JP5105237B2 - Compositions and kits for nucleic acid extraction and use thereof - Google Patents

Description

  The present invention relates to a technique for easily obtaining information on microorganisms in soil, and more particularly, to a technique for easily extracting nucleic acids from soil in a high yield.

  In order to know the distribution and dynamics of substances in growing plants, techniques for imaging the substance dynamics in plants have been developed.

  It is considered that the environment at the time of imaging in a plant approximates the original growth environment for the plant in order to know the original material dynamics in the plant. It has long been known that physicochemical properties of soil, such as soil pH and inorganic salt concentrations, and microorganisms present in the soil have a great influence on plant growth. Substances contained in the air (particularly microorganisms) can also affect plant growth. In order to know the original growth environment for a plant, it is considered that an excellent technique for analyzing information on the soil in which the plant grows and the atmosphere in which the plant is placed is indispensable.

Microorganisms are very much present in the soil (10 ⁸ to 10 ⁹ per 1 g of soil). The types are extremely diverse, and it is said that a considerable number of naturally occurring microorganisms are distributed in the soil. However, the microorganisms present in the soil are very difficult to culture, and less than 10% of the total soil microorganisms can be cultured. Thus, most soil microorganisms are unknown.

  In recent years, it has become possible to obtain information on soil microorganisms by directly extracting nucleic acids from soil and performing analysis using molecular biological techniques. That is, information on microorganisms in the soil is obtained by detecting nucleic acids (DNA or RNA) derived from microorganisms from the soil.

  A number of techniques for detecting or separating microbial DNA from soil have been reported so far. DNA extraction technology from soil facilitates the acquisition of genes for soil microorganisms that are difficult to cultivate. It made it possible to supply, and it contributed greatly to academic and industrial aspects.

  However, volcanic ash soil, which is unique to Japan, contains amorphous aluminum (allophane), so these substances strongly adsorb nucleic acids in the solution in the nucleic acid extraction process, Nucleic acid extraction from volcanic ash soil was difficult.

The present inventors have developed a technique that inactivates the allophane adsorption ability by using EDTA and a phosphoric acid solution at a high concentration and easily extracts DNA (see Patent Document 1).
WO2005 / 073377 (International publication date: August 11, 2005)

  DNA is a relatively stable substance and tends to remain in the soil. However, DNA may have evaluated even dead microorganisms. On the other hand, RNA is chemically unstable and is much easier to degrade than DNA. When a microorganism dies, it is rapidly degraded by RNase or the like. Therefore, if RNA can be extracted and analyzed, living microorganisms in the soil can be accurately evaluated.

  However, RNA is adsorbed to allophane more powerfully and more rapidly than DNA, and therefore the technique described in Patent Document 1 cannot efficiently perform RNA extraction. As for RNA extraction, a technique for extracting RNA from soil in high yield has not been developed yet.

  The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to easily obtain information on microorganisms in soil. Specifically, nucleic acid (especially RNA) is obtained in high yield from soil. It is to realize a technique for simple and easy extraction.

  In order to efficiently extract nucleic acid (DNA or RNA) from soil containing allophane such as volcanic ash soil, allophane that can adsorb nucleic acid is effectively destroyed, or DNA or RNA (particularly adsorbed to allophane is adsorbed). It is necessary to block the allophane adsorption site before it is adsorbed. As a result of trial and error, the present inventors have found that nucleic acid can be efficiently extracted by adding a fluorine compound to the extract.

The composition according to the present invention is for preparing a nucleic acid extract, and is characterized by containing at least one compound selected from a fluorinated compound and a silicate compound. The above _{compound, NaF, Na 2 SiF 6,} K 2 SiF 6, it is _{(NH 4) 2 SiF 6,} NaPF 6, KPF 6 and a compound selected from the group consisting of _NH 4 PF ₆ preferably, Na ₂ and more preferably SiF _6. In addition, it is still more preferable that the nucleic acid extract prepared using the present invention contains Na ₂ SiF ₆ at a concentration in the range of ₅ to 50 g / L. Therefore, the composition according to the present invention preferably contains Na ₂ SiF ₆ so as to be supplied into the nucleic acid extract at a concentration in the range of 5 to 50 g / L.

