° C for 1 hour, followed by centrifugation at 12000 × g at 25 ° C for 5 minutes. To obtain a supernatant. (For the activated sludge sample, the extraction operation was performed by adding an equal amount of 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6) as described above.) After adding vortex, the mixture was centrifuged at 12000 X g at 25 ° C for 20 minutes. Collect the aqueous phase, add 0.6 volumes of 2-propanol, vortex, centrifuge at 20,000 X g at 4 ° C for 20 minutes, collect the DNA precipitate, wash and dry with 70% ethanol, and add TE buffer ( pH 8.0) to give a compost DNA solution and an activated sludge DNA solution.
BeadsBeating法、加熱抽出法、従来法により得られた堆肥 DNAの収量につい ての結果を図 46、 純度についての結果を図 47に示す。 また活性汚泥サンプルの 重量についての結果を図 48、 純度についての結果を図 49に示す。  Figure 46 shows the results for the yield of compost DNA obtained by the BeadsBeating method, the heat extraction method, and the conventional method, and Figure 47 shows the results for the purity. Fig. 48 shows the results for the weight of the activated sludge sample, and Fig. 49 shows the results for the purity.
(2) 結果と考察 (2) Results and discussion
図 44より、 BeadsBeating法では 1% SDS 1 100 mM Tris-HCl 1 100 mM EDTA I 100 mM Na2HP04 (pH 8.6) , 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6)による抽出量が多かった。 加熱抽出法ではそれほど大きな差はなかったが、 1% SDS I 100 mM Tris-HCl I 100 mM EDTA I 100 mM Na2HPO4 (pH 8.6)の抽 出量が最も多かった。 From FIG. 44, according to the 1% SDS 1 100 mM Tris- HCl 1 100 mM EDTA I 100 mM Na 2 HP0 4 (pH 8.6), 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6) at BeadsBeating method The amount of extraction was large. There was no significant difference in the heat extraction method, but the extraction amount of 1% SDS I 100 mM Tris-HCl I 100 mM EDTA I 100 mM Na 2 HPO 4 (pH 8.6) was the largest.
図 46より、 収量において従来法よりも今回開発した BeadsBeating法、 加熱 抽出法による堆肥からの DNA の抽出量は高いことが明らかとなった。 特に BeadsBeating法による抽出量は多く、 従来法との比較では 3倍から 6倍近い収 量が得られた。  From Fig. 46, it was clarified that the amount of DNA extracted from compost by the newly developed BeadsBeating method and heating extraction method was higher than that of the conventional method. In particular, the amount of extraction by the BeadsBeating method was large, and compared with the conventional method, the yield was almost three to six times.
図 48より、活性汚泥からの DNA抽出量は従来の方法とほとんど変わらないこ とが示された。  From Fig. 48, it was shown that the amount of DNA extracted from activated sludge was almost the same as the conventional method.
図 47より、 従来法に比較して、 今回開発した BeadsBeating法、 加熱抽出法 で堆肥から抽出した DNAの純度ははるかに高いことが明らかとなった。  From Fig. 47, it is clear that the purity of DNA extracted from compost by the BeadsBeating method and the heat extraction method developed this time is much higher than the conventional method.
図 49 より、 活性汚泥から抽出した DNA の純度についても今回開発した BeadsBeating法、加^抽出法により、 はるかに純度の高い DNAが得られること が明らかとなった。 From Fig. 49, the purity of DNA extracted from activated sludge was also developed this time. It was revealed that much higher purity DNA can be obtained by the BeadsBeating method and the extraction method.
以上のことから、 今回開発した DNAの精製、 沈殿方法は堆肥サンプル、 活性 汚泥サンプルから高収量で DNA を抽出することが可能であり、 選択的に DNA のみを抽出できる'ことが示された。  From the above, it was shown that the newly developed DNA purification and sedimentation method can extract DNA from compost samples and activated sludge samples with high yield, and can selectively extract only DNA.
〔実施例 22〕 湖底堆積物からの DNA抽出 [Example 22] DNA extraction from lake bottom sediments
本実施例では、 湖底堆積物サンプルから DNA抽出を行った。  In this example, DNA was extracted from a lake bottom sediment sample.
サンプルは 6種類 上野公園 不忍池の 3 ケ所 ， 東京大学構内 三四郎池 ， 東大農学部圃場 池 2ケ所より採取したものを使用した。  Six types of samples were collected from three locations at Ueno Park Shinobazu Pond, Sanshiro Pond on the premises of the University of Tokyo, and two farm ponds at the University of Tokyo Faculty of Agriculture.
湖底堆積物サンプルは懸濁状態であつたので 500 l をサンプルとし、 BeadsBeating処理、 加熱抽出時に 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6)となるように、 500 zlの 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6)を添加し、 BeadsBeating処理、 加熱処理を行って DNAを抽出した。  Since the lake bottom sediment sample was in a suspended state, 500 l of the sample was used.BeadsBeating treatment and 500 l of 500 zl were performed so that 1% SDS I 100 mM Tris-HCl I 50 mM EDTA (pH 8.6) was obtained during heat extraction. % SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6) was added, followed by BeadsBeating treatment and heat treatment to extract DNA.
BeadsBeatingによる方法は 4m / secで 30秒間の BeadsBeating処理を行い、 その後 12000 Xg25t 5分間の遠心により上清を得た。 この上清 750 1に対し 10% CTAB溶液 250 lぉょび3.33M CH3COONa/ l·67MNaCl溶液を 250 l 添加し、 vortex後等量のクロ口ホルムを添加し、 vortex後 12000 Xg 25°C 20 分間の遠心を行った。 水相を回収し等量の 12 % PEG I 1.5 M Tris-HCl (pH 8.6) を添加し、 vortex後、 20000X^4^： 20分間遠心を行い、 DNAの沈殿を回収し、 70% エタノールで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、 湖底堆積 物 DNA溶液とした。 In the method using BeadsBeating, BeadsBeating treatment was performed at 4 m / sec for 30 seconds, and then a supernatant was obtained by centrifugation at 12000 × g25t for 5 minutes. Add 250 l of 10% CTAB solution and 250 l of 3.33M CH 3 COONa / l67M NaCl solution to 750 1 of this supernatant, add vortex and equal amount of clonal form, and after vortex 12000 Xg 25 ° C Centrifugation was performed for 20 minutes. Collect the aqueous phase, add an equal volume of 12% PEG I 1.5 M Tris-HCl (pH 8.6), vortex, and centrifuge at 20000X ^ 4 ^: 20 minutes to collect the DNA precipitate. After washing and drying, the DNA was dissolved in TE buffer (pH 8.0) to obtain a lake bottom sediment DNA solution.
加熱処理法による抽出は 65°Cにおいて 1時間ィンキュベート処理を行い、その 後 12000Xg25°C 5分間の遠心により上清を得た。この上清 750 Uに対し 10% CTAB溶液 250 lぉょび3.33M CH3COONa/ 1.67MNaCI溶液を 250 l添加 し、 vortex後等量のクロ口ホルムを添加し、 vortex後 12000Xg 25°C 20分間 の遠心を行った。 水相を回収し等量の 12 % PEG I 1.5 M Tris-HCl (pH 8.6)を添 加し、 vortex後、 20000 Xg 4 20分間遠心を行い、 DNAの沈殿を回収し、 70% ェタノ一ルで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、湖底堆積物 DNA 溶液とした。 Extraction by the heat treatment method was performed by incubating at 65 ° C for 1 hour, and then centrifuged at 12000Xg at 25 ° C for 5 minutes to obtain a supernatant. Add 250 l of 10% CTAB solution and 250 l of 3.33 M CH 3 COONa / 1.67 M NaCI solution to 750 U of this supernatant, add vortex and equal volume of clonal form, and after vortex 12000 Xg 25 ° C 20 Centrifugation was performed for minutes. Collect the aqueous phase, add an equal volume of 12% PEG I 1.5 M Tris-HCl (pH 8.6), vortex, and centrifuge at 20000 Xg for 20 minutes to collect the DNA precipitate and collect 70% After washing with ethanol and drying, the residue was dissolved in TE buffer (pH 8.0) to obtain a lake bottom sediment DNA solution.
得られた湖底堆積物 DNAについての電気泳動の結果を図 50に示す。  Figure 50 shows the results of electrophoresis of the obtained lake sediment DNA.
また従来法と純度、 収量の点で比較を行った。  In addition, a comparison was made with the conventional method in terms of purity and yield.
従来法は上記ど同じくサンプル 500 lに 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6)を等量添加して、 65°C 1時間の加熱操作により抽出操作を 行った。 この上清に対し、 等量のクロ口ホルムを添加し、 vortex 後 12000 X g 25°C 20分間の遠心を行った。 水相を回収し 0.6倍量の 2-プロパノールを添加 し、 vortex後、 20000 X g 4°C 20分間遠心を行い、 DNAの沈殿を回収し、 70% ェ 夕ノールで洗浄および乾燥後、 TE buffer (pH 8.0)に溶解させ、 湖底堆積物 DNA 溶液とした。  In the conventional method, an extraction operation was performed by adding an equal amount of 2% SDS / 200 mM Tris-HCl / 100 mM EDTA (pH 8.6) to 500 l of the sample as described above and heating at 65 ° C. for 1 hour. To this supernatant, an equal amount of black-mouthed form was added. After vortexing, centrifugation was performed at 12000 X g at 25 ° C for 20 minutes. Collect the aqueous phase, add 0.6 volumes of 2-propanol, vortex, centrifuge at 20000 X g at 4 ° C for 20 minutes, collect the DNA precipitate, wash and dry with 70% ethanol, and add TE buffer (pH 8.0) to give a lake bottom sediment DNA solution.
BeadsBeating法、加熱抽出法、従来法により得られた湖底堆積物 DNAの収量 についての結果を図 51、 純度についての結果を図 52に示す。 (2) 結果と考察  Figure 51 shows the results for the yield of lake sediment DNA obtained by the BeadsBeating method, the heat extraction method, and the conventional method, and Figure 52 shows the results for the purity. (2) Results and discussion
図 51より、 収量において従来法よりも今回開発した BeadsBeating法、 加熱 抽出法による湖底堆積物からの DNAの抽出量は高いことが明らかとなった。 特 に BeadsBeating法による抽出量は多く、 従来法との比較では 3倍から 7倍近い 収量が得られた。  From Fig. 51, it is clear that the yield of DNA from lake sediments by the newly developed BeadsBeating method and the heat extraction method is higher than that of the conventional method. In particular, the amount of extraction by the BeadsBeating method was large, and the yield was nearly three to seven times that of the conventional method.
図 52より、 従来法に比較して、 今回開発した BeadsBeating法、 加熱抽出法 で堆肥から抽出した DNAの純度ははるかに高いことが明らかとなった。  From Fig. 52, it is clear that the purity of the DNA extracted from compost by the newly developed BeadsBeating method and the heat extraction method is much higher than that of the conventional method.
これにより今回開発した DNA抽出法は従来法よりも高収量で湖底堆積物から DNAを抽出可能であり、 精製、 沈殿方法は湖底堆積物サンプルから得た DNA抽 出液から選択的に DNAのみを取り出すことに極めて有効であることが示された。 以上のように今回開発した DNAの抽出精製法は土壌以外の環境サンプルから も DNAを高収量で抽出することが可能であり、 得られた DNAは従来法と比較 して極めて純度の高いものが得られ、 環境サンプル全般からの DNA抽出に適し ていると考えられた。 As a result, the newly developed DNA extraction method can extract DNA from lake sediment at higher yields than the conventional method, and the purification and sedimentation method selectively extracts only DNA from the DNA extract obtained from lake sediment samples. It was shown to be very effective for removal. As described above, the DNA extraction and purification method developed this time can extract DNA from environmental samples other than soil in high yields, and the obtained DNA has a much higher purity than the conventional method. And suitable for DNA extraction from all environmental samples Was thought to be.
〔実施例 23〕 DGGE解析 (Example 23) DGGE analysis
本実施例では、 今回開発した最終的な抽出精製方法を用いて、 土壌、 糞便、 堆 肥、活性汚泥、湖底'堆積物から BeadsBeating法、加熱抽出法により抽出した DNA サンプルについて DGGE解析を行った。  In this example, DGGE analysis was performed on DNA samples extracted from soil, feces, compost, activated sludge, and lake bottom sediments using the BeadsBeating method and heat extraction method using the final extraction and purification method developed this time. .
DNA溶液 l lをを铸型として、 16S rRNA遺伝子の V3領域を PCRにより増 幅し、 これを DGGEにより解析した。 PCR反応の条件および使用したプライマ ―、 DGGEの条件を以下に示す。  The V3 region of the 16S rRNA gene was amplified by PCR using the DNA solution 11 as type III, and this was analyzed by DGGE. The conditions of the PCR reaction and the primers used and the conditions of DGGE are shown below.
(1-1) PCR (1-1) PCR
プライマーセット 341FGC-534R  Primer set 341FGC-534R
16SrRNA遺伝子の V3領域を増幅  Amplifies V3 region of 16S rRNA gene
341FGC： 341FGC:
CGCCCGCCGC GCCCCGCGCC CGGCCCGCCG CCCCCGCCCC CCTACGGGAG GCAGCAG (配列番号 3 )  CGCCCGCCGC GCCCCGCGCC CGGCCCGCCG CCCCCGCCCC CCTACGGGAG GCAGCAG (SEQ ID NO: 3)
534R： 534R:
ATTACCGCGGCTGCTGGCAC (配列番号 4 )  ATTACCGCGGCTGCTGGCAC (SEQ ID NO: 4)
反応条件は、まず 94°Cで 2.5分反応させた後、次に 94°C 30秒の変性、 55°C 30 秒のァニーリング及び 72で 1分の伸長反応を 1サイクルとしてこれを 25サイク ル行い、 最後に、 72°Cで 10分反応させた。 反応液組成は以下の通りである。  The reaction conditions were as follows: reaction at 94 ° C for 2.5 minutes, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and elongation at 72 for 1 minute. The reaction was finally performed at 72 ° C for 10 minutes. The composition of the reaction solution is as follows.
BSAを終濃度 400ng/ lで含む。  Contains BSA at a final concentration of 400 ng / l.
Taq DNA Polymerase (sigma) 2.5 unit  Taq DNA Polymerase (sigma) 2.5 unit
反応液 50 1  Reaction liquid 50 1
(1-2) DGGE  (1-2) DGGE
ゲル濃度 8%  Gel concentration 8%
変性剤濃度勾配 35%〜65%  Denaturant concentration gradient 35% to 65%
Buffer温度 60°C  Buffer temperature 60 ° C
電圧 100V 泳動時間 10時間 土壌 DNAの DGGE解析結果について図 53に示す。 Voltage 100V Fig. 53 shows the results of DGGE analysis of soil DNA for 10 hours.
糞便 DNAの DGGE解析結果について図 54に示す。  Figure 54 shows the results of DGGE analysis of fecal DNA.
堆肥 DNA、 活性汚泥 DNAの DGGE解析結果について図 55に示す。  Figure 55 shows the results of DGGE analysis of compost DNA and activated sludge DNA.
湖底堆積物 DNAの DGGE解析結果について図 56に示す。 参照文献  Figure 56 shows the results of DGGE analysis of lake sediment DNA. References
Aasta Agersborg, Reidun Dahl and Iciar Martinez 1997 Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods International Journal of Food Microbiology 35:(3) 275-280  Aasta Agersborg, Reidun Dahl and Iciar Martinez 1997 Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods International Journal of Food Microbiology 35: (3) 275-280
A. Frostegard, A. Tunlid and E. Baath 1996 Changes in Microbial Community A. Frostegard, A. Tunlid and E. Baath 1996 Changes in Microbial Community
Structure during Long-Term Incubation in Two Soils Experimentally Contaminated with Metals Soil Biology and Biochemistry 28;(1)55·63 Structure during Long-Term Incubation in Two Soils Experimentally Contaminated with Metals Soil Biology and Biochemistry 28; (1) 55
