WO2005073377A1 - Methode d’extraction de l’adn depuis un echantillon environnemental - Google Patents

Methode d’extraction de l’adn depuis un echantillon environnemental Download PDF

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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
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Prior art keywords
dna
extract
edta
soil
concentration
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PCT/JP2004/011956
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English (en)
Japanese (ja)
Inventor
Hiroki Rai
Shigeto Ohtsuka
Masaya Nishiyama
Keishi Senoo
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Toudai Tlo, Ltd.
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Priority to JP2005517375A priority Critical patent/JP4665124B2/ja
Publication of WO2005073377A1 publication Critical patent/WO2005073377A1/fr

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    • 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

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Méthode d’extraction de l’ADN d’un échantillon environnemental qui se caractérise par le fait qu’elle inclut le traitement d’échantillons environnementaux par broyage à billes en présence d’un extrait d'ADN ne contenant pas plus de 5% d'un surfactant, idem après chauffage.
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JP2008092835A (ja) * 2006-10-10 2008-04-24 Ebara Jitsugyo Co Ltd 核酸抽出方法
JP2009082066A (ja) * 2007-09-28 2009-04-23 Japan Science & Technology Agency 土壌から抽出したｒｎａの精製法
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 (fr) * 2017-01-30 2018-08-02 Safeguard Biosystems Holdings Ltd. Tube de battage de billes pour l'extraction d'acide désoxyribonucléique et/ou d'acide ribonucléique des micro-organismes
KR20190100315A (ko) * 2016-12-29 2019-08-28 쇼어라인 바이옴, 엘엘씨 포괄적인 세포 용해를 위한 조합된 용해 프로토콜
CN110592072A (zh) * 2019-09-11 2019-12-20 北京百迈客生物科技有限公司 一种植物基因组dna的提取方法及其应用
CN110636903A (zh) * 2017-01-30 2019-12-31 保障生物系统控股有限公司 用于从微生物中提取脱氧核糖核酸和/或核糖核酸的珠粒撞击管和方法
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
JP7511829B2 (ja) 2020-02-28 2024-07-08 国立大学法人 宮崎大学 核酸吸着材

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Cited By (14)

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Publication number Priority date Publication date Assignee Title
JP2008092835A (ja) * 2006-10-10 2008-04-24 Ebara Jitsugyo Co Ltd 核酸抽出方法
JP2009082066A (ja) * 2007-09-28 2009-04-23 Japan Science & Technology Agency 土壌から抽出したｒｎａの精製法
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 (ko) 2016-12-29 2021-07-22 쇼어라인 바이옴, 엘엘씨 포괄적인 세포 용해를 위한 조합된 용해 프로토콜
KR20190100315A (ko) * 2016-12-29 2019-08-28 쇼어라인 바이옴, 엘엘씨 포괄적인 세포 용해를 위한 조합된 용해 프로토콜
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 (ja) * 2016-12-29 2020-01-30 ショアライン バイオミー エルエルシー 細胞を完全に溶解するための併用式溶解プロトコル
CN110636903A (zh) * 2017-01-30 2019-12-31 保障生物系统控股有限公司 用于从微生物中提取脱氧核糖核酸和/或核糖核酸的珠粒撞击管和方法
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 (fr) * 2017-01-30 2018-08-02 Safeguard Biosystems Holdings Ltd. Tube de battage de billes pour l'extraction d'acide désoxyribonucléique et/ou d'acide ribonucléique des micro-organismes
CN110592072B (zh) * 2019-09-11 2021-08-13 北京百迈客生物科技有限公司 一种植物基因组dna的提取方法及其应用
CN110592072A (zh) * 2019-09-11 2019-12-20 北京百迈客生物科技有限公司 一种植物基因组dna的提取方法及其应用
JP7511829B2 (ja) 2020-02-28 2024-07-08 国立大学法人 宮崎大学 核酸吸着材

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