  The composition according to the present invention may further contain EDTA and / or phosphoric acid. When EDTA is used in the present invention, the nucleic acid extract prepared using the present invention preferably contains EDTA at a concentration in the range of 50 to 100 mM. Therefore, the composition according to the present invention preferably contains EDTA so as to be supplied to the nucleic acid extract at a concentration in the range of 50 to 100 mM. Moreover, when phosphoric acid is used for this invention, it is preferable that the nucleic acid extract prepared using this invention contains phosphoric acid in the density | concentration within the range of 100-300 mM. Therefore, the composition according to the present invention preferably contains phosphoric acid so as to be supplied into the nucleic acid extract at a concentration in the range of 100 to 300 mM.

  The composition according to the present invention may further contain a surfactant. When a surfactant is used in the present invention, the nucleic acid extract prepared using the present invention preferably contains a surfactant at a concentration of 5% or less. Therefore, the composition according to the present invention preferably contains a surfactant so as to be supplied into the nucleic acid extract at a concentration of 5% or less. The surfactant usable in the present invention is preferably selected from the group consisting of SDS, CTAB, Triton X-100 and N-lauroyl sarcosine sodium.

  The kit according to the present invention is for preparing a nucleic acid extract, and may be in a form in which the components of the composition described above are separately provided. That is, the kit according to the present invention includes at least one compound selected from a fluorinated compound and a silicate compound, and further includes EDTA, phosphoric acid and / or a surfactant. Also good.

  The composition and kit according to the present invention are used for preparing a nucleic acid extract for extracting nucleic acid from a sample derived from at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge and feces. It is preferred that

  The nucleic acid extraction method according to the present invention is characterized by using the above-mentioned nucleic acid extract, and specifically, a sample in an extract containing at least one compound selected from a fluorinated compound and a silicate compound. A step of extracting the nucleic acid from.

  The nucleic acid extraction method according to the present invention is preferably used for extracting nucleic acid from a sample derived from at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge and feces.

  The method for producing a nucleic acid extract according to the present invention includes a step of preparing a solution containing at least one compound selected from a fluorinated compound and a silicate compound, and the solution contains EDTA. A step of adding a phosphoric acid to the solution, or a step of adding a surfactant to the solution.

  By using the present invention, not only DNA but also RNA can be extracted from soil with high yield.

[1: Composition and kit for preparing nucleic acid extract]
The present invention provides compositions and kits for preparing nucleic acid extracts. The composition and kit according to the present invention are used for preparing a nucleic acid extract for extracting nucleic acid from a sample derived from at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge and feces. It is preferable to be used for preparing a nucleic acid extract that extracts nucleic acid from a sample derived from volcanic ash soil.

  As used herein, the term “composition” is intended to be a form in which various components are contained in one substance, and the term “kit” is intended to mean that at least one of the various components is a separate substance. It is intended to be in a state contained in (for example, a container).

  As described above, in order to efficiently extract nucleic acid from soil containing allophane such as volcanic ash soil, allophane capable of adsorbing nucleic acid is effectively destroyed, or DNA or RNA (particularly adsorbed by allophane is adsorbed). It is necessary to block the allophane adsorption site before it is adsorbed. The present inventors have developed a technique that inactivates the allophane adsorption ability by using EDTA and a phosphoric acid solution at a high concentration and easily extracts DNA (see Patent Document 1). This technique is very excellent as a technique for efficiently extracting DNA from soil containing allophane. However, since RNA is insolubilized and precipitated under high salt concentration conditions, it is not suitable as a technique for RNA extraction.

  The present inventors have made further studies in order to develop RNA extraction technology from soil, but the technology for extracting RNA from soil in high yield is not yet known. There was no indicator, and trial and error were repeated. As a result, it has been found that if a specific compound is used, the adsorption ability of allophane can be inactivated even under a low salt concentration condition.

  The composition according to the present invention is for preparing a nucleic acid extract, and is characterized by containing at least one compound selected from a fluorinated compound and a silicate compound. The kit according to the present invention is characterized by comprising at least one compound selected from a fluorinated compound and a silicate compound.