Aimo Saano and Knstina Lmdstrom 1995 Small scale extraction of DNA from soil with spun column cleanup Molecular Microbial Ecology Manual 1.3. 1-6 Akira Hirais i 1999 Isoprenoid Quinone s as Biomarkers of Microbial Populations in the Environment Journal of Bioscience and Bioengineering 88；(5)449~460 Akira Hiraishi 1990 キノンプロファイル法による活性汚泥細菌の生態学的研究 Yousui to Haisui 32;(12)13-24 Aimo Saano and Knstina Lmdstrom 1995 Small scale extraction of DNA from soil with spun column cleanup Molecular Microbial Ecology Manual 1.3. 1-6 Akira Hirais i 1999 Isoprenoid Quinone s as Biomarkers of Microbial Populations in the Environment Journal of Bioscience and Bioengineering 88; (5 449-460 Akira Hiraishi 1990 Ecological study of activated sludge bacteria by quinone profile method Yousui to Haisui 32; (12) 13-24
Akira Hiraishi 1988 Respiratory Quinone Profiles as Tools lor Identifying differentAkira Hiraishi 1988 Respiratory Quinone Profiles as Tools lor Identifying different
Bacterial Populations in Activated Sludge Journal of General and AppliedBacterial Populations in Activated Sludge Journal of General and Applied
Microbiology 34;39-56 Microbiology 34; 39-56
Akira Hiraishi and Kenji Kato 1999 Quinone profiles in lake sediments- Implications for microbial diversity and community structures Journal of General and AppliedAkira Hiraishi and Kenji Kato 1999 Quinone profiles in lake sediments- Implications for microbial diversity and community structures Journal of General and Applied
Microbiology 45;221'227 Microbiology 45; 221'227
Akira Hiraishi, iyoshi asamune and Hiroshi Kitamura 1989 Akira Hiraishi, iyoshi asamune and Hiroshi Kitamura 1989
Characterization of the Bacterial Population Structure in an Anaerobic-Aerobic Characterization of the Bacterial Population Structure in an Anaerobic-Aerobic
Activated Sludge System on the Basis of Respiratory Quinone Profiles Applied andActivated Sludge System on the Basis of Respiratory Quinone Profiles Applied and
Environmental Microbiology 55；(4)897~901 Environmental Microbiology 55; (4) 897-901
Akira Hiraishi, Yoko Ueda, Junko Ishihara and Tadahiro Mori 1996 ComparativeAkira Hiraishi, Yoko Ueda, Junko Ishihara and Tadahiro Mori 1996 Comparative
Lipoquinone Analysis of Influent Sewage and Activated Sludge by High-PerformanceLipoquinone Analysis of Influent Sewage and Activated Sludge by High-Performance
Liquid Chromatography and P otodiode Array Detection Journal of General and Applied Microbiology 42；457-469 Liquid Chromatography and Photodiode Array Detection Journal of General and Applied Microbiology 42; 457-469
A.M. Ibekwe, A.C. Kennedy, RS. Frohne, S.K. Papiernik, C H. Yang, D.E. Crowley 2002 Microbial diversity along a transect of agronomic zones FEMSA.M.Ibekwe, A.C.Kennedy, RS.Frohne, S.K.Papiernik, C.H.Yang, D.E.Crowley 2002 Microbial diversity along a transect of agronomic zones FEMS
Microbiology Ecology 39; 183- 191 Microbiology Ecology 39; 183- 191
Anders Tunlid and David C. White 1992 Biochemical Analysis of Biomass, Community Anders Tunlid and David C. White 1992 Biochemical Analysis of Biomass, Community
Structure, Nutritional Status, and Metabolic Activity of Microbial Communities inStructure, Nutritional Status, and Metabolic Activity of Microbial Communities in
Soil Soil Biochemistry 7；229~262 Soil Soil Biochemistry 7; 229 ~ 262
荒尾知人、 岡野正豪、 金森哲夫 1998 土壌リン脂質と微生物バイオマス ·群集構造土と微 生物 51;49-58 荒尾知人、 岡野正豪、 金森哲夫 1998 淡色黒ポク土畑土壌におけるリン脂質脂肪酸組成 の解析および微生物バイオマスとの関係 日本土壌肥料科学雑誌 69;(1)38-46 荒尾知人、 岡野正豪、 金森哲夫 1998 各種土壌におけるリン脂質脂肪酸組成の解析 日本土壌肥料科学雑誌 69;(1)47-52 Tomoto Arao, Masago Okano, Tetsuo Kanamori 1998 Soil Phospholipids and Microbial Biomass ・ Community Structure Soil and Microorganisms 51; 49-58 Tomoto Arao, Masago Okano, Tetsuo Kanamori 1998 Analysis of phospholipid fatty acid composition in light-colored black pork soil and its relationship to microbial biomass Japanese Journal of Soil Fertilizer Science 69; (1) 38-46 Tomoto Arao, Masago Okano, Tetsuo Kanamori 1998 Analysis of phospholipid fatty acid composition in various soils Japanese Journal of Soil Fertilizer Science 69; (1) 47-52
Arata Katayama 2000 土壌中の農薬分解に関与する微生物群の構造と挙動 nippcm Noyakugak kais i 25;300·309 Arata Katayama 2000 Structure and behavior of microorganisms involved in pesticide degradation in soil nippcm Noyakugak kais i 25; 300 · 309
Arata Katayama, Keiko Funasaka and Koichi Fujie 2001 Changes in the respiratory quinone profile of a soil treated with pesticides Biology and Fertility of SoilsArata Katayama, Keiko Funasaka and Koichi Fujie 2001 Changes in the respiratory quinone profile of a soil treated with pesticides Biology and Fertility of Soils
33J454-459 33J454-459
Arata Katayama, Hong"Ying Hu， Mamie Nozawa, Haruyoshi Yamakawa and Koichi Fujie 1998 Long-Term Changes in Microbial Community Structure in SoilsArata Katayama, Hong "Ying Hu, Mamie Nozawa, Haruyoshi Yamakawa and Koichi Fujie 1998 Long-Term Changes in Microbial Community Structure in Soils
Subjected to Different Fertilizing Practices Revealed by Quinone Profile Analysis Soil Science and Plant Nutrition 44;(4)559_569 Subjected to Different Fertilizing Practices Revealed by Quinone Profile Analysis Soil Science and Plant Nutrition 44; (4) 559_569
Arjen G. C. L. Speksnijder, George A. Kowalchuk, Sander de jong, Elizabeth Kline, John R. Stephen and Hendrikus J. Laanbroek 2001 Microvariation Aritifacts Introduced by PGR and Cloning of Closely Related 16S rRNA Gene Sequences Applied and Environmental Microbiology 67;(l)469"472  Arjen GCL Speksnijder, George A. Kowalchuk, Sander de jong, Elizabeth Kline, John R. Stephen and Hendrikus J. Laanbroek 2001 Microvariation Aritifacts Introduced by PGR and Cloning of Closely Related 16S rRNA Gene Sequences Applied and Environmental Microbiology 67; (l) 469 "472
Bakken LR， jumdahl V Recovery of bacterial cells from soil Nucleic Acids in Environment  Bakken LR, jumdahl V Recovery of bacterial cells from soil Nucleic Acids in Environment
Bell KS， Kuyukina MS, Heidbrink S， Philp JC? Aw dwj, Ivshina IB and Christofi N 1999 Identification and environmental detection of Rhodococcus species by 16S rDNA-targeted PCR Journal of Industrial Microbiology 87:472-480 Bell KS, Kuyukina MS, Heidbrink S, Philp JC ? Aw dwj, Ivshina IB and Christofi N 1999 Identification and environmental detection of Rhodococcus species by 16S rDNA-targeted PCR Journal of Industrial Microbiology 87: 472-480
Berry AE, Chiocchmi C， Selby T， et al. 2003 Isolation oi high molecular weight DNA from soil for cloning into BAC vectors FEMS Microbiology Letters 223: (1) 15-20 Berry AE, Chiocchmi C, Selby T, et al. 2003 Isolation oi high molecular weight DNA from soil for cloning into BAC vectors FEMS Microbiology Letters 223: (1) 15-20
B.Bolin and R.B.Cook The major biogeochemical cycles and their interactions Fhon Wiley & Sons 1983  B.Bolin and R.B.Cook The major biogeochemical cycles and their interactions Fhon Wiley & Sons 1983
Bonnet R， Suau A， Dore J， et al. 2002 Differences in rDNA libraries of faecal bacteria derived from lOand 25-cycle PCRs Internaional Journal of Systematic and Evolutionary Microbiology 52: 757-763  Bonnet R, Suau A, Dore J, et al. 2002 Differences in rDNA libraries of faecal bacteria derived from lOand 25-cycle PCRs Internaional Journal of Systematic and Evolutionary Microbiology 52: 757-763
Boon N， De Windt W, Verstraete W， et al. 2002 Evaluation of nested PCR-DGGE Boon N, De Windt W, Verstraete W, et al. 2002 Evaluation of nested PCR-DGGE
(denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants(denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants
FEMS Microbiology Ecology 39: (2) 101-112 FEMS Microbiology Ecology 39: (2) 101-112
Chandler DP, Fredrickson JK, Brockman FJ 1997 Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone librariesChandler DP, Fredrickson JK, Brockman FJ 1997 Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries
Molecular Ecology 6： (5) 475-482 Molecular Ecology 6: (5) 475-482
D N. Miller,* J. E. Bryant, E. L. Madsen, and W. C. Ghiorse 1999 Evaluation andD N. Miller, * J. E. Bryant, E. L. Madsen, and W. C. Ghiorse 1999 Evaluation and
Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples Applied and Environmental Microbiology 65:(11) 4715-4724 Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples Applied and Environmental Microbiology 65: (11) 4715-4724
Darrell P. Chandler, Randall W. Schreckhise, Jeffrey L. Smith and Harvey Bolton Jr.  Darrell P. Chandler, Randall W. Schreckhise, Jeffrey L. Smith and Harvey Bolton Jr.
1997 Electroelution to remove humic compounds from soil DNA and RNA extracts Journal of Microbiological Methods 28:(1) 11-19  1997 Electroelution to remove humic compounds from soil DNA and RNA extracts Journal of Microbiological Methods 28: (1) 11-19
David B. Hedric and David C. White 1986 Microbaial respiratory quinones in the environment Journal of Microbiological Methods 5J243-254  David B. Hedric and David C. White 1986 Microbaial respiratory quinones in the environment Journal of Microbiological Methods 5J243-254
E. G. Bligh and W. J. Dyer 1959 A RAPID OF TOTAL LIPID EXTRACTION AND E. G. Bligh and W. J. Dyer 1959 A RAPID OF TOTAL LIPID EXTRACTION AND
PURIFICATION Canadian Journal of Biochemistry and Physiology 37;911·917 E. Molina Gi,ima， A. Robles Medina, A. Gimenez Gimenez, J.A. Sanchez Perez. F. Garcia Cmamacho and J.L. Garcia Sanchez 1994 Comparison Between Extraction ofPURIFICATION Canadian Journal of Biochemistry and Physiology 37; 911 917 E. Molina Gi, ima, A. Robles Medina, A. Gimenez Gimenez, JA Sanchez Perez. F. Garcia Cmamacho and JL Garcia Sanchez 1994 Comparison Between Extraction of
Lipids and Fatty Acids from Microalgal Biomass Journal of the American OilLipids and Fatty Acids from Microalgal Biomass Journal of the American Oil
Chemists' Society 71;(9) 955-959 Chemists' Society 71; (9) 955-959
Esther M. Gabor, Erik J. de Vries and Dick B. Janssen 2003 Efficient recovery of environmental DNA for expression cloning by indirect extraction methods FEMSEsther M. Gabor, Erik J. de Vries and Dick B. Janssen 2003 Efficient recovery of environmental DNA for expression cloning by indirect extraction methods FEMS
Microbiology Ecology 44:(2) 153-163 Microbiology Ecology 44: (2) 153-163
Ezra S. Abrams and Vincent P. Stanton, JR. 1992 Use of Denaturing Gradient Gel Ezra S. Abrams and Vincent P. Stanton, JR. 1992 Use of Denaturing Gradient Gel
Electrophoresis' to Study Conformational Transitions in Nucleic Acids Methods in enzymology 212；71~104 Electrophoresis' to Study Conformational Transitions in Nucleic Acids Methods in enzymology 212; 71-104
Foppe Smedes and Torsten K. Askland 1999 Revisiting the Development of the Bligh and Dyer Total Lipid Determination Method Marine Pollution BulletinFoppe Smedes and Torsten K. Askland 1999 Revisiting the Development of the Bligh and Dyer Total Lipid Determination Method Marine Pollution Bulletin
38;(3)193-201 38; (3) 193-201
Foppe Smedes and Torsten K. Thomasen 1996 Evaluation of the Bligh & Dyer Lipid Foppe Smedes and Torsten K. Thomasen 1996 Evaluation of the Bligh & Dyer Lipid
Determination Method Marine Pollution Bulletin 32;(8，9) 681.688 Determination Method Marine Pollution Bulletin 32; (8, 9) 681.688
Frostegard A and Baath E 1996 The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil Biol. Fertil Soils 22:59-65 Frostegard A and Baath E 1996 The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil Biol. Fertil Soils 22: 59-65
Frostegard A, Tunlid A and Baath E 1991 Microbaial biomass measured as total lipid phosphate in soils of different organic content Journal of Microbiological MethodsFrostegard A, Tunlid A and Baath E 1991 Microbaial biomass measured as total lipid phosphate in soils of different organic content Journal of Microbiological Methods
14:151-163 14: 151-163
藤間充、 三枝正彦 1997 石膏による稲わらおよび稲わら堆肥の分解調節 日本土壌肥料科学 雑誌 68；(6)645-650 Mitsuru Fujima, Masahiko Saegusa 1997 Degradation control of rice straw and rice straw compost by gypsum Japan Soil Fertilizer Science Magazine 68; (6) 645-650
Futoslii Kurisu, mroyasu Satoh, Takashi Mino， Tomonori Matsuo 2002 Microbial community analysis of thermophilic contact oxiaation process by using robosomalFutoslii Kurisu, mroyasu Satoh, Takashi Mino, Tomonori Matsuo 2002 Microbial community analysis of thermophilic contact oxiaation process by using robosomal
RNA approaches ans the quinone profile method Water Eesearch 36J429-438 G. Lloyd -Jones and D. W. F. Hunter 2001 Comparison of rapid DNA extraction methods applied to contrasting New Zealand soils Soil Biology and Biochemistry 33:(15)RNA approaches ans the quinone profile method Water Eesearch 36J429-438 G. Lloyd -Jones and D.W.F.Hunter 2001 Comparison of rapid DNA extraction methods applied to contrasting New Zealand soils Soil Biology and Biochemistry 33: (15)
2053-2059 2053-2059
Gabor EM, de Vries EJ， Janssen DB 2003 Efficient recovery of environmental DNA for expression cloning by indirect extraction methods FEMS Microbiology Ecology 44： (2) 153-163  Gabor EM, de Vries EJ, Janssen DB 2003 Efficient recovery of environmental DNA for expression cloning by indirect extraction methods FEMS Microbiology Ecology 44: (2) 153-163
Gerard Muyzer 1999 DGGE/TGGE a method for identifying genes from natural ecosystems Current Opinion in Microbiology 2;317-322  Gerard Muyzer 1999 DGGE / TGGE a method for identifying genes from natural ecosystems Current Opinion in Microbiology 2; 317-322
Gerard Muyzer and Ellen C. de Waal 1994 Determination of the genetic diversity of micorbial communities using DGGE analysis of PCR-amplified 16S r NA NATOGerard Muyzer and Ellen C. de Waal 1994 Determination of the genetic diversity of micorbial communities using DGGE analysis of PCR-amplified 16S r NA NATO
ASI Series G 35;207-214 ASI Series G 35; 207-214
Gerard Muyzer and Kornelia Smalla 1998 Application of denaturing gradient gel electrophoresis(DGGE) and temperature gradient gel electrophoresis(TGGE) in microbial ecology Antonie van Leeuwenhoek 73; 127- 141 Gerard Muyzer and Kornelia Smalla 1998 Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology Antonie van Leeuwenhoek 73; 127-141
Grace C._Y. Wang and Yue Wang 1997 Frequency of Formation of Chimeric Molecules as a Consequence of PCR Coamplification of 16S rRNA Genes from Mixed BacterialGrace C. _ Y.Wang and Yue Wang 1997 Frequency of Formation of Chimeric Molecules as a Consequence of PCR Coamplification of 16S rRNA Genes from Mixed Bacterial
Genomes Applied and Environmental Microbiology 63;(12)4645·4650 Genomes Applied and Environmental Microbiology 63; (12) 4645
Hasebe Akira 2003 土は宝の山か？— eDNAの利用による土壌微生物資源へのァプロ ーチ Nippon Nogeikagaku kaishi 77:(12) 1230 - 1239 Hasebe Akira 2003 Is Soil a Treasure Mountain? — An approach to soil microbial resources using eDNA Nippon Nogeikagaku kaishi 77: (12) 1230-1239
Helmut Burgmann, Manuel Pesaro, Franco ^idmer and Josei Zeyer 2001 A strategy for optimizing quality and quantity of DNA extracted from soil Journal ofHelmut Burgmann, Manuel Pesaro, Franco ^ idmer and Josei Zeyer 2001 A strategy for optimizing quality and quantity of DNA extracted from soil Journal of
Microbiological Methods 45:(1) 7*20 Microbiological Methods 45: (1) 7 * 20
樋口太重 1981 緩衝液による勇気窒素および土壌有機態窒素の抽出特性 日本土壌肥料科学 雑誌 52;(6)481-489 Holger Heuer, Martin Krsek, Paul Baker, Kornelia Smalla, and Elizabeth M. H.Higuchi, Taishi 1981 Extraction characteristics of courage nitrogen and soil organic nitrogen by buffer solution Japanese Journal of Soil Fertilizer Science 52; (6) 481-489 Holger Heuer, Martin Krsek, Paul Baker, Kornelia Smalla, and Elizabeth MH
Wellington 1997 Analysis of Actinomycete Communities by Specific Amplification ofWellington 1997 Analysis of Actinomycete Communities by Specific Amplification of
Genes Encodin 16S rRNA and Gel-Electrophoretic Separation in DenatruingGenes Encodin 16S rRNA and Gel-Electrophoretic Separation in Denatruing
Gradients Applied and Environmental Microbiology 63;(8)3233_3241 Gradients Applied and Environmental Microbiology 63; (8) 3233_3241
Hongoh Y， Yuzawa H， Ohkuma M， et al. 2003 Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment FEMSHongoh Y, Yuzawa H, Ohkuma M, et al. 2003 Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment FEMS
Microbiology Letters 221： (2) 299-304 Microbiology Letters 221: (2) 299-304
Hong -Ying Hu， Koichi Fujie and Kohei urano 1999 Development of a Novel Solid Phase Hong -Ying Hu, Koichi Fujie and Kohei urano 1999 Development of a Novel Solid Phase
Extraction Method for the Analysis of Bacterial Quinones in Activated Sludge with aExtraction Method for the Analysis of Bacterial Quinones in Activated Sludge with a
Higher Reliability Journal of Bioscience and Bioengineering 87；(3)378~382 Higher Reliability Journal of Bioscience and Bioengineering 87; (3) 378-382
Hugenholtz P, Huber T 2003 Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases Internaional Journal of Systematic andHugenholtz P, Huber T 2003 Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases Internaional Journal of Systematic and
Evolutionary Microbiology 53: 289-293 Evolutionary Microbiology 53: 289-293
Is ii K， Fukui M 2001 Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitem late PCR Applied and EnvironmentalIs ii K, Fukui M 2001 Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitem late PCR Applied and Environmental
Microbiology 67: (8) 3753-3755 Microbiology 67: (8) 3753-3755
石井浩介、 中川達功、 福井学 2000微生物生態学への変性剤濃度勾配ゲル電気泳動法の応用Kosuke Ishii, Tatsunori Nakagawa, Manabu Fukui 2000 Application of denaturing gradient gel electrophoresis to microbial ecology
Microbes and Environments lo?(l)59-73 Microbes and Environments lo ? (L) 59-73
Jacek Kozdroj, Jan Dirk van Elsas 2001 Structual diversity of microbial communities in arable soils of a heavily industrialised area determined by PCR'DGGE fingerprinting and FAME profiling Applied Soil Ecology 17,31-42 Jacek Kozdroj, Jan Dirk van Elsas 2001 Structual diversity of microbial communities in arable soils of a heavily industrialized area determined by PCR'DGGE fingerprinting and FAME profiling Applied Soil Ecology 17,31-42
Jacek Kozdroj, Jan Dirk van Elsas 2000 Response of the bacterial community to root exudates in soil polluted with heavy r etals assessed by molecular and culutural approaches Soil Biology and Biochemistry 32; 1405- 1417 Jacek Kozdroj, Jan Dirk van Elsas 2000 Response of the bacterial community to root exudates in soil polluted with heavy r etals assessed by molecular and culutural approaches Soil Biology and Biochemistry 32; 1405-1417
Jackson CR, Harper JP， Willoughby D， Roden EE, Churchill PF 1997 A simple, efficient method for the separation of humic substances and DNA from environmental samples Applied and Environmental Microbiology 63:(12) 4993.4995 Jackson CR, Harper JP, Willoughby D, Roden EE, Churchill PF 1997 A simple, efficient method for the separation of humic substances and DNA from environmental samples Applied and Environmental Microbiology 63: (12) 4993.4995
J. A. McKeague, J. E. Brydon and N. M. Miles 1971 Differentiation of Forms ofJ. A. McKeague, J. E. Brydon and N. M. Miles 1971 Differentiation of Forms of
Extractable Iron and Aluminium in Soils Soil Science Society of AmericanExtractable Iron and Aluminum in Soils Soil Science Society of American
Proceeding 35;33'38 Proceeding 35; 33'38
James B. Guckert? Christopher P. Antworth, Peter D. Nichols and David C. White 1985 Phospholipid, ester-linked fatty acid profiles as reproduciole assays for changes in prokaryotic community structure of estuarine sediments FEMS Microbiology Ecology 31 147-158 James B. Guckert ? Christopher P. Antworth, Peter D. Nichols and David C. White 1985 Phospholipid, ester-linked fatty acid profiles as reproduciole assays for changes in prokaryotic community structure of estuarine sediments FEMS Microbiology Ecology 31 147-158
Jan Dirk Van Elsas and Kornelia Smalla 1995 Extraction of microbial community DNA from soils Molecular Microbial Ecology Manual 1.3.3 1-11  Jan Dirk Van Elsas and Kornelia Smalla 1995 Extraction of microbial community DNA from soils Molecular Microbial Ecology Manual 1.3.3 1-11
Janelle R. Thompson, Luisa A. Marcelino and Martin F. Polz 2002 Heteroduplexes in mixed-template amplifications: fomation, consequence and elimination byJanelle R. Thompson, Luisa A. Marcelino and Martin F. Polz 2002 Heteroduplexes in mixed-template amplifications: fomation, consequence and elimination by
'recoditioning PCR' Nucleic Acids Research 30;(9)2083_2088 'recoditioning PCR' Nucleic Acids Research 30; (9) 2083_2088
John T.Lis 1980 Fractionation of DNA Fragments by Polyethlene Glycol InducedJohn T. Lis 1980 Fractionation of DNA Fragments by Polyethlene Glycol Induced
Precipitation Methods in enzymology 65 347-353 Precipitation Methods in enzymology 65 347-353
Jorg Gerke 1994 Kinetiks of soil phosphate desorption as affected by citric acid Jorg Gerke 1994 Kinetiks of soil phosphate desorption as affected by citric acid
Z.Pflanzenernahr. Bodenk.. 157；17-22 Z. Pflanzenernahr. Bodenk .. 157; 17-22
J.R. Lawrence, M.J. Hendry, L.I. Wassenaar, J.J. Germida, G.M. Wolfaardt, N. Fortin,J.R. Lawrence, M.J.Hendry, L.I.Wassenaar, J.J.Germida, G.M.Wolfaardt, N. Fortin,
C.W. Greer 2000 Distribution and Biogeochemical Importance of BacterialC.W.Greer 2000 Distribution and Biogeochemical Importance of Bacterial
Populations in a Thick Clay-Rich Aquitard System Microbial Ecology 40;273'291 J. Ruhlmann 1999 A new approach to estimating the pool of stable organic matter in soil using data from long-term neid experiments Plant and Soil 213;149-1わ 0 J. Tamaoka, Y. Katayama-Fujimura and H, Kuraishi 1983 Analysis of bacterial menaquinone mixtures by high performance liquid chromatography Journal of Applied Bacteriology 54J31-56 Populations in a Thick Clay-Rich Aquitard System Microbial Ecology 40; 273'291 J. Ruhlmann 1999 A new approach to estimating the pool of stable organic matter in soil using data from long-term neid experiments Plant and Soil 213; 149-1 0 J. Tamaoka, Y. Katayama-Fujimura and H, Kuraishi 1983 Analysis of bacterial menaquinone mixtures by high performance liquid chromatography Journal of Applied Bacteriology 54J31-56
Julie Tans-Kersten, Yanfen Guan， and Caitilyn Allen 1998 Ralstonia  Julie Tans-Kersten, Yanfen Guan, and Caitilyn Allen 1998 Ralstonia
solanacearum Pectin Methylesterase Is Required for Growth on Methylated Pectin but Not for Bacterial Wilt Virulence Applied and Environmental Microbiology 64；(12)4918-4923  solanacearum Pectin Methylesterase Is Required for Growth on Methylated Pectin but Not for Bacterial Wilt Virulence Applied and Environmental Microbiology 64; (12) 4918-4923
Katsuaki Saitou, Koichi Fujie and Arata Katayama 1999 Detection of Microbial Katsuaki Saitou, Koichi Fujie and Arata Katayama 1999 Detection of Microbial
Groups Metabolizing a Substrate in Soil Based on the [l4C]Quinone Profile Soil Science and Plant Nutrition 45;(3)669_679 Groups Metabolizing a Substrate in Soil Based on the [l4C] Quinone Profile Soil Science and Plant Nutrition 45; (3) 669_679
Kazunori Yamamoto, Rituko Murakami, Yoshichika Takamura 1998 Isoprenoid quinone, cellular fatty acid composition and diaminopimelic acid isomers of newly classified thermophilic anaerobic Gram-positive bacteria FEMS Microbiology Letters 16;(2)351-358  Kazunori Yamamoto, Rituko Murakami, Yoshichika Takamura 1998 Isoprenoid quinone, cellular fatty acid composition and diaminopimelic acid isomers of newly classified thermophilic anaerobic Gram-positive bacteria FEMS Microbiology Letters 16; (2) 351-358
Kazuya Watanabe, Yumiko Kodama and Shigeaki Harayama 2001 Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting Journal of Microbiological Methods 44:(3) 253*262 Kazuya Watanabe, Yumiko Kodama and Shigeaki Harayama 2001 Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting Journal of Microbiological Methods 44: (3) 253 * 262
Koichi Fujie, Hong-Ying Hu， Hajime Tanaka, Kohei Urano, Katsuaki Saitou and Arata Katayama 1998 Analysis of Respiratory Quinones in Soil for Characterization of Microbiota Soil Science and Plant Nutrition 44;(3)393'404 Koichi Fujie, Hong-Ying Hu, Hajime Tanaka, Kohei Urano, Katsuaki Saitou and Arata Katayama 1998 Analysis of Respiratory Quinones in Soil for Characterization of Microbiota Soil Science and Plant Nutrition 44; (3) 393'404
Koji Wada and Teruo Higashi 1976 The Categolies of Aluminium- and Iron-Humus Complexes in Ando Soils Determined by Selective Dissolution Journal of Soil Koji Wada and Teruo Higashi 1976 The Categolies of Aluminum- and Iron-Humus Complexes in Ando Soils Determined by Selective Dissolution Journal of Soil
Science 27 ;357-687 Science 27; 357-687
Kozdroj J.， Elsas JD 2000 Application of polymerase chein reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting Biol. Fertil Soils 31:372-378 Kozdroj J., Elsas JD 2000 Application of polymerase chein reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting Biol. Fertil Soils 31: 372-378
Krsek M， Wellington EMH 1999 Comparison of different methods for the isolation and purification of total community DNA from soil Journal of Microbiological Methods 39: (1) 1-16 Krsek M, Wellington EMH 1999 Comparison of different methods for the isolation and purification of total community DNA from soil Journal of Microbiological Methods 39: (1) 1-16
久保田徹、 箱石正、 高橋茂 1986 ヒドロキシアルミニウム処理による堆肥の分解抑制 Toru Kubota, Tadashi Hakoishi, Shigeru Takahashi 1986 Suppression of compost decomposition by hydroxyaluminum
日本土壌肥料科学雑誌 57;(2)155_160  Japanese Journal of Soil Fertilizer Science 57; (2) 155_160
倉石 衍 1985 9 化学分類学的技法による活性汚泥微生物相の類別化 微生物の生態 13Takashi Kuraishi 1985 9 Classification of activated sludge microflora by chemical taxonomic techniques Microbial ecology 13
—化学的手法をめぐって— 125-138 —On the chemical method— 125-138
倉石 衍 1982 2. 脂質 微生物の化学分類実験法 87-143 Takashi Kuraishi 1982 2. Experimental method for chemical classification of lipid microorganisms 87-143
Kurien BT， Kaufman KM, harley JB, et al, 2001 ellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction Analytical Kurien BT, Kaufman KM, harley JB, et al, 2001 ellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction Analytical
Biochemistry 296: (2) 162-166 Biochemistry 296: (2) 162-166