Examples of the compound selected from the fluorinated compound and the silicate compound include NaF, Na ₂ SiF ₆ , K ₂ SiF ₆ , (NH ₄ ) ₂ SiF ₆ , NaPF ₆ , KPF ₆ , NH ₄ PF _{6, and the} like. However, it is not limited to these. Demonstrate the invention using _Na 2 SiF ₆ in the embodiment described _below, Na 2 SiF ₆ is a low solubility indicates a higher reactivity with allophane. Since RNA is insolubilized and precipitated when the salt concentration is high, it is difficult to use high-concentration EDTA and phosphoric acid solutions that increase the salt concentration. However, it has been found that when Na ₂ SiF ₆ is mixed with soil in an aqueous solution, the reaction proceeds gradually and allophane can be inactivated at a relatively low salt concentration. A person skilled in the art who has read this specification will easily understand that a compound having such an effect is not limited to Na ₂ SiF ₆ .

_{If Na} 2 SiF ₆ is used in the present invention, _Na 2 SiF ₆ is preferably in a nucleic acid extract prepared in 1 to 100 g / L, more preferably 5 to 50 g / L, more preferably 25 to 50 g / L May be included at concentrations within the range of. Therefore, the composition according to the present invention is preferably supplied into the nucleic acid extract at a concentration in the range of 1 to 100 g / L, more preferably 5 to 50 g / L, and even more preferably 25 to 50 g / L. Contains Na ₂ SiF ₆ .

  As disclosed in Patent Document 1, EDTA is also effective for inactivating allophane. Therefore, the composition according to the present invention may further contain EDTA. However, since EDTA having a high concentration as used in Patent Document 1 does not have the desired effect, when EDTA is used in the present invention, EDTA is preferably 10 to 300 mM in the prepared nucleic acid extract. More preferably, it may be contained at a concentration in the range of 50 to 200 mM, more preferably 50 to 100 mM. Therefore, the composition according to the present invention preferably contains EDTA so as to be supplied in the nucleic acid extract at a concentration within the range of 10 to 300 mM, more preferably 50 to 200 mM, and even more preferably 50 to 100 mM. . In consideration of DNase inactivation, the EDTA concentration in the extract is preferably 100 mM or less. Therefore, it is still more preferable that the composition according to the present invention contains EDTA so as to be supplied in the nucleic acid extract at 100 mM or less.

  As used herein, the term “phosphate” includes phosphate buffer or phosphate buffer. Phosphate buffers are well known in the art and are often used in DNA extraction. As disclosed in Patent Document 1, phosphoric acid is also effective for inactivating allophane. Therefore, the composition according to the present invention may further contain phosphoric acid. Although the intended effect can be obtained even if the concentration is not as high as that used in Patent Document 1, it is preferable that the phosphoric acid concentration is high as shown in the Examples described later. When phosphoric acid is used in the present invention, phosphoric acid may be contained in the prepared nucleic acid extract at a concentration within the range of 10 to 500 mM, more preferably 50 to 300 mM, and even more preferably 200 to 300 mM. Therefore, the composition according to the present invention preferably contains phosphoric acid so as to be supplied into the nucleic acid extract at a concentration within the range of 10 to 500 mM, more preferably 50 to 300 mM, and even more preferably 200 to 300 mM. Yes. The form of phosphoric acid contained in the composition according to the present invention is not particularly limited, and may be solid or liquid and can be easily understood by those skilled in the art. In carrying out the present invention, phosphoric acid may be used in combination with EDTA or used alone.

  When nucleic acids are extracted from bacteria or fungi, it is necessary to denature cellular proteins with a surfactant to destroy the cell structure. Therefore, the composition according to the present invention may further contain a surfactant. When a surfactant is used in the present invention, the surfactant may be contained at a concentration of preferably 5% or less, more preferably 3% or less, and even more preferably 2% or less in the prepared nucleic acid extract. Therefore, the composition according to the present invention preferably contains a surfactant so as to be supplied into the nucleic acid extract at a concentration of 5% or less, more preferably 3% or less, and even more preferably 2% or less. Is preferred. The surfactant usable in the present invention is preferably selected from the group consisting of SDS, CTAB, Triton X-100 and N-lauroyl sarcosine sodium, but is not limited thereto.

  The pH of the nucleic acid extract is preferably 7 or higher, more preferably 7.5 or higher, and still more preferably in the range of 8.0 to 9.0. Therefore, the composition according to the present invention may further include a buffer (or a buffering agent) so that the pH environment is provided in the prepared nucleic acid extract.