Kurien BT， Sconeld RH 2002 Extraction of nucleic acid fragments from gels Analytical Kurien BT, Sconeld RH 2002 Extraction of nucleic acid fragments from gels Analytical
Biochemistry 302: (l) 1-9 Biochemistry 302: (l) 1-9
Kuske CR， Banton KL, Adorada DL, et al, 1998 Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil Applied andKuske CR, Banton KL, Adorada DL, et al, 1998 Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil Applied and
Environmental Microbiology 64: (7) 2463-2472 Environmental Microbiology 64: (7) 2463-2472
Kusukawa N， Umemori T， Asada K and Kato I 1990 DNA Sequencing Report Rapid and Reliable Protocol for Direct Squencing of Material Amplified by the PolymeraseKusukawa N, Umemori T, Asada K and Kato I 1990 DNA Sequencing Report Rapid and Reliable Protocol for Direct Squencing of Material Amplified by the Polymerase
Chain Reactio Biotechniques 9:(1) 66- & Chain Reactio Biotechniques 9: (1) 66- &
K. W. Perrot 1978 The Influence of Organic Matter Extracted from Humified Clover on the Properties of Amorphous Aluminosilicates. I Surface Charge AustralianK. W. Perrot 1978 The Influence of Organic Matter Extracted from Humified Clover on the Properties of Amorphous Aluminosilicates.I Surface Charge Australian
Journal of Soil Reserch 16；327~346 Liesack W, Weyland H，' St Ackerandt E 1991 Potential risks of gene amplification byJournal of Soil Reserch 16; 327-346 Liesack W, Weyland H, 'St Ackerandt E 1991 Potential risks of gene amplification by
PGR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria Molecular Microbial Ecology Manual 21: (3) 191^198 PGR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria Molecular Microbial Ecology Manual 21: (3) 191 ^ 198
Lowell JL， Klein DA 2000 Heteroduplex resolution using T7 endonuclease I in microbial community analyses BIOTECHNIQUES 28: (4) 676-+ APR 2000 Lowell JL, Klein DA 2000 Heteroduplex resolution using T7 endonuclease I in microbial community analyzes BIOTECHNIQUES 28: (4) 676- + APR 2000
L. 0vreas， V. Torsvik 1998 Microbial Diversity and Community Structures in TwoL. 0vreas, V. Torsvik 1998 Microbial Diversity and Community Structures in Two
Different Agricultural Soil Communities Microbial Ecology 36;303-315 Different Agricultural Soil Communities Microbial Ecology 36; 303-315
L. Zelles and Q.Y. Bai 1993 Fractionation of Fatty Acids Derived from Soil Lipids by Solid Phase Extraction and Their Quantitative Analysis by GC-MS Soil biology and biochemistry 25；(4)495-507 L. Zelles and Q.Y. Bai 1993 Fractionation of Fatty Acids Derived from Soil Lipids by Solid Phase Extraction and Their Quantitative Analysis by GC-MS Soil biology and biochemistry 25; (4) 495-507
Matthew D. Collins and Dorothy Jones 1981 Distribution of Isoprenoid Q noneMatthew D. Collins and Dorothy Jones 1981 Distribution of Isoprenoid Q none
Structural Types in Bacteria and Their Taxonomic Im ications Microbiological eviesws 45；(2)316-354 Structural Types in Bacteria and Their Taxonomic Immunications Microbiological eviesws 45; (2) 316-354
M, D. Collins Analysis of Isoprenoid Quinone s Methods in Microbiology 18;329·366 M, G. LaMontagne, R C. Michel, Jr.， P. A, Holden and C, A. Reddy 2002 M, D. Collins Analysis of Isoprenoid Quinones Methods in Microbiology 18; 329366 M, G. LaMontagne, R C. Michel, Jr., PA, Holden and C, A. Reddy 2002
Evaluation of extraction and purification methods for obtaining P CR- am lifiable Evaluation of extraction and purification methods for obtaining P CR- am lifiable
DNA from compost for microbial community analysis Journal of MicrobiologicalDNA from compost for microbial community analysis Journal of Microbiological
Methods 49:(3) 255-264 Methods 49: (3) 255-264
Manirakiza P， Covaci A, Sc epens P 2001 Comparative study on total lipid determination using Soxhlet，Roese'Gottlied，Bligh & Dyer，and modified Bligh &Manirakiza P, Covaci A, Sc epens P 2001 Comparative study on total lipid determination using Soxhlet, Roese'Gottlied, Bligh & Dyer, and modified Bligh &
Dyer extraction methods Journal of Food Composition and Analysis 14:93-100 Michael Howeler, William C. Ghiorse and Larry R Walker 2003 A quantitative analysis of DNA extraction and purification from compost Journal of MicrobiologicalDyer extraction methods Journal of Food Composition and Analysis 14: 93-100 Michael Howeler, William C. Ghiorse and Larry R Walker 2003 A quantitative analysis of DNA extraction and purification from compost Journal of Microbiological
Methods 54:(1) 37-45 Methods 54: (1) 37-45
Miller DN 2001 Evaluation of gel filtration resins for the removal of PC -inhibitory  Miller DN 2001 Evaluation of gel filtration resins for the removal of PC -inhibitory
Mirbod F， Mori S， Nozawa Y 1993 Methods for phospholipid extraction in Candida albicans *an extraction method with high efficacy Journal of Medical andMirbod F, Mori S, Nozawa Y 1993 Methods for phospholipid extraction in Candida albicans * an extraction method with high efficacy Journal of Medical and
Veterinary Mycology 31-405-409 Veterinary Mycology 31-405-409
Mituru Iwasaki and Akira Hiraishi 1998 A New Approach to Numerical Analysis ofMituru Iwasaki and Akira Hiraishi 1998 A New Approach to Numerical Analysis of
Microbial Quinone Pronles in the Environment Microbes and EnvironmentsMicrobial Quinone Pronles in the Environment Microbes and Environments
13；(2)67-76 13; (2) 67-76
Moreira D 1998 Efficient removal of PCR inhibitors using agarose*embedded DNA preparations Nucleic Acids Research 26: (13) 3309-3310  Moreira D 1998 Efficient removal of PCR inhibitors using agarose * embedded DNA preparations Nucleic Acids Research 26: (13) 3309-3310
M. Russell, R, L. Parfitt and G. G. C. Claridge 1981 Estimation of the Amounts of Allop ane and Other Materials in the Clay Fraction of an Egmont Loam Profile and Other Volcanic Ash Soils, New Zealand Australian Journal of Soil Reserch 19；185"95  M. Russell, R, L. Parfitt and GGC Claridge 1981 Estimation of the Amounts of Allop ane and Other Materials in the Clay Fraction of an Egmont Loam Profile and Other Volcanic Ash Soils, New Zealand Australian Journal of Soil Reserch 19; 185 "95
MT Suzuki and SJ Giovannoni 1996 Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR Applied and Environmental MT Suzuki and SJ Giovannoni 1996 Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR Applied and Environmental
Microbiology 62:(2) 625.630 Microbiology 62: (2) 625.630
Murray MG， Thompson WF 1980 Rapid isolation of high molecular-weight plant DNAMurray MG, Thompson WF 1980 Rapid isolation of high molecular-weight plant DNA
Nucleic Acids Research 8： (19) 4321-4325 Nucleic Acids Research 8: (19) 4321-4325
Paithankar K , Prasad KSN 1991 Precipitation of DNA by polyethylene-glycol and ethanal Nucleic Acids Research 19: (6) 1346-1346 Paithankar K, Prasad KSN 1991 Precipitation of DNA by polyethylene-glycol and ethanal Nucleic Acids Research 19: (6) 1346-1346
Per Wikstrom, Anna Wiklund? Ann-Christin Andersson and Mats Forsman 1996Per Wikstrom, Anna Wiklund ? Ann-Christin Andersson and Mats Forsman 1996
DNA recovery and FC quantification of catechol 2,3-dioxygenase genes from different soil types Journal of Biotechnology 52:(2) 107-120 P. Garbeva, L.S. van Overbeek, J.W.L. van Vuurde, J.D. van Elsas 2001 Analysis of Endophytic Bacterial Communities of Potato by Plating and Denaturing Gradient Gel Electrophoresis(DGGE) of 16S rDNA Based PCR Fragments Microbial Ecology 41J369-383 DNA recovery and FC quantification of catechol 2,3-dioxygenase genes from different soil types Journal of Biotechnology 52: (2) 107-120 P. Garbeva, LS van Overbeek, JWL van Vuurde, JD van Elsas 2001 Analysis of Endophytic Bacterial Communities of Potato by Plating and Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA Based PCR Fragments Microbial Ecology 41J369-383
Pitcher DG, Saunders NA, Owen RJ 1989 Rapid extraction of bacterial genomic DNA with guanidium thiocyanate Letters in Applied Microbiology 8: (4) 151-156 Pitcher DG, Saunders NA, Owen RJ 1989 Rapid extraction of bacterial genomic DNA with guanidium thiocyanate Letters in Applied Microbiology 8: (4) 151-156
P.J. Loveland and P. Digby 1984 the extractions of Fe and Al by 0.1 M pyrophosphate solutions-a comparison of some techniques Journal of Soil Science 35；243"250 P. Marschner, C.-H. Yang, R. Lieberei, D.E. Crowley 2001 Soil and plant specific effects on bacterial community composition in the rhizosphere Soil Biology andPJ Loveland and P. Digby 1984 the extractions of Fe and Al by 0.1 M pyrophosphate solutions-a comparison of some techniques Journal of Soil Science 35; 243 "250 P. Marschner, C.-H. Yang, R. Lieberei, DE Crowley 2001 Soil and plant specific effects on bacterial community composition in the rhizosphere Soil Biology and
Biochemistry 33;1437'1445 Biochemistry 33; 1437'1445
Polz MF， Cavanaug CM 1998 Bias in template-to-product ratios in multitemplate PCRPolz MF, Cavanaug CM 1998 Bias in template-to-product ratios in multitemplate PCR
Applied and Environmental Microbiology 64： (10) 3724*3730 Applied and Environmental Microbiology 64: (10) 3724 * 3730
Porteous LA, Armstrong JL, Seialer RJ， et al. 1994 An effective method to extractPorteous LA, Armstrong JL, Seialer RJ, et al. 1994 An effective method to extract
DNA from environmental-samples for polymerase chain-reaction amplification andDNA from environmental-samples for polymerase chain-reaction amplification and
DNA fingerprint analysis Current Microbiology 29: (5) 301-307 DNA fingerprint analysis Current Microbiology 29: (5) 301-307
Porteous LA, Seidler RJ, Watrud LS 1997 An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applicationsPorteous LA, Seidler RJ, Watrud LS 1997 An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applications
Molecular Ecology 6:(8) 787*791 Molecular Ecology 6: (8) 787 * 791
Qiu XY， Wu LY， Huang HS, et al. 2001 Evaluation of PCR-generated chimeras: Mutations, and heteroduplexes with 16S rRNA gene-based cloning Applied andQiu XY, Wu LY, Huang HS, et al. 2001 Evaluation of PCR-generated chimeras: Mutations, and heteroduplexes with 16S rRNA gene-based cloning Applied and
Environmental Microbiology 67： (2) 880,887 Environmental Microbiology 67: (2) 880,887
Richard J. Ellis, Barry Isieish, Marcus W. Trett, J. George Best, Andrew J. Weightman,Richard J. Ellis, Barry Isieish, Marcus W. Trett, J. George Best, Andrew J. Weightman,
Philip Morgan, Jonn C. Fry 2001 Comparison of microbial and meiofauna丄 community analyses for determining impact of heavy metal contamination Journal of Microbiological Methods 45; 171- 185 Philip Morgan, Jonn C. Fry 2001 Comparison of microbial and meiofauna 丄 community analyzes for determining impact of heavy metal contamination Journal of Microbiological Methods 45; 171-185
Richard M.Myers, Stuart G.Fischer, Leonard S.Lerman and Tom Maniatis 1985Richard M. Myers, Stuart G. Fischer, Leonard S. Lerman and Tom Maniatis 1985
Modification of the melting properties of duplex DNA by attachment of a GC-richModification of the melting properties of duplex DNA by attachment of a GC-rich
DNA sequence as determined by denaturing gradient gel electrophoresis Nucleic Acids Research 13;(9)3111_3129 DNA sequence as determined by denaturing gradient gel electrophoresis Nucleic Acids Research 13; (9) 3111_3129
Richard M.Myers, Stuart . Fischer, Tom Maniatis and Leonard S.Lerman 1985 Nearly all sigle base substitutions in DNA fragments joined to a GC.clamp can be detected by denaturing gradient gel electrophoresis Nucleic Acids ResearchRichard M. Myers, Stuart .Fischer, Tom Maniatis and Leonard S. Lerman 1985 Nearly all sigle base substitutions in DNA fragments joined to a GC.clamp can be detected by denaturing gradient gel electrophoresis Nucleic Acids Research
13;(9)3131-3145 13; (9) 3131-3145
R, L. Parfitt 1992 Potassium-Calcium Exchange in Some New Zealand Soils Australian R, L. Parfitt 1992 Potassium-Calcium Exchange in Some New Zealand Soils Australian
Journal of Soil Reserch 30;145_58 Journal of Soil Reserch 30; 145_58
R. L. Parfitt 1992 Surface Charge in Some New Zealand Soils Measured at Typical IonicR.L.Parfitt 1992 Surface Charge in Some New Zealand Soils Measured at Typical Ionic
Strength Australian Joui-nal of Soil Keserch 30;331-41 Strength Australian Joui-nal of Soil Keserch 30; 331-41
R. L. Parfitt 1990 AUophane in New Zealand-A Review Australian Journal of SoilR. L. Parfitt 1990 AUophane in New Zealand-A Review Australian Journal of Soil
Reserch 28;343·60 Reserch 28; 343
R. L. Parfitt, M. Russel and G. E. Orbell 1988 Estimation of Forms of Fe and Al: A R. L. Parfitt, M. Russel and G. E. Orbell 1988 Estimation of Forms of Fe and Al: A
Review, and Analysis of Contrasting Soils by Dissolution and Moessbauer MethodsReview, and Analysis of Contrasting Soils by Dissolution and Moessbauer Methods
Soil Chemistry and Mineralogy 26；121~44 Soil Chemistry and Mineralogy 26; 121-44
R. L. Parfitt and M. Saigusa 1983 Weathering Sequence of Soils from Volcanic AshR. L. Parfitt and M. Saigusa 1983 Weathering Sequence of Soils from Volcanic Ash
Involving AUophane and Halloysite, New Zealand ieoderma 29;41-57 Involving AUophane and Halloysite, New Zealand ieoderma 29; 41-57
R. L. Parfitt and C. W. Childs 1985 AUophane and Humus -Aluminum in Spodosols and Andepts Formed from the Same Volcanic Ash Beds in New Zealand SonR. L. Parfitt and C. W. Childs 1985 AUophane and Humus -Aluminum in Spodosols and Andepts Formed from the Same Volcanic Ash Beds in New Zealand Son
Science 139;(2)149'155 Rogstad SH 1992 Saturated NaCl'CTAB solution as. a means of field preservation of leaves forDNA analyses Taxon 41:701.708 Science 139; (2) 149'155 Rogstad SH 1992 Saturated NaCl'CTAB solution as.a means of field preservation of leaves for DNA analyzes Taxon 41: 701.708
Rondon MR， Goodman RM， Handelsman J 1999 The Earth's bounty: assessing and accessing soil microbial diversity Trends in Biotechnology 17: (10) 403*409 Rondon MR, Goodman RM, Handelsman J 1999 The Earth's bounty: assessing and accessing soil microbial diversity Trends in Biotechnology 17: (10) 403 * 409
Saano A， Tas E， Pippola S, Lindstrom K and van Elsas JD Extraction and analysis of microbial DNA from soil Nucleic Acids in Environment Saano A, Tas E, Pippola S, Lindstrom K and van Elsas JD Extraction and analysis of microbial DNA from soil Nucleic Acids in Environment
三枝正彦、 松山信彦 1996 東北地方の耕地黒ポク土の活性アルミニウムとそれに関わる 2， Masahiko Saegusa, Nobuhiko Matsuyama 1996 Activated aluminum of cultivated black soil in Tohoku region
3の化学性 日本土壌肥料科学雑誌 67;(2)174-Γ78  Chemical properties of 3 Japan soil fertilizer science journal 67; (2) 174-Γ78
Sarah Macnaughton, John R. Stephen, Yun-duan Chang, Aaron Peacock, Cecily A. Sarah Macnaughton, John R. Stephen, Yun-duan Chang, Aaron Peacock, Cecily A.
Flemming, KamTin Leung, and David C. White 1999 Characterization of metal-resisitant soil eubacteria by polymerase chain reaction - denaturing gradient gel electrophoresis with isolation of resistatnt strains Canadian Journal of Flemming, KamTin Leung, and David C. White 1999 Characterization of metal-resisitant soil eubacteria by polymerase chain reaction-denaturing gradient gel electrophoresis with isolation of resistatnt strains Canadian Journal of
Microbiology 45; 116· 124 Microbiology 45; 116
Sarah J. Macnaughton, John R. Stephen, Albert D. Venosa, Gregory A. Davis, Yun-JuanSarah J. Macnaughton, John R. Stephen, Albert D. Venosa, Gregory A. Davis, Yun-Juan
Chang, and David C. White 1999 Microbial Population Changes duringChang, and David C. White 1999 Microbial Population Changes during
Bioremediation of an Experimental Oil Spill Applied and EnvironmentalBioremediation of an Experimental Oil Spill Applied and Environmental
Microbiology 65;(8)3566'3574 Microbiology 65; (8) 3566'3574
Schnitzer M. and S.I>M Skinner 2000 A Lifetime Perspective on the Chemistry of SoilSchnitzer M. and S.I> M Skinner 2000 A Lifetime Perspective on the Chemistry of Soil
Organic Matter Advances in Agronomy 68;l-58 Organic Matter Advances in Agronomy 68; l-58
Schnitzer M. 1966 OrgancMetallic Interactions in Soils: 5. Stability Constants of Cu++ -，Schnitzer M. 1966 Organc Metallic Interactions in Soils: 5. Stability Constants of Cu ++-,
FE++ -， and Z N++ -， Fulvic Acid Complexes Soil Sceince 102；(6)361-365 FE ++-, and Z N ++-, Fulvic Acid Complexes Soil Sceince 102; (6) 361-365
Shin onda, Akira Hiraishi, Yoshitaka Matsuo, and Susumu Takii 2002 Polyphasic approaches to the identification of predominant polyp hosphate-accumulating organisms in a laboratoryscale anaerobic/aerobic activated sludge system Journal of General and Applied Microbiology 48；43-54 Shin onda, Akira Hiraishi, Yoshitaka Matsuo, and Susumu Takii 2002 Polyphasic approaches to the identification of predominant polyp hosphate-accumulating organisms in a laboratoryscale anaerobic / aerobic activated sludge system Journal of General and Applied Microbiology 48; 43-54
Smedes F， Thomasen TK 1996 Evaluation of Blign & Dyer lipid determination methodSmedes F, Thomasen TK 1996 Evaluation of Blign & Dyer lipid determination method
Marine Pollution Bulletin32:681-688 ' Sneh Goyal, Kazunorj Sakamoto and Kazuyuki Inubushi 2000 Microbial Biomass andMarine Pollution Bulletin32: 681-688 'Sneh Goyal, Kazunorj Sakamoto and Kazuyuki Inubushi 2000 Microbial Biomass and
Activities along an Andosol Profile in Relation to Soil Organic Mtatter LevelActivities along an Andosol Profile in Relation to Soil Organic Mtatter Level
Microbes and Environments 15;(3)143-150 Microbes and Environments 15; (3) 143-150
Soren O. Petersen and Michael J. Klug 1994 Effects of Sieving, Storage, and IncubationSoren O. Petersen and Michael J. Klug 1994 Effects of Sieving, Storage, and Incubation
Temperature on the Phospholipid Fatty Acid Profile of a Soil Microbial Community Applied and Environmental Microbiology 60;(7)2421-2430 Temperature on the Phospholipid Fatty Acid Profile of a Soil Microbial Community Applied and Environmental Microbiology 60; (7) 2421-2430
S. P. Mathur 1971 Characterization of Soil Humus throug Enzymatic Degradation SoilS. P. Mathur 1971 Characterization of Soil Humus throug Enzymatic Degradation Soil
Sceince 111;(3)147_157 Sceince 111; (3) 147_157
S.R. Kehrmeyer, B.M. Applegate, H.C. Pinkart, D.B. Hedrick, D.C. White and G.S. S.R.Kehrmeyer, B.M.Applegate, H.C.Pinkart, D.B.Hedrick, D.C.White and G.S.
Sayler 1996 Combined丄 lpid/DNA extraction method for environmental samples Sayler 1996 Combined 丄 lpid / DNA extraction method for environmental samples
Journal of Microbiological Methods 25; 153· 163 Journal of Microbiological Methods 25; 153
Sven Becker, Peter Boger, Ralfli Oehlmann, and Anneliese Ernst 2000 PCR Bias inSven Becker, Peter Boger, Ralfli Oehlmann, and Anneliese Ernst 2000 PCR Bias in
Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities Applied and Environmental Microbiology 66；(11)4945-4953 Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities Applied and Environmental Microbiology 66; (11) 4945-4953
Tillmann Lueders and Michael W. Friedrich 2003 Evaluation of PCR Amplification Bias by Terminal Restriciton Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts Applied and Environmental Microbiology 69;(l)320-326  Tillmann Lueders and Michael W. Friedrich 2003 Evaluation of PCR Amplification Bias by Terminal Restriciton Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts Applied and Environmental Microbiology 69; (l) 320-326
ToUeson, T.S. and McKercher, R.B. 1983 The degradation of "Olabelede phosphatidyl choline in soil. Soil Biology Biochemistry 15; 145- 148 ToUeson, TS and McKercher, RB 1983 The degradation of "Olabelede phosphatidyl choline in soil.Soil Biology Biochemistry 15; 145-148
Torsvik V, Daae FL， Goksoyr J Extraction, purification, and analysis of DNA from soil bacteria Nucleic Acids in Environment Torsvik V, Daae FL, Goksoyr J Extraction, purification, and analysis of DNA from soil bacteria Nucleic Acids in Environment
Torsvik V， Sorheim R, Goksoyr J 1996 Total bacterial diversity in soil and sediment communities-a review Journal of Industrial Microbiology 17:170178 Torsvik V, Sorheim R, Goksoyr J 1996 Total bacterial diversity in soil and sediment communities-a review Journal of Industrial Microbiology 17: 170178
Tsai YL， Olson BH 1992 Rapid method for separation of bacterial'DNA from humic substances in sediments for polymerase chain -re action Applied and Environmental Tsai YL, Olson BH 1992 Rapid method for separation of bacterial 'DNA from humic substances in sediments for polymerase chain -reaction Applied and Environmental
Microbiology · 58: (7) 2292-2295 Microbiology58: (7) 2292-2295
宇智田奈津代、平舘俊太郎、故 井上克弘 2000ァロフェン， フェリハイドライト， 微粒炭， 活性炭の腐食酸吸着特性の比較 日本土壌肥料科学雑誌 71;(1)1-7 Natshiro Uchida, Shuntaro Hiradate, late Katsuhiro Inoue 2000 Comparison of adsorption properties of corrosive acid by Aarofen, ferrihydrite, pulverized coal and activated carbon Japan Soil Fertilizer Science 71; (1) 1-7
V Farrelly, FA Rainey and E Stackebrandt 1995 Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species Applied and Environmental Microbiology 61:(7) 2798-2801  V Farrelly, FA Rainey and E Stackebrandt 1995 Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species Applied and Environmental Microbiology 61: (7) 2798-2801
Velegraki A, Kambouris M， Kostourou A; Chalevelakis G， Legakis NJ 1999 Rapid extraction of fungai DNA from clinical samples for PCR amplification MedicalVelegraki A, Kambouris M, Kostourou A ; Chalevelakis G, Legakis NJ 1999 Rapid extraction of fungai DNA from clinical samples for PCR amplification Medical
Mycology 37:(1) 69-73 Mycology 37: (1) 69-73
Vigdis Lid Torsvik 1980 Isolation or Bacterial DNA from soil Soil Biology and Vigdis Lid Torsvik 1980 Isolation or Bacterial DNA from soil Soil Biology and
Biochemistry 12:(1) 15.21 Biochemistry 12: (1) 15.21
Watson RJ， Blackwell B 2000 Purification and characterization of a common soil component which inhibits the polymerase chain reaction Canadian Journal ofWatson RJ, Blackwell B 2000 Purification and characterization of a common soil component which inhibits the polymerase chain reaction Canadian Journal of
Microbiology 46： (7) 633-642 Microbiology 46: (7) 633-642
White DC, Davis WM, Nickels JS， King JD and Bobbie RJ 1979 Determination of the sedimentary microbial biomass by extractiole lipid phosphate Oecologia 40:51-62 White, D.C. and Tucker, A.N. 1969 Phospholipid metabolism during bacterial growth.  White DC, Davis WM, Nickels JS, King JD and Bobbie RJ 1979 Determination of the sedimentary microbial biomass by extractiole lipid phosphate Oecologia 40: 51-62 White, D.C. and Tucker, A.N. 1969 Phospholipid metabolism during bacterial growth.
Journal of Lipid Research 19; 115- 123  Journal of Lipid Research 19; 115- 123
Yu-li Tsai and Betty H. Olson 1991 Rapid Method for Direct Extraction of DNA from Yu-li Tsai and Betty H. Olson 1991 Rapid Method for Direct Extraction of DNA from
Soil and Sediments Applied and Environmental Microbiology 57:(4) 1070-1074 Yu-li Tsai, MarieJ. Park and Betty H. Olson 1991 Rapid Method for Direct Extraction of mRNA from Seeded Soils Applied and Environmental Microbiology 57:(3) 765-768 Soil and Sediments Applied and Environmental Microbiology 57: (4) 1070-1074 Yu-li Tsai, MarieJ. Park and Betty H. Olson 1991 Rapid Method for Direct Extraction of mRNA from Seeded Soils Applied and Environmental Microbiology 57: (3) 765- 768
Yun-Hwei Shen 1999 Sorption of Humic Acia to Soil: the Role of Soil Mineral Yun-Hwei Shen 1999 Sorption of Humic Acia to Soil: the Role of Soil Mineral
Composition C emosphere 38 ;(11)2489-2499 Composition C emosphere 38; (11) 2489-2499
Yutaka Oba 1996 火山灰土壌との 40年 肥料科学 18;45_65  Yutaka Oba 1996 40 years with volcanic ash soil Fertilizer science 18; 45_65
Zelles L. 1997 Phospholipid Fatty Acid Profiles in Selected Members of Soil Microbial Communities Chemosphere 35;(1, 2)275.294  Zelles L. 1997 Phospholipid Fatty Acid Profiles in Selected Members of Soil Microbial Communities Chemosphere 35; (1, 2) 275.294
Zelles L. 1999 Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review Biology and Fertility of Soils 29;111-129  Zelles L. 1999 Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review Biology and Fertility of Soils 29; 111-129
Zhu H， Q F, Zhu LH 1993 Isolation of genomic DNAs from plants, fungi and bacteria using benzyl-chloride Nucleic Acids Research 21: (22) 5279-5280 Zhu H, Q F, Zhu LH 1993 Isolation of genomic DNAs from plants, fungi and bacteria using benzyl-chloride Nucleic Acids Research 21: (22) 5279-5280
Hong Wang, Susanne E. Konalmi and Adrian J. Cutler An Improved Method for Polymerase Chain Reaction Using Whole Yeast Cells 3232 産業上の利用可能性 Hong Wang, Susanne E. Konalmi and Adrian J. Cutler An Improved Method for Polymerase Chain Reaction Using Whole Yeast Cells 3232 Industrial applicability
本発明により、 土壌から DNAを高収率で、 しかも高純度で回収し得る方法が 提供される。 本発明の方法により得られた DNAにはほとんど夾雑物が混入して おらず、 抽出および IS製時の DNAの損失を最小限に抑えることができている。 また、 本発明の実施に際し、 特殊な技術を必要とせず、 簡便でコストがかからな い。 従って、 本発明の方法は土壌微生物の群集構造解析および学問分野として土 壌微生物生態学、 コンビナトリアル生物学 (Combinatorial Biology)又はコンビナ トリアル遺伝学 (Combinatorial Genetics)など環境 DNAよりの新規遺伝子探索 への利用が可能である点で極めて有用である。 配列表フリーテキスト The present invention provides a method for recovering DNA from soil with high yield and high purity. Almost all contaminants are mixed in the DNA obtained by the method of the present invention. No DNA loss during extraction and IS production has been minimized. Further, the present invention does not require special techniques, and is simple and inexpensive. Therefore, the method of the present invention is useful for analyzing the microbial community structure of soil microorganisms and searching for novel genes from environmental DNA such as soil microbial ecology, combinatorial biology or combinatorial genetics as a discipline. This is extremely useful in that it is possible. Sequence listing free text
配列番号 1 ：合成 DNA  Sequence number 1: Synthetic DNA
配列番号 2 ：合成 DNA  Sequence number 2: Synthetic DNA
配列番号 3 ：合成 DNA  Sequence number 3: Synthetic DNA
配列番号 4 ：合成 DNA  SEQ ID NO: 4: Synthetic DNA