  The kit according to the present invention may be in a form in which the components of the composition described above are separately provided in addition to at least one compound selected from a fluorinated compound and a silicate compound. That is, the kit according to the present invention includes at least one compound selected from a fluorinated compound and a silicate compound, and further includes EDTA, phosphoric acid and / or a surfactant. Also good. In addition, a reagent or instrument necessary for performing nucleic acid extraction may be further provided, and when purification is performed after extraction, a reagent or instrument necessary for performing purification may be further provided.

  As used herein, the term “kit” intends a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material. Instructions for using each of the reagents or instruments are preferably provided. As used herein, in the aspect of a kit, “provided” is intended to mean a state in which a reagent or the like is included in any of the individual containers constituting the kit. The The “instructions” may be printed on paper or other media, or may be recorded on electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, and the like.

[2: Nucleic acid extract and production method thereof]
The method for producing a nucleic acid extract according to the present invention includes a step of preparing a solution containing at least one compound selected from a fluorinated compound and a silicate compound, and the solution contains EDTA. A step of adding a phosphoric acid to the solution, or a step of adding a surfactant to the solution.

  Various nucleic acid extracts are known in the art. That is, the method for producing a nucleic acid extract according to the present invention can be produced by mixing (adding) a solution containing at least one compound selected from a fluorinated compound and a silicate compound to a known nucleic acid extract. . Moreover, when the well-known nucleic acid extract used does not contain EDTA, phosphoric acid, and / or surfactant, it can be said that these may be further mixed (added).

  Thus, the nucleic acid extract according to the present invention contains at least one compound selected from a fluorinated compound and a silicate compound, and may further contain EDTA, phosphoric acid and / or a surfactant. . By using the present invention, not only DNA but also RNA can be extracted from soil with high yield.

[3: Nucleic acid extraction method]
The nucleic acid extraction method according to the present invention is characterized by using the above-mentioned nucleic acid extract, and specifically, a sample in an extract containing at least one compound selected from a fluorinated compound and a silicate compound. A step of extracting the nucleic acid from.

  The nucleic acid extraction method according to the present invention is preferably used for extracting nucleic acid from a sample derived from at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge and feces.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to these Examples. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

[Soil sample]
Two types of soil shown in FIG. 1 were used. Yayoi compost area is a light-colored black soil with relatively advanced accumulation of organic matter because compost is input once a year. The field soil of the Gunma Agricultural Experiment Station is a light-colored black soil with little organic matter accumulation.

[Extraction of soil nucleic acid solution]
20 ml of LB medium (tryptone 10 g, yeast extract 5 g / 1 L) was added to 50 g of the air-dried fine soil of the above two types of soil and incubated at 30 ° C. for 2 days. 0.25 g of the incubated soil is taken into a 2 ml screw cap tube, to which 1 g of silica / zirconia beads (0.1 mm diameter and 0.5 mm diameter mixed in 2: 1) and 5 mm diameter are mixed. One glass bead was added. The tube _Na 2 SiF ₆ solution (0.5 g / ml) to 10 [mu] l, 25 [mu] l, was added 50μl or 100 [mu] l, and further extracts [I] ~ [III] or _Na 2 SiF ₆ was added to the above solution of A total of 1250 μl was added. The extract contains ATA (aurintricarboxylic acid) as an RNase inhibitor.

  The samples in these tubes were subjected to Beads Beating treatment at 3000 rpm for 30 seconds with a bead crusher (Micro Smash MS-100 Tommy Seiko), and then the samples were incubated at 65 ° C. for 30 minutes. Subsequently, the mixture was centrifuged at 10,000 × g and 25 ° C. for 5 minutes, and the supernatant was collected. An equal volume of chloroform was added to 750 μl of this supernatant, then vortexed and centrifuged at 12,000 × g, 25 ° C. for 20 minutes to recover 500 μl of supernatant. The supernatant was diluted after an appropriate amount of 2-propanol, then vortexed and centrifuged at 20000 × g for 20 minutes at 4 ° C. to recover DNA and RNA as precipitates. The precipitate was washed with 70% ethanol and then dried. The dried precipitate was dissolved in 50 μl of TE buffer (pH 8.0) to obtain a soil nucleic acid solution.