Claims

I . 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを処理す ることを特徴とする、 環境サンプルから DNAを抽出する方法。 I. A method for extracting DNA from an environmental sample, comprising treating the environmental sample in the presence of a DNA extract containing 5% or less of a surfactant.
2 . 界面活性剤が SDS、 CTAB、 Triton X-100及び N-ラウロイルサルコシンナ トリゥムからなる群から選ばれるいずれかのものである請求項 1記載の方 法。 2. The method according to claim 1, wherein the surfactant is any one selected from the group consisting of SDS, CTAB, Triton X-100, and N-lauroyl sarcosine sodium.
3 . DNA抽出液が、さらにリン酸一一口緩衝液及び/又は EDTAを含むものである請求 項 1記載の方法。  3. The method according to claim 1, wherein the DNA extract further contains a one-piece phosphate buffer and / or EDTA.
4 . DNA抽出液の pHが 7以上である請求項 1記載の方法。 4. The method according to claim 1, wherein the pH of the DNA extract is 7 or more.
5 . リン酸緩衝液の濃度が lOOmM〜： l500mM囲である請求項 3記載の方法。 5. The method according to claim 3, wherein the concentration of the phosphate buffer is in the range of 100 mM to 500 mM.
6 . EDTAの濃度が 50mM〜600mMである請求項 3記載の方法。 6. The method according to claim 3, wherein the concentration of EDTA is 50 mM to 600 mM.
7 . リン酸緩衝液の濃度が 100mM〜750mM であり、 かつ、 EDTA の濃度が 7. The concentration of the phosphate buffer is 100 mM to 750 mM, and the concentration of EDTA is
50mM〜600mMである請求項 3記載の方法。 4. The method according to claim 3, wherein the amount is 50 mM to 600 mM.
8 . 環境サンプルが、 土壌、 堆肥、 水系堆積物、 活性汚泥及び糞便からなる群か ら選ばれる少なくとも 1つである請求項 1記載の方法。 8. The method according to claim 1, wherein the environmental sample is at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge, and feces.
9 . 環境サンプルの処理が、環境サンプルを beads-beating処理及び/又は加熱処 理するものである請求項 1記載の方法。 9. The method according to claim 1, wherein the processing of the environmental sample is performed by beads-beating processing and / or heating processing of the environmental sample.
1 0 . 環境サンプルから DNAを抽出する方法であって、 10. A method for extracting DNA from environmental samples, comprising:
(a) 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 遠心後上清を採取し、  (c) Collect the supernatant after centrifugation,
(d) 上清の採取後の残存環境サンプルについて、上記 (a)〜(c)工程を 1回〜 4回繰り返す  (d) Repeat steps (a) to (c) once to four times for the remaining environmental sample after collecting the supernatant
工程を含む前記方法。  The method comprising the steps of:
I I . DNA抽出液が、さらにリン酸緩衝液及び/又は EDTAを含むものである請 求項 10記載の方法。 11. The method according to claim 10, wherein the DNA extract further contains a phosphate buffer and / or EDTA.
1 2 . EDTAの濃度 50〜600mMである請求項 11記載の方法。 12. The method according to claim 11, wherein the concentration of EDTA is 50 to 600 mM.
1 3 . リン酸緩衝液の濃度が 100〜2000mMである請求項 11記載の方法。 13. The method according to claim 11, wherein the concentration of the phosphate buffer is 100 to 2000 mM.
1 4 . 環境サンプルから DNAを抽出する方法であって、 1 4. A method for extracting DNA from environmental samples,
(a) 5 %以下の界面活性剤及び 50〜600mMの EDTAを含む DNA抽出液 の存在下で瑗境サンプルを beads-beating処理及び/又は加熱処理し、  (a) beads-beating and / or heat treatment of the environmental sample in the presence of a DNA extract containing 5% or less of surfactant and 50 to 600 mM EDTA,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心して上清を採 取し、  (b) The extract after beads-beating treatment and / or heat treatment is centrifuged and the supernatant is collected.
(c) 採取された上清と 600〜： LlOOmMの EDTAとを混合する  (c) Mix the collected supernatant with 600 ~: LlOOmM EDTA
工程を含む前記方法。  The method comprising the steps of:
1 5 . 環境サンプルから DNAを抽出する方法であって、 1 5. A method for extracting DNA from environmental samples,
(a) 5 %以下の界面活性剤及び 50〜600mMの EDTAを含む DNA抽出液 の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 mM EDTA, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清と 600〜1100mMの EDTAとの混合物を加熱処理する 工程を含む前記方法。  (c) The above method, comprising a step of heat-treating a mixture of the obtained supernatant and 600 to 1100 mM EDTA.
1 6 . 環境サンプルから DNAを抽出する方法であって、  1 6. A method for extracting DNA from environmental samples,
5 %以下の界面活性剤及び 250〜2000mMのリン酸緩衝液を含む DNA抽 出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理する 工程を含む前記方法。  The above method comprising the step of subjecting an environmental sample to beads-beating treatment and / or heat treatment in the presence of a DNA extract containing 5% or less of a surfactant and a phosphate buffer of 250 to 2000 mM.
1 7 . 環境サンプルから DNAを抽出する方法であって、 1 7. A method for extracting DNA from environmental samples,
5 %以下の界面活性剤、 100〜800mMの EDTA及び 250〜2000mMのリ ン酸緩衝液を含む DNA抽出液の存在下で環境サンプルを beads-beating処 理及び/又は加熱処理する  Treat environmental samples with beads-beating and / or heat in the presence of DNA extract containing 5% or less of surfactant, 100-800 mM EDTA and 250-2000 mM phosphate buffer
工程を含む前記方法。  The method comprising the steps of:
1 8 . EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある請求項 17記載の方法。 18. The method according to claim 17, wherein the concentration of EDTA is 400 mM, and the concentration of the phosphate buffer is 750 mM.
1 9 . 環境サンプルから DNAを抽出する方法であって、 1 9. A method for extracting DNA from environmental samples,
(a) 5 %以下の界面活性剤を含む DNA抽出液 Iの存在下で環境サンプル を beads-beating処理及び/又は加熱処理し、 (a) Environmental sample in the presence of DNA Extract I containing 5% or less of surfactant Is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液 Iを、 75〜： 1200mM の EDTA、 250〜3000mMのリン酸緩衝液、 又は前記 EDTAとリン酸緩衝 液との混合物と混合して抽出液 IIを調製し、  (b) The extract I after beads-beating treatment and / or heat treatment is mixed with 75 to: 1200 mM EDTA, 250 to 3000 mM phosphate buffer, or a mixture of the above EDTA and phosphate buffer. Prepare Extract II,
(c) 前記抽出液 IIから DNAを抽出する  (c) extracting DNA from the extract II
工程を含む前記方法。 The method comprising the steps of:
抽出液 Iと混合する EDTAの濃度が 400〜800mMである請求項 19記載 の方法。  The method according to claim 19, wherein the concentration of EDTA to be mixed with Extract I is 400 to 800 mM.
抽出液 I と混合するリン酸緩衝液の濃度が 750〜： L500mMである請求項 19記載の方法。  20. The method according to claim 19, wherein the concentration of the phosphate buffer mixed with the extract I is 750 to: L500 mM.
抽出液 Iと混合する EDTAの濃度が 400mMであり、 かつ、抽出液 Iと混 合するリン酸緩衝液の濃度が 750 mMである請求項 19記載の方法。  20. The method according to claim 19, wherein the concentration of EDTA mixed with the extract I is 400 mM, and the concentration of the phosphate buffer mixed with the extract I is 750 mM.
環境サンプルから DNAを抽出する方法であって、  A method of extracting DNA from an environmental sample,
(a) 5 %以下の界面活性剤、 400mM以下の EDTA及び 250mM以下のリ ン酸緩衝液を含む DNA抽出液 IIIの存在下で土壌サンプルを beads-beating 処理し、  (a) beads-beating the soil sample in the presence of DNA extract III containing 5% or less of surfactant, 400 mM or less of EDTA and 250 mM or less of phosphate buffer,
(b) beads-beating処理後の抽出液 IIIを、 400〜： lOOOmMの EDTA、 750 〜2050mMのリン酸緩衝液、 又は前記 EDTAとリン酸緩衝液との混合物と 混合して抽出液 IVを調製し、  (b) Extract III after beads-beating treatment is mixed with 400-: 100 mM EDTA, 750-2050 mM phosphate buffer, or a mixture of EDTA and phosphate buffer to prepare extract IV And
(c) 前記抽出液 IVから DNAを抽出する  (c) extracting DNA from the extract IV
工程を含む前記方法。 The method comprising the steps of:
DNA .を抽出する工程が、抽出液 IVを加熱処理した後、遠心するものであ る請求項 23記載の方法。  24. The method according to claim 23, wherein the step of extracting DNA comprises subjecting the extract IV to heat treatment and then centrifuging.
抽出液 IIIにおいて、 EDTAの濃度が 300mMであり、 かつ、 リン酸緩衝 液の濃度が lOOmMである請求項 23記載の方法。  24. The method according to claim 23, wherein in the extract III, the concentration of EDTA is 300 mM, and the concentration of the phosphate buffer is 100 mM.
抽出液 IIIと混合する EDTAの濃度が 400mMであり、 かつ、 抽出液 III と混合するリン酸緩衝液の濃度が 750mMである請求項 23記載の方法。 環境サンプルから DNAを抽出する方法であって、 (a) 100〜400mM の EDTA及び 250〜1500mM のリン酸緩衝液を含む DNA抽出液 Vの存在下で環境サンプルを加熱処理し、 24. The method according to claim 23, wherein the concentration of EDTA mixed with Extract III is 400 mM, and the concentration of phosphate buffer mixed with Extract III is 750 mM. A method of extracting DNA from an environmental sample, (a) heat-treating the environmental sample in the presence of a DNA extract V containing 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer,
(b) 加熱処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after the heat treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の界面活性剤、 400mM 以下の EDTA及び 250mM以下のリン酸緩衝液を含む DNA抽出液 IIIの存在下で beads-beating処理し、  (c) beads-beating treatment of the remaining environmental sample in the presence of DNA extract III containing 5% or less of a surfactant, 400 mM or less of EDTA and 250 mM or less of a phosphate buffer,
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取する 工程を含む前記方法。  (d) The above method comprising a step of centrifuging the extract III after beads-beating treatment and collecting a supernatant.
環境サンプルから DNAを抽出する方法であって、  A method of extracting DNA from an environmental sample,
(a) 100〜400mM の EDTA及び 250〜： L500mM のリン酸緩衝液を含む (a) 100 to 400 mM EDTA and 250 to: L500 mM phosphate buffer
DNA抽出液 Vの存在下で環境サンプルを beads-beating処理し、 Beads-beating treatment of the environmental sample in the presence of DNA extract V,
(b) beads-beating処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after beads-beating treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の界面活性剤、 400mM 以下の EDTA及び 250πιΜ以下のリン酸緩衝液を含む DNA抽出液 IIIの存在下で beads-beating処理.し、  (c) Treat the remaining environmental sample with beads-beating treatment in the presence of DNA extract III containing 5% or less of surfactant, 400 mM or less of EDTA, and 250 πιΜ or less of phosphate buffer,
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取する 工程を含む前記方法。  (d) The above method comprising a step of centrifuging the extract III after beads-beating treatment and collecting a supernatant.
環境サンプルから DNAを抽出する方法であって、  A method of extracting DNA from an environmental sample,
(a) 200~800mM の EDTA及び 250〜2000mM のリン酸緩衝液を含む DNA抽出液の存在下で環境サンプルを第一加熱処理し、  (a) first heat treating the environmental sample in the presence of a DNA extract containing 200-800 mM EDTA and 250-2000 mM phosphate buffer,
(b) 第一加熱処理後の環境サンプルと、 5 %以下の界面活性剤とを混合し て当該混合物を第二加熱処理する  (b) Mix the environmental sample after the first heat treatment with 5% or less of the surfactant and heat-treat the mixture with the second heat treatment
工程を含む前記方法。 The method comprising the steps of:
環境サンプルから DNAを抽出する方法であって、  A method of extracting DNA from an environmental sample,
(a) 100〜400mM の EDTA及び 250〜1500mM のリン酸緩衝液を含む (a) Contains 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer
DNA抽出液の存在下で環境サンプルを beads-beating処理し、 Beads-beating treatment of environmental samples in the presence of DNA extract,
(b) beads-beating処理後の環境サンプルと 5 %以下の界面活性剤とを混 合して当該混合物を加熱処理する 工程を含む前記方法。 (b) Mix the environmental sample after beads-beating treatment with 5% or less of surfactant and heat-treat the mixture. The method comprising:
3 1 . 抽出液 Vにおいて、 EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝 液の濃度が 750mMである請求項 27又は 28記載の方法。  31. The method according to claim 27 or 28, wherein the concentration of EDTA in the extract V is 400 mM, and the concentration of the phosphate buffer is 750 mM.
3 2 . EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある請求項 29又は 30記載の方法。 32. The method according to claim 29, wherein the concentration of EDTA is 400 mM, and the concentration of the phosphate buffer is 750 mM.
3 3 . 環境サンプル由来の DNAを、 陽イオン界面活性剤及び塩の存在下で精製 することを特徴とする DNAの精製方法。  33. A method for purifying DNA, comprising purifying DNA derived from an environmental sample in the presence of a cationic surfactant and a salt.
3 4 . 環境サンプル由来の DNAが、 請求項 1〜32のいずれか 1項に記載の方 法により抽出された DNAである請求項 33記載の方法。 34. The method according to claim 33, wherein the DNA derived from the environmental sample is DNA extracted by the method according to any one of claims 1 to 32.
3 5 . 陽イオン界面活性剤が CTABである請求項 33又は 34記載の方法。 35. The method according to claim 33 or claim 34, wherein the cationic surfactant is CTAB.
3 6 . 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモニゥ ム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからなる群 から選択される少なくとも 1つである請求項 33又は 34記載の方法。  36. The method according to claim 33, wherein the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate. .
3 7 . 陽イオン界面活性剤の濃度が 1〜3%である請求項 33又は 34記載の方法。 3 8 . 塩の濃度が 0.7〜2.1Mである請求項 33又は 34記載の方法。 37. The method according to claim 33 or 34, wherein the concentration of the cationic surfactant is 1 to 3%. 38. The method according to claim 33 or claim 34, wherein the salt concentration is 0.7 to 2.1M.
3 9 . 陽イオン界面活性剤の濃度が 2〜3 %であり、 かつ、 塩の濃度が 1.0Mで ある請求項 33又は 34記載の方法。  39. The method according to claim 33 or 34, wherein the concentration of the cationic surfactant is 2-3%, and the concentration of the salt is 1.0M.
4 0 . pH7.0未満の条件で精製することを特徴とする請求項 33又は 34記載の 方法。 '  35. The method according to claim 33 or 34, wherein the purification is performed under a condition of 40 or less than pH 7.0. '
4 1 . 請求項 33〜40のいずれか 1項に記載の方法により精製された DNAを、 2-プロパノール、 エタノール又はポリエチレンダリコールの存在下で沈殿さ せることを特徴とする DNAの回収方法。 41. A method for recovering DNA, comprising precipitating DNA purified by the method according to any one of claims 33 to 40 in the presence of 2-propanol, ethanol, or polyethylenedaricol.
4 2 . pH7.0以上の条件で沈殿させることを特徴とする請求項 41記載の方法。 4 3 . ポリエチレングリコ一ルの濃度が 5〜7.5%である請求項 41記載の方法。 4 4 . 請求項 1〜 9のいずれか 1項に記載の方法により抽出された DNAを、 2- プロパノール、エタノール又はポリエチレングリコールの存在下で沈殿させ ることを特徴とする DNAの回収方法。 42. The method according to claim 41, wherein the precipitation is performed under a condition of pH 7.0 or higher. 43. The method according to claim 41, wherein the concentration of the polyethylene glycol is 5 to 7.5%. 44. A method for recovering DNA, comprising precipitating DNA extracted by the method according to any one of claims 1 to 9 in the presence of 2-propanol, ethanol, or polyethylene glycol.
4 5 . 環境サンプルから DNAを回収する方法であって、 (a) 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、 4 5. A method for recovering DNA from environmental samples, (a) In the presence of a DNA extract containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清から DNAを回収する  (c) recover DNA from the resulting supernatant
工程を含む M記方法。  M notation including process.
4 6 . DNA抽出液が、さらにリン酸緩衝液及び/又は EDTAを含むものである請 求項 45記載の方法。  46. The method according to claim 45, wherein the DNA extract further contains a phosphate buffer and / or EDTA.
4 7 . EDTAの濃度が 50〜600mMである請求項 46記載の方法。 47. The method according to claim 46, wherein the concentration of EDTA is 50-600 mM.
4 8 . リン酸緩衝液の濃度が 100〜2000mMである請求項 46記載の方法。 4 9 . 環境サンプルから DNAを回収する方法であって、  48. The method according to claim 46, wherein the concentration of the phosphate buffer is 100 to 2000 mM. 4 9. A method for recovering DNA from environmental samples,
(a) 5 %以下の界面活性剤を含む DNA抽出液の存在下で環境サンプルを beads-beating処理又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant, an environmental sample is subjected to beads-beating treatment or heat treatment,
(b) beads-beating処理又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment or heat treatment,
(c) 得られる上清から DNAを回収し、  (c) recover the DNA from the resulting supernatant,
(d) DNA回収後の残存環境サンプルについて、 上記 (a)〜(c)工程を 1回〜 (d) For the remaining environmental sample after DNA recovery, perform the above steps (a) to (c) once
4回繰り返す Repeat 4 times
工程を含む前記方法。  The method comprising the steps of:
5 0 . DNA抽出液が、 さらにリン酸緩衝液及び EDTAを含むものである請求項 50. The DNA extract further comprises a phosphate buffer and EDTA.
49記載の方法。 49. The method of claim 49.
5 1 . EDTAの濃度が 50 mM〜600mMである請求項 50記載の方法。 51. The method according to claim 50, wherein the concentration of EDTA is 50 mM to 600 mM.
5 2 . リン酸緩衝液の濃度が 100〜2000mMである請求項 50記載の方法。 5 3 . 環境サンプルから DNAを回収する方法であって、  52. The method according to claim 50, wherein the concentration of the phosphate buffer is 100 to 2000 mM. 5 3. A method for recovering DNA from environmental samples,
(a) 5 %以下の界面活性剤及び 50〜600mMの EDTAを含む DNA抽出液 の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 mM EDTA, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心して上清を採 取し、  (b) The extract after beads-beating treatment and / or heat treatment is centrifuged and the supernatant is collected.
(c) 採取された上清と 600〜1100mMの EDTAとを混合して当該混合物を ¾s心し、 (d) 得られる上清から DNAを回収する (c) The collected supernatant is mixed with 600 to 1100 mM EDTA, and the mixture is centrifuged. (d) Recover DNA from the resulting supernatant
工程を含む前記方法。The method comprising the steps of:
. 環境サンプルから DNAを回収する方法であって、 A method of recovering DNA from an environmental sample, comprising:
(a) 5 %以下の界面活性剤及び 50〜600mMの EDTAを含む DNA抽出液 の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant and 50 to 600 mM EDTA, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清と 600〜： LlOOmMの EDTAとを混合して当該混合物を加 熱処理し、  (c) The obtained supernatant is mixed with 600 ~: LlOOmM EDTA, the mixture is heat-treated,
(d) 加熱後の抽出液を遠心し、  (d) Centrifuge the heated extract,
(e) 得られる上清から DNAを回収する  (e) Recover DNA from the resulting supernatant
工程を含む前記方法。 The method comprising the steps of:
環境サンプルから DNAを回収する方法であって、  A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤及び 250〜2000mM のリン酸緩衝液を含む DNA抽出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処 理し、  (a) In the presence of a DNA extract containing 5% or less of a surfactant and a phosphate buffer of 250 to 2000 mM, an environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清から DNAを回収する  (c) recover DNA from the resulting supernatant
工程を含む前記方法。 The method comprising the steps of:
環境サンプルから DNAを回収する方法であって、  A method for recovering DNA from an environmental sample,
(a) 5 %以下の界面活性剤、 100〜800mMの EDTA及び 250〜2000mM のリ ン酸緩衝液を含む DNA 抽出液の存在下で環境サンプルを beads-beating処理及び/又は加熱処理し、  (a) Beads-beating and / or heat treatment of the environmental sample in the presence of a DNA extract containing 5% or less of surfactant, 100 to 800 mM EDTA and 250 to 2000 mM phosphate buffer,
(b) beads-beating処理及び/又は加熱処理後の抽出液を遠心し、  (b) centrifuging the extract after beads-beating treatment and / or heat treatment,
(c) 得られる上清から DNAを回収する  (c) recover DNA from the resulting supernatant
工程を含む前記方法。 The method comprising the steps of:
EDTAの濃度が 400raMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある請求項 56記載の方法。  57. The method according to claim 56, wherein the concentration of EDTA is 400 raM, and the concentration of the phosphate buffer is 750 mM.
環境サンプルから DNAを.回収する方法であって、 (a) 5 %以下の界面活性剤を含む DNA抽出液 Iの存在下で環境サンプル を beads-beating処理及び/又は加熱処理し、 A method of recovering DNA from an environmental sample, (a) In the presence of DNA extract I containing 5% or less of surfactant, the environmental sample is subjected to beads-beating treatment and / or heat treatment,
(b) beads-beating処理及び/又は加熱処理後の抽出液 Iを、 75〜： l200mM の EDTA、 250〜3000mMのリン酸緩衝液、 又は前記 EDTAとリン酸緩衝 液との混合物と混合して抽出液 IIを調製し、  (b) Extract I after beads-beating and / or heat treatment is mixed with 75 to: 200 mM EDTA, 250 to 3000 mM phosphate buffer, or a mixture of EDTA and phosphate buffer. Prepare Extract II,
(c) 抽出液 IIを遠心し、  (c) Centrifuge Extract II,
(d) 得られる上清から DNAを回収する  (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
5 9 . 抽出液 Iと混合する EDTAの濃度が 400〜800mMである請求項 58記載 の方法。  59. The method according to claim 58, wherein the concentration of EDTA to be mixed with the extract I is 400 to 800 mM.
6 0 . 抽出液 I と混合するリン酸緩衝液の濃度が 750〜1500mMである請求項 60. The concentration of the phosphate buffer mixed with the extract I is 750 to 1500 mM.
58記載の方法。 58. The method of paragraph 58.
6 1 . 抽出液 Iと混合する EDTAの濃度が 400mMであり、 かつ、抽出液 Iと混 合するリン酸緩衝液の濃度が 750 mMである請求項 58記載の方法。  61. The method according to claim 58, wherein the concentration of EDTA mixed with Extract I is 400 mM, and the concentration of phosphate buffer mixed with Extract I is 750 mM.
6 2 . 環境サンプルから DNAを回収する方法であって、 6 2. A method for recovering DNA from environmental samples,
(a) 5 %以下の界面活性剤、 400mM以下の EDTA及び 250mM以下のリ ン酸緩衝液を含む DNA抽出液 IIIの存在下で環境サンプルを beads-beating 処理し、  (a) Beads-beating the environmental sample in the presence of DNA extract III containing 5% or less of surfactant, 400 mM or less of EDTA and 250 mM or less of phosphate buffer,
(b) beads-beating処理後の抽出液 IIIを、 400〜： lOOOmMの EDTA、 750 〜2050mMのリン酸緩衝液、 又は前記 EDTAとリン酸緩衝液との混合物と 混合して抽出液 IVを調製し、  (b) Extract III after beads-beating treatment is mixed with 400-: 100 mM EDTA, 750-2050 mM phosphate buffer, or a mixture of EDTA and phosphate buffer to prepare extract IV And
(c) 抽出液 IVを遠心じ、  (c) Centrifuge Extract IV,
(d) 得られる上清から DNAを回収する  (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
6 3 . 抽出液 IVを加熱処理した後、 遠心することを特徴とする請求項 62記載 の方法。 63. The method according to claim 62, wherein the extract IV is heated and then centrifuged.
6 4 . 抽出液 IIIにおいて、 EDTAの濃度が 300mMであり、 かつ、 リン酸緩衝 液の濃度が lOOmMである請求項 62又は 63記載の方法。 64. The method according to claim 62 or 63, wherein the extract III has an EDTA concentration of 300 mM and a phosphate buffer concentration of 100 mM.
. 抽出液 IIIと 合する EDTAの濃度が 400mMであり、 かつ、 抽出液 III と混合するリン酸緩衝液の濃度が 750mMである請求項 62又は 63記載の方 法。64. The method according to claim 62 or 63, wherein the concentration of EDTA combined with Extract III is 400 mM, and the concentration of phosphate buffer mixed with Extract III is 750 mM.
. 環境サンプルから DNAを回収する方法であって、 A method of recovering DNA from an environmental sample, comprising:
(a) 100〜4'00mM の EDTA及び 250〜： !500mM のリン酸緩衝液を含む (a) 100 ~ 4'00mM EDTA and 250 ~: contains! 500mM phosphate buffer
DNA抽出液 Vの存在下で環境サンプルを加熱処理し、 Heat-treat the environmental sample in the presence of DNA extract V,
(b) 加熱処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after the heat treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の界面活性剤、 400mM 以下の EDTA及び 250mM以下のリン酸緩衝液を含む DNA抽出液 IIIの存在下で beads-beating処 し、  (c) beads-beating the remaining environmental sample in the presence of DNA extract III containing 5% or less of surfactant, 400 mM or less of EDTA and 250 mM or less of phosphate buffer,
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取し、  (d) Centrifuge Extract III after beads-beating treatment and collect supernatant,
(e) 工程 (b)及び/又は (d)において得られた上清から DNAを回収する 工程を含む前記方法。  (e) The above method, comprising the step of collecting DNA from the supernatant obtained in step (b) and / or (d).
環境サンプルから DNAを回収する方法であって、  A method for recovering DNA from an environmental sample,
(a) 100〜400mM の EDTA及び 250〜1500mM のリン酸緩衝液を含む (a) Contains 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer
DNA抽出液 Vの存在下で環境サンプルを beads-beating処理し、 Beads-beating treatment of the environmental sample in the presence of DNA extract V,
(b) beads-beating処理後の抽出液 Vを遠心して上清を採取し、  (b) The extract V after beads-beating treatment is centrifuged to collect the supernatant,
(c) 残存した環境サンプルを、 5 %以下の SDS、 400mM以下の EDTA及 び 250mM 以下のリン酸緩衝液を含む DNA 抽出液 III の存在下で beads-beating処理し、  (c) beads-beating the remaining environmental sample in the presence of DNA extract III containing 5% or less of SDS, 400 mM or less of EDTA, and 250 mM or less of phosphate buffer,
(d) beads-beating処理後の抽出液 IIIを遠心して上清を採取し、  (d) Centrifuge Extract III after beads-beating treatment and collect supernatant,
(e) 工程 (b)及び/又は (d)において得られた上清から DNAを回収する 工程を含む前記方法。  (e) The above method, comprising the step of collecting DNA from the supernatant obtained in step (b) and / or (d).
環境サンプルから DNAを回収する方法であって、  A method for recovering DNA from an environmental sample,
(a) 200〜800mM の EDTA及び 250〜2000mM のリン酸緩衝液を含む (a) Contains 200 to 800 mM EDTA and 250 to 2000 mM phosphate buffer
DNA抽出液の存在下で環境サンプルを第一加熱処理し、 First heat treatment of the environmental sample in the presence of the DNA extract,
(b) 5 %以下の界面活性剤と環境サンプルとを混合して当該混合物を第二 加熱処理し、 (c) 第二加熱処理後の抽出液を遠心し、 (b) 5% or less of a surfactant and an environmental sample are mixed and the mixture is subjected to a second heat treatment, (c) centrifuging the extract after the second heat treatment,
(d) 得られる上清から DNAを回収する  (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
6 9 . 環境サンプルから DNAを回収する方法であって、  6 9. A method for recovering DNA from environmental samples,
(a) 100〜400mM の EDTA及び 250〜1500mM のリン酸緩衝液を含む (a) Contains 100 to 400 mM EDTA and 250 to 1500 mM phosphate buffer
DNA抽出液の存在下で環境サンプルを beads-beating処理し、 Beads-beating treatment of environmental samples in the presence of DNA extract,
(b) beads-beating処理後の環境サンプルと 5 %以下の界面活性剤とを混 合して当該混合物を加熱処理し、  (b) The environment sample after beads-beating treatment is mixed with 5% or less of a surfactant, and the mixture is heat-treated,
(c) 加熱処理後の抽出液を遠心し、  (c) centrifuging the extract after the heat treatment,
(d) 得られる上清から DNAを回収する  (d) Recover DNA from the resulting supernatant
工程を含む前記方法。  The method comprising the steps of:
7 0 . 抽出液 Vにおいて、 EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝 液の濃度が 750mMである請求項 66又は 67記載の方法。  70. The method according to claim 66 or 67, wherein the concentration of EDTA in the extract V is 400 mM, and the concentration of the phosphate buffer is 750 mM.
' 7 1 . EDTAの濃度が 400mMであり、 かつ、 リン酸緩衝液の濃度が 750mMで ある請求項 68又は 69記載の方法。 71. The method according to claim 68, wherein the concentration of EDTA is 400 mM, and the concentration of the phosphate buffer is 750 mM.
7 2 . DNAの回収が、 遠心後の上清と陽イオン界面活性剤及び塩とを混合して 7 2. The DNA was recovered by mixing the supernatant after centrifugation with a cationic surfactant and salt.
DNAを精製する工程を含む請求項 45〜57のいずれか 1項に記載の方法。 7 3 . DNAの回収が、抽出液 IIと陽イオン界面活性剤及び塩とを混合して DNA を精製する工程を含む請求項 58〜61のいずれか 1項に記載の方法。 58. The method according to any one of claims 45 to 57, comprising a step of purifying the DNA. 73. The method according to any one of claims 58 to 61, wherein recovering the DNA comprises a step of mixing the extract II with a cationic surfactant and a salt to purify the DNA.
7 4 . DNA の回収が、 抽出液 IV と陽イオン界面活性剤及び塩とを混合して DNAを精製する工程を含む請求項 62〜65のいずれか 1項に記載の方法。 7 5 . DNAの回収が、加熱処理後の抽出液 V及び/又は beads-beating後の抽出 液 IIIと陽イオン界面活性剤及び塩とを混合して DNAを精製する工程を含 む請求項 66記載の方法。  74. The method according to any one of claims 62 to 65, wherein recovering the DNA comprises a step of purifying the DNA by mixing the extract IV with a cationic surfactant and a salt. 75. 70. The method for recovering DNA, comprising the step of mixing the extract V after heat treatment and / or the extract III after beads-beating with a cationic surfactant and a salt to purify the DNA. The described method.
7 6 . DNAの回収が、 beads-beating後の抽出液 III及び/又は beads-beating 後の抽出液 Vと陽イオン界面活性剤及び塩とを混合して DNAを精製するェ 程を含む請求項 67記載の方法。  76 6. The method for recovering DNA includes a step of mixing the extract III after beads-beating and / or the extract V after beads-beating with a cationic surfactant and a salt to purify the DNA. 67. The method of claim 67.
7 7 . DNAの回収が、 第二加熱後の抽出液と陽イオン界面活性剤及び塩とを混 合して DNAを If製する工程を含む請求項 68記載の方法。 7 7. The DNA was recovered by mixing the extract after the second heating with a cationic surfactant and salt. 69. The method according to claim 68, further comprising the step of producing DNA if.
7 8 . DNAの回収が、 加熱処理後の抽出液と陽イオン界面活性剤及び塩とを混 合して DNAを精製する工程を含む請求項 69記載の方法。 78. The method according to claim 69, wherein recovering the DNA comprises a step of mixing the extract after the heat treatment with a cationic surfactant and a salt to purify the DNA.
7 9 . 陽イオン界面活性剤が CTABである請求項 72〜78のいずれか 1項に記載 の方法。 '  79. The method according to any one of claims 72 to 78, wherein the cationic surfactant is CTAB. '
8 0 . 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモニゥ ム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからなる群 から選択される少なくとも 1つである請求項 72〜78のいずれか 1項に記載 の方法。  80. The salt according to any one of claims 72 to 78, wherein the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate. The method described in paragraph 1.
8 1 . 混合液中における陽イオン界面活性剤の濃度が 1〜3%である請求項 72〜 78のいずれか 1項に記載の方法。 ' 81. The method according to any one of claims 72 to 78, wherein the concentration of the cationic surfactant in the mixture is 1 to 3%. '
8 2 . 混合液中における塩の濃度が 0.7〜2.1Mである請求項 72〜78のいずれか 1項に記載の方法。 82. The method according to any one of claims 72 to 78, wherein the concentration of the salt in the mixture is 0.7 to 2.1M.
8 3 . 混合液中における陽イオン界面活性剤の濃度が 2〜3 %であり、 かつ、 混 合液中における塩の濃度が 1.0Mである請求項 72〜78のいずれか 1項に記 載の方法。  83. The method according to any one of claims 72 to 78, wherein the concentration of the cationic surfactant in the mixture is 2 to 3%, and the concentration of the salt in the mixture is 1.0M. the method of.
8 4 . pH7.0未満の条件で精製することを特徴とする請求項 72〜78のいずれか 1項に記載の方法。  84. The method according to any one of claims 72 to 78, wherein the purification is performed under conditions of a pH of less than 7.0.
8 5 . DNA の回収が、 2-プロパノール、 エタノール又はポリエチレングリコ一 ルの存在下で DNAを沈殿させる工程を含む請求項 45〜84のいずれか 1項 に記載の方法。  85. The method according to any one of claims 45 to 84, wherein recovering the DNA comprises precipitating the DNA in the presence of 2-propanol, ethanol or polyethylene glycol.
8 6 . ポリエチレングリコールの濃度が 5.0〜7.5%である請求項 85記載の方法。 8 7 . pH7.0以上の条件で DNAを沈殿させることを特徴とする請求項 85記載 の方法。  86. The method of claim 85, wherein the concentration of polyethylene glycol is between 5.0 and 7.5%. 87. The method according to claim 85, wherein the DNA is precipitated under conditions of pH 7.0 or higher.
8 8 . 環境サンプルから DNAを回収する方法であって、 8 8. A method of recovering DNA from environmental samples,
(a) 1%の界面活性剤、 400mMの EDTA及び 750mMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを処理して DNAを抽出し、  (a) extracting the DNA by treating the environmental sample in the presence of a DNA extract containing 1% detergent, 400 mM EDTA and 750 mM phosphate buffer,
(b) DNA抽出液と 2%の CTAB及び 1 Mの塩とを混合して DNAを精製し、 (d) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記方法。 (b) DNA is purified by mixing the DNA extract with 2% CTAB and 1 M salt, (d) The method comprising a step of precipitating the purified DNA in the presence of polyethylene glycol.
環境サンプルから DNAを回収する方法であって、  A method for recovering DNA from an environmental sample,
(a) 1%の界面活性剤、 300mMの EDTA及び lOOmMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを beads-beating処理し、  (a) beads-beating the environmental sample in the presence of a DNA extract containing 1% surfactant, 300 mM EDTA and 100 mM phosphate buffer,
(b) beads-beating処理後の抽出液と、 600mMの EDTA及び 2050mMの リン酸緩衝液とを混合し、  (b) Mix the extract after beads-beating treatment with 600 mM EDTA and 2050 mM phosphate buffer,
(c) 工程 (b)により得られる抽出液を遠心し、  (c) centrifuging the extract obtained in step (b),
(d) 得られる上清と、 2%の CTAB及び 1 Mの塩とを混合して DNAを精 製し、  (d) The resulting supernatant is mixed with 2% CTAB and 1 M salt to purify DNA,
(e) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記方法。  (e) the method comprising a step of precipitating the purified DNA in the presence of polyethylene glycol;
環境サンプルから DNAを回収する方法であって、  A method for recovering DNA from an environmental sample,
(a) 1 %の界面活性剤、 300mMの EDTA及び lOOmMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを beads-beating処理し、  (a) beads-beating the environmental sample in the presence of a DNA extract containing 1% surfactant, 300 mM EDTA and 100 mM phosphate buffer,
(b) beads-beating処理後の抽出液と、 600mMの EDTA及び 2050mMの リン酸緩衝液とを混合して加熱処理し、  (b) The extract after beads-beating treatment is mixed with 600 mM EDTA and 2050 mM phosphate buffer and heat-treated.
(c) 加熱処理後の抽出液を遠心し、  (c) centrifuging the extract after the heat treatment,
(d) 得られる上清と、 2%の CTAB及び 1 Mの塩とを混合して DNAを精 製し、  (d) The resulting supernatant is mixed with 2% CTAB and 1 M salt to purify DNA,
(e) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記方法  (e) precipitating the purified DNA in the presence of polyethylene glycol.
環境サンプルから DNAを回収する方法であって、  A method for recovering DNA from an environmental sample,
(a) 1 %の界面活性剤、 300mMの EDTA及び lOOmMのリン酸緩衝液を含 む DNA抽出液の存在下で環境サンプルを beads-beating処理し、  (a) beads-beating the environmental sample in the presence of a DNA extract containing 1% surfactant, 300 mM EDTA and 100 mM phosphate buffer,
(b) beads-beating処理後の抽出液を遠心し、  (b) Centrifuge the extract after beads-beating treatment,
(c) 得られる上清と、 2% CTAB及び 1 Mの塩とを混合して DNAを精製し、 (c) The resulting supernatant is mixed with 2% CTAB and 1 M salt to purify DNA,
(d) 精製された DNAを、 ポリエチレングリコールの存在下で沈殿させる 工程を含む前記 法。 (d) Precipitating the purified DNA in the presence of polyethylene glycol The above method comprising the steps of:
9 2 . 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモニゥ ム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからなる群 から選択される少なくとも 1つである請求項 88〜91のいずれか 1項に記載 の方法。 '  92. The salt according to any one of claims 88 to 91, wherein the salt is at least one selected from the group consisting of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate, and ammonium phosphate. The method described in paragraph 1. '
9 3 . ポリエチレングリコールの濃度が 5.0〜7.5%である請求項 88〜91のいず れか 1項に記載の方法。  93. The method according to any one of claims 88 to 91, wherein the concentration of the polyethylene glycol is 5.0 to 7.5%.
9 4。 pH7.0以上の条件で DNAを沈殿させることを特徴とする請求項 88〜91 のいずれか 1項に記載の方法。 9 4. The method according to any one of claims 88 to 91, wherein DNA is precipitated under conditions of pH 7.0 or more.
9 5 . 5 %以下の界面活性剤、又は前記界面活性剤と beads-beating用ビーズと の組合せを含む、 環境サンプルからの DNA抽出用キット。 A kit for extracting DNA from an environmental sample, comprising 95.5% or less of a surfactant or a combination of the surfactant and beads for beads-beating.
9 6 . アルカリ性緩衝液並びに EDTA及び/又はリン酸緩衝液をさらに含む請求 項 95記載のキット。 96. The kit according to claim 95, further comprising an alkaline buffer and an EDTA and / or phosphate buffer.
9 7 . アル力リ性緩衝液が Tris緩衝液である請求項 96記載のキット。 97. The kit of claim 96, wherein the alkaline buffer is a Tris buffer.
9 8 . EDTAの濃度が 50mM〜1200mMの範囲から選ばれるいずれかのもので ある請求項 96記載のキット。 98. The kit according to claim 96, wherein the EDTA concentration is any one selected from the range of 50 mM to 1200 mM.
9 9 . リン酸緩衝液の濃度が 50mM〜3000mMの範囲から選ばれるいずれかの ものである請求項 96記載のキット。  99. The kit according to claim 96, wherein the concentration of the phosphate buffer is any one selected from the range of 50 mM to 3000 mM.
1 0 0 . DNA抽出液の pHを 7.0以上に調整することができる請求項 96記載の キット。  100. The kit according to claim 96, wherein the pH of the DNA extract can be adjusted to 7.0 or more.
1 0 1 . 酸性側の pKaを有する pH緩衝液を含む塩溶液、陽イオン界面活性剤、 又は当該塩溶液と陽イオン界面活性剤との混合物を含む、環境サンプルから の DNA精製用キット。  101. A kit for purifying DNA from an environmental sample, comprising a salt solution containing a pH buffer having an acidic pKa, a cationic surfactant, or a mixture of the salt solution and a cationic surfactant.
1 0 2 . 酸性側の pKaを有する pH緩衝液が酢酸緩衝液、 リン酸緩衝液、 塩酸 緩衝液又は硫酸緩衝液である請求項 101記載のキット。  102. The kit according to claim 101, wherein the pH buffer having an acidic pKa is an acetate buffer, a phosphate buffer, a hydrochloric acid buffer, or a sulfate buffer.
1 0 3 . 陽イオン界面活性剤が CTABである請求項 101記載のキット。  103. The kit of claim 101, wherein the cationic surfactant is CTAB.
1 0 4 . 塩が、 塩化ナトリウム、 酢酸ナトリウム、 酢酸カリウム、 酢酸アンモ 二ゥム、 リン酸ナトリウム、 リン酸カリウム及びリン酸アンモニゥムからな る群から選択される少なくとも 1つである請求項 101記載のキット。 104. The salt consists of sodium chloride, sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate and ammonium phosphate. The kit according to claim 101, which is at least one selected from the group consisting of:
105. DNA含有溶液の pHを 7.0未満に調整することができる請求項 101記 載のキット。 105. The kit of claim 101, wherein the pH of the DNA-containing solution can be adjusted to less than 7.0.
106. アルカリ性緩衝液を含む、 環境サンプルからの DNA回収用キット。 1 07. アル力リ性の緩衝液が Tris緩衝液である請求項 106記載のキット。 108. さらに 2-プロパノール、 エタノール又はポリエチレングリコールを含 む、 請求項 106記載のキット。  106. Kit for recovering DNA from environmental samples, including alkaline buffer. 107. The kit of claim 106, wherein the alkaline buffer is a Tris buffer. 108. The kit of claim 106, further comprising 2-propanol, ethanol or polyethylene glycol.
109. DNA含有溶液の pHを 7.0以上に調整することができる請求項 106記 載のキッ卜。 109. The kit according to claim 106, wherein the pH of the DNA-containing solution can be adjusted to 7.0 or more.
1 10. 請求項 95〜： L00のいずれか 1項に記載の DNA抽出用キット、 請求項 101〜105のいずれか 1項に記載の DNA精製用キット、 及び請求項 106〜 109のいずれか 1項に記載の DNA回収用キットからなる群から選ばれる少 なくとも 2組のキットを含む、 環境サンプルからの DNA取得用キットセッ 卜。  1 10. Claim 95-: The DNA extraction kit according to any one of L00, the DNA purification kit according to any one of claims 101-105, and any one of claims 106-109 A kit for obtaining DNA from an environmental sample, comprising at least two sets of kits selected from the group consisting of the kits for DNA recovery described in (1).
PCT/JP2004/011956 2004-01-28 2004-08-13 Method of collecting dna from environmental sample WO2005073377A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005517375A JP4665124B2 (en) 2004-01-28 2004-08-13 Method for recovering DNA from environmental samples