[Quantification of DNA and RNA]
After separation of DNA and RNA by 1.5% agarose gel electrophoresis (30 min at 50 V in x0.5 TAE buffer), the gel was stained with SYBR Green II for 1 hour. Using BAS3000 (Fuji Photo Film Co., Ltd.), DNA and RNA were quantitatively detected by fluorescence under conditions of 473 nm excitation and 532 nm cut. Luminance was measured by Image Gauge using a portion having a size larger than 5000 bp near the upper end of the gel as DNA and two rRNA bands of 23S rRNA and 16S rRNA as RNA. In each gel, 0.05 μg to 0.25 μg of λDNA HindIII digest was simultaneously electrophoresed as a standard of 4 to 5 steps, and the amount of DNA and the amount of DNA were determined based on a calibration curve based on the HindIII digest of λDNA. The amount of RNA was quantified.

〔result〕
The results are shown in FIGS. It can be seen that the amount of DNA and RNA extracted from the soil increased with the addition of Na ₂ SiF ₆ . The amount of Na ₂ SiF ₆ was studied in the range of 5mg~50mg per soil 250 mg. The required amount of Na ₂ SiF ₆ added was different depending on the soil, but good results were obtained when about 50 mg was added to 250 mg of soil. Although there was a concern that the yield would decrease with a decrease in pH caused by adding a large amount of Na ₂ SiF ₆ , there was no particular problem. In addition, for Gunma Agricultural Experiment Station soil (light colored black soil with low organic matter content), where extraction of DNA has been very difficult, the extraction amount is greatly increased by adding 50 mg of Na ₂ SiF ₆ to 250 mg of soil. Improvement was obtained. In addition, the extraction amount improved as the concentration of phosphoric acid in the extract increased, and the extract [III] showed the best results.

  INDUSTRIAL APPLICABILITY The present invention is useful as a technique for extracting RNA from soil, as well as a technique for extracting DNA from soil, and can accurately evaluate microorganisms that are active in the soil. Can detect the ecology of As described above, the present invention can be expected to utilize RNA extraction technology from soil in a wide range of fields such as plant diseases and specific gene expression analysis, and also to clarify the ecology of soil microorganisms in Japan. It is a very useful technique.

It is a figure which shows the property of the soil used in the Example. It is a graph which shows the quantity of DNA extracted from Yayoi compost area soil. It is a graph which shows the quantity of RNA extracted from Yayoi compost area soil. It is a graph which shows the quantity of DNA extracted from the field soil of Gunma Agricultural Experiment Station.

Claims (15)

A composition for preparing a nucleic acid extract having a pH of 7-9 ,
A composition comprising Na ₂ SiF ₆ and phosphoric acid so as to be supplied into a nucleic acid extract at a concentration in the range of 5 to 50 g / L.   The composition of claim 1, further comprising EDTA. The composition according to claim 2 , comprising EDTA so as to be supplied in the nucleic acid extract at a concentration in the range of 50 to 100 mM. The composition according to claim 1 , comprising phosphoric acid so as to be supplied into the nucleic acid extract at a concentration in the range of 100 to 300 mM.   The composition according to claim 1, further comprising a surfactant. 6. The composition of claim 5 , wherein the surfactant is selected from the group consisting of SDS, CTAB, Triton X-100 and N-lauroyl sarcosine sodium. The composition according to claim 5 , comprising a surfactant so as to be supplied in the nucleic acid extract at a concentration of 5% or less.   2. The method according to claim 1, wherein the nucleic acid extract is used to prepare a nucleic acid extract that extracts nucleic acid from a sample derived from at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge, and feces. The composition as described. Nucleic acid extraction with a pH of 7-9, comprising Na ₂ SiF ₆ and a phosphoric acid so as to have a concentration in the range of 5-50 g / L in the prepared nucleic acid extract Kit for preparing liquid. The kit according to claim 9 , further comprising EDTA. The kit according to claim 9 , further comprising a surfactant. A step of extracting nucleic acid from a sample in an extract containing Na ₂ SiF ₆ at a concentration in the range of 5 to 50 g / L and having a pH of 7 to 9 containing phosphoric acid. Extraction method. Nucleic acid extraction with a pH of 7-9, comprising the steps of preparing a solution containing Na ₂ SiF ₆ at a concentration in the range of 5-50 g / L, and adding phosphoric acid to the solution Liquid manufacturing method. The method according to claim 13 , further comprising a step of including EDTA in the solution. The method according to claim 13 , further comprising a step of adding a surfactant to the solution.

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