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004-020498 2004-01-28
JP2004020498 2004-01-28

Publications (1)

Publication Number Publication Date
WO2005073377A1 true WO2005073377A1 (en) 2005-08-11

Family

ID=34823754

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/011956 WO2005073377A1 (en) 2004-01-28 2004-08-13 Method of collecting dna from environmental sample

Country Status (2)

Country Link
JP (3) JP4665124B2 (en)
WO (1) WO2005073377A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008092835A (en) * 2006-10-10 2008-04-24 Ebara Jitsugyo Co Ltd Method for extracting nucleic acid
JP2009082066A (en) * 2007-09-28 2009-04-23 Japan Science & Technology Agency Method for refining rna extracted from soil
US10036054B2 (en) 2016-01-30 2018-07-31 Safeguard Biosystems Holdings Ltd. Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
WO2018138363A1 (en) * 2017-01-30 2018-08-02 Safeguard Biosystems Holdings Ltd. Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
KR20190100315A (en) * 2016-12-29 2019-08-28 쇼어라인 바이옴, 엘엘씨 Combined Lysis Protocol for Comprehensive Cell Lysis
CN110592072A (en) * 2019-09-11 2019-12-20 北京百迈客生物科技有限公司 Extraction method and application of plant genome DNA
CN110636903A (en) * 2017-01-30 2019-12-31 保障生物系统控股有限公司 Bead impact tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
US11149246B2 (en) 2016-12-29 2021-10-19 Shoreline Biome, Llc Methods for cell lysis and preparation of high molecular weight DNA from modified cells

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005073377A1 (en) * 2004-01-28 2005-08-11 Toudai Tlo, Ltd. Method of collecting dna from environmental sample
JP2017099376A (en) * 2015-11-19 2017-06-08 株式会社日吉 Pretreatment method of environmental water sample, pretreatment agent for environmental water sample and estimation method of amount of biota or organisms
CN112646804A (en) * 2020-12-28 2021-04-13 生态环境部环境规划院 Method for extracting environmental DNA in soil

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU781961B2 (en) * 1999-11-29 2005-06-23 Aventis Pharma S.A. Method for obtaining nucleic acids from an environment sample, resulting nucleic acids and use in synthesis of novel compounds
JP2005073643A (en) * 2003-09-02 2005-03-24 Kubota Corp Method for extracting dna originating from microorganism
WO2005073377A1 (en) * 2004-01-28 2005-08-11 Toudai Tlo, Ltd. Method of collecting dna from environmental sample

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CULLEN D.W. ET AL.: "Simple and rapid method for direct extraction of microbila DNA from soil for PCR", SOIL. BIOL. BIOCHEM., vol. 30, no. 8/9, 1998, pages 983 - 993, XP002983301 *
KABIR S. ET AL.: "Quantitative measurements of fungal DNA extracted by three different methods using real-time polymerase chain reaction", J. BIOSCI. BIOENG., vol. 96, no. 4, 2003, pages 337 - 343, XP002983000 *
KOWALCHUK G.A. ET AL.: "Community analysis of ammonia-oxydising bacteria, in relation to oxygen availability in soils and root-oxygenated sediments, using PCR, DGGE and oligonucleotide probe hybridisation", FEMS MICROBIOLOGY ECOLOGY, vol. 27, no. 4, 1998, pages 339 - 350, XP002982998 *
KUSKE C.R. ET AL.: "Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil", APPL. ENVIRON. MICROBIOL., vol. 64, no. 7, 1998, pages 2463 - 2472, XP002226433 *
VOLOSSIOUK T. ET AL.: "Direct DNA extraction for PCR-mediated assays of soil organisms", APPL. ENVIRON. MICROBIOL., vol. 61, no. 11, 1995, pages 3972 - 3976, XP002982999 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008092835A (en) * 2006-10-10 2008-04-24 Ebara Jitsugyo Co Ltd Method for extracting nucleic acid
JP2009082066A (en) * 2007-09-28 2009-04-23 Japan Science & Technology Agency Method for refining rna extracted from soil
US10036054B2 (en) 2016-01-30 2018-07-31 Safeguard Biosystems Holdings Ltd. Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
US11952614B2 (en) 2016-01-30 2024-04-09 Safeguard Biosystems Holdings Ltd. Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
KR102281213B1 (en) 2016-12-29 2021-07-22 쇼어라인 바이옴, 엘엘씨 Combined lysis protocol for comprehensive cell lysis
KR20190100315A (en) * 2016-12-29 2019-08-28 쇼어라인 바이옴, 엘엘씨 Combined Lysis Protocol for Comprehensive Cell Lysis
US11149246B2 (en) 2016-12-29 2021-10-19 Shoreline Biome, Llc Methods for cell lysis and preparation of high molecular weight DNA from modified cells
JP2020503068A (en) * 2016-12-29 2020-01-30 ショアライン バイオミー エルエルシー Combined lysis protocol for complete lysis of cells
CN110636903A (en) * 2017-01-30 2019-12-31 保障生物系统控股有限公司 Bead impact tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
US11667884B2 (en) 2017-01-30 2023-06-06 Safeguard Biosystems Holdings Ltd. Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
WO2018138363A1 (en) * 2017-01-30 2018-08-02 Safeguard Biosystems Holdings Ltd. Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms
CN110592072B (en) * 2019-09-11 2021-08-13 北京百迈客生物科技有限公司 Extraction method and application of plant genome DNA
CN110592072A (en) * 2019-09-11 2019-12-20 北京百迈客生物科技有限公司 Extraction method and application of plant genome DNA

Also Published As

Publication number Publication date
JP2011045386A (en) 2011-03-10
JP2013135681A (en) 2013-07-11
JP4665124B2 (en) 2011-04-06
JPWO2005073377A1 (en) 2008-01-10

Similar Documents

Publication Publication Date Title
JP2013135681A (en) Method for recovering dna from environmental sample
Roose-Amsaleg et al. Extraction and purification of microbial DNA from soil and sediment samples
EP1756136B1 (en) Kits and processes for removing contaminants from nucleic acids in environmental and biological samples
Dong et al. Removal of humic substances from soil DNA using aluminium sulfate
Mahuku A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA
Rajendhran et al. Strategies for accessing soil metagenome for desired applications
Jacobsen Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay
Schneegurt et al. Direct extraction of DNA from soils for studies in microbial ecology
US7893251B2 (en) Methods for selective isolation of nucleic acids from microbial cells present in samples containing higher eukaryotic cells and/or tissues
US9145553B2 (en) Method for isolating nucleic acids
US20170321209A1 (en) Method for isolating dna
Zammit et al. The recovery of nucleic acid from biomining and acid mine drainage microorganisms
CN101712953B (en) DNA extracting method for evaluating community diversity of the intestinal microorganisms of animals
Ranjard et al. A single procedure to recover DNA from the surface or inside aggregates and in various size fractions of soil suitable for PCR-based assays of bacterial communities
Gutiérrez-Lucas et al. Strategies for the extraction, purification and amplification of metagenomic DNA from soil growing sugarcane
WO2015154013A1 (en) System and method for dna extraction from samples containing humic acids
Wechter et al. A rapid, cost-effective procedure for the extraction of microbial DNA from soil
HOSHINO et al. Skim milk drastically improves the efficacy of DNA extraction from andisol, a volcanic ash soil
JP2006067890A (en) Method for extracting nucleic acid and nucleic acid-extracting kit
CN112646806A (en) Rapid extraction method and kit for soil DNA
Chattopadhyay et al. Purification of quality DNA from citrus plant using iron oxide nanoparticle as solid based support
EP4365292A1 (en) Aptamer for the detection of the microorganism bacillus subtilis
Kaushal et al. Purification of Metagenomic DNA Using Novel Nanocomposite Titanium Dioxide-polyaniline Tin (1V) Antimonophosphate, Insights into the Mechanism Underlying Purification Process
JP5277497B2 (en) Purification of RNA extracted from soil

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2005517375

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase