Nucleic acid separation method, detection method, nucleic acid purification column and method for producing same

Abstract

Disclosed is a method for, when a nucleic acid in an extremely minute amount of a sample is to be separated to detect the nucleic acid, reducing variation in measurement data by removing the nucleic acid mixed into a nucleic acid purification column. The method for removing a nucleic acid from a sample containing the same comprises step (1) for bringing the target nucleic acid-containing sample into contact with a nucleic acid-binding solid phase carrier which is capable of adsorbing the nucleic acid, and step (2) for eluting the nucleic acid from the nucleic acid-binding solid phase carrier on which the nucleic acid is adsorbed in step (1), wherein the nucleic acid-binding solid phase carrier is contacted with a solution containing a chaotropic agent prior to step (1).

Description

[Technical field]

[0001] The present invention relates to a method for separating nucleic acid from a sample containing nucleic acid, a method for detecting the separated nucleic acid, a nucleic acid purification column for separating nucleic acid, and a method for producing the nucleic acid purification column. [Background technology]

[0002] Technologies for separating and purifying nucleic acids such as DNA and RNA from samples such as biological samples are important in biotechnology. In order to obtain reproducible and highly accurate experimental results in nucleic acid detection, hybridization, amplification, sequencing, etc., it is necessary to remove impurities other than the target nucleic acid from the sample and separate or isolate the nucleic acid. When the sample containing nucleic acid is a biological sample, examples of impurities include proteins, carbohydrates, lipids, and polyphenols.

[0003] Various approaches have been used to separate nucleic acids from biological samples, including extraction methods using organic solvents such as ethanol precipitation, nucleic acid dialysis, purification using ultrafiltration membranes, and separation methods using nucleic acid-binding solid phase carriers.

[0004] Among these, the ethanol precipitation method is a widely used method that can separate nucleic acids with a simple operation. However, the recovery rate of nucleic acids is generally low, and the recovery rate varies depending on the operation method. Purification methods using dialysis and ultrafiltration membranes are also used, but there are limitations on the molecular weight and the samples that can be applied.

[0005] In recent years, a commonly used method for separation using the above-mentioned nucleic acid-binding solid phase carrier is a method for separating a target nucleic acid in a sample by binding it to a solid phase carrier in the presence of a chaotropic agent (Patent Document 1, Non-Patent Document 1). This method is called the Boom method, and is a method for purifying nucleic acid that utilizes the property of nucleic acid binding to a solid phase carrier such as silica in the presence of a chaotropic agent such as guanidinium salt or urea, and the property of nucleic acid bound to the solid phase carrier being eluted with water. The procedure for purifying nucleic acid using the Boom method involves contacting a solution containing nucleic acid with a solid phase carrier such as silica in the presence of a chaotropic agent to adsorb the nucleic acid in the sample to the solid phase carrier, then passing a washing solution through the carrier to wash and remove excess components such as salt, and then eluting the nucleic acid with water or the like.

[0006] When a target nucleic acid is isolated from a biological sample using the above-mentioned existing method, if only a small amount of the biological sample is available, the amount of nucleic acid obtained by the isolation will be extremely small. Therefore, in order to analyze or detect the extremely small amount of nucleic acid isolated in this manner, it is necessary to use a highly sensitive analytical method. For example, when quantification using ultraviolet absorbance or fluorescence is technically difficult, for example, when the amount of nucleic acid is below the detection limit (for example, less than 1 ng), nucleic acid amplification techniques such as amplification by polymerase chain reaction (PCR) are applied, and then detection by sequencing or electrophoresis, detection by highly sensitive nucleic acid microarrays, etc. are performed. In addition, the LC / MS / MS method (Patent Document 2), which directly analyzes nucleic acid molecules by mass spectrometry, is used.

[0007] Non-Patent Document 2 reports that when RNA obtained by purifying a human sample using a commercially available nucleic acid purification column was analyzed, nucleic acids not derived from the human sample were detected. The authors of Non-Patent Document 2 revealed that the nucleic acid was contaminated in the nucleic acid purification column product, and reported that they attempted to remove it, but that it remained after washing with water alone. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 6,027,945 [Patent Document 2] JP 2017-223469 A [Non-patent literature]

[0009] [Non-Patent Document 1] R Boom et al., 1990, J Clin Microbiol, 28(3):495-503 [Non-Patent Document 2] A Heintz-Buschart et al., 2018, BMC Biology, 16:52 Summary of the Invention [Problem to be solved by the invention]

[0010] When attempting to detect a trace amount of the target nucleic acid as described above, if the nucleic acid purification column used to purify the nucleic acid is contaminated with nucleic acids as impurities, there is a problem that the measurement data of the target nucleic acid will vary greatly. In particular, when applying the above-mentioned nucleic acid amplification technology or using a highly sensitive nucleic acid microarray, reducing the variation in data between experiments is extremely important for performing a highly reliable analysis of nucleic acids. The present invention aims to provide a method for reducing the variation in measurement data by removing the nucleic acids contaminating the nucleic acid purification column when attempting to separate and detect a very small amount of sample, for example, an amount of nucleic acid of about 1 ng. [Means for solving the problem]

[0011] In order to solve the above problems, the present inventors conducted extensive research and found that when separating (purifying) a target nucleic acid from a sample containing the nucleic acid using a nucleic acid purification column having a nucleic acid-binding solid phase carrier, it is possible to remove nucleic acids contaminating the nucleic acid purification column by contacting the nucleic acid-binding solid phase carrier with a solution containing a chaotropic agent in advance, thereby reducing measurement variability when measuring the target nucleic acid after separation.

[0012] That is, the present invention provides the following. 1. The following steps (1) and (2) (1) contacting a sample containing a target nucleic acid with a nucleic acid-binding solid phase carrier capable of adsorbing the nucleic acid; and (2) a step of eluting the nucleic acid from the nucleic acid-binding solid phase carrier to which the nucleic acid has been adsorbed in the step (1); A method for isolating a nucleic acid from a sample containing the nucleic acid, comprising: The method comprises, prior to step (1), contacting the nucleic acid-binding solid phase carrier with a solution containing a chaotropic agent. 2. The method according to claim 1, further comprising the step of washing the nucleic acid-binding solid phase carriers with water or an aqueous alcohol solution after contacting the nucleic acid-binding solid phase carriers with a solution containing the chaotropic agent. 3. The method according to item 1 or 2, wherein the nucleic acid-binding solid phase carrier is a silica membrane. 4. The method according to any one of items 1 to 3, wherein the chaotropic agent is any one of a guanidinium salt, urea, an iodide salt, a chlorate, a perchlorate, a thiocyanate, and an isothiocyanate. 5. The method according to any one of items 1 to 4, wherein the solution containing a chaotropic agent contains an alcohol. 6. The method according to claim 5, wherein the alcohol is ethanol. 7. The method according to any one of items 1 to 6, wherein the nucleic acid is DNA or RNA. 8. The following steps (1), (2) and (3) (1) contacting a sample containing a target nucleic acid with a nucleic acid-binding solid phase carrier capable of adsorbing the nucleic acid; (2) eluting the nucleic acid from the nucleic acid-binding solid phase carrier to which the nucleic acid has been adsorbed in the step (1); and (3) labeling and detecting the eluted nucleic acid A method for detecting a nucleic acid comprising: The method comprises, prior to step (1), contacting the nucleic acid-binding solid phase carrier with a solution containing a chaotropic agent. 9. A nucleic acid purification column having a hollow body provided with an inlet opening and an outlet opening, and a nucleic acid-binding solid phase carrier, A nucleic acid purification column produced by a method including a step A of passing a solution containing a chaotropic agent through a nucleic acid-binding solid phase carrier and then drying the nucleic acid-binding solid phase carrier. 10. A method for producing a nucleic acid purification column having a hollow body provided with an inlet opening and an outlet opening and a nucleic acid-binding solid phase carrier, comprising the steps of: A production method comprising a step A of passing a solution containing a chaotropic agent through a nucleic acid-binding solid phase carrier and then drying the nucleic acid-binding solid phase carrier. 11. The method according to claim 10, further comprising the step of washing the nucleic acid-binding solid phase carriers with water or an aqueous alcohol solution after passing a solution containing the chaotropic agent through the nucleic acid-binding solid phase carriers. 12. The method according to item 10 or 11, wherein the step A is carried out in a state where the nucleic acid-binding solid carrier is filled in a hollow body. 13. The method according to any one of items 10 to 12, wherein the nucleic acid-binding solid phase carrier is a silica membrane. 14. The method according to any one of items 10 to 13, wherein the chaotropic agent is a guanidinium salt, urea, an iodide salt, a chlorate, a perchlorate, a thiocyanate, or an isothiocyanate. Effect of the Invention

[0013] According to the present invention, it is possible to reduce variability in the measurement of a target nucleic acid that has been separated and purified from a sample containing the nucleic acid. [Brief description of the drawings]

[0014] [Figure 1] FIG. 2 is a diagram showing the steps of treating a nucleic acid-binding solid carrier with a solution containing a chaotropic agent in the method of the present invention. [Diagram 2] FIG. 1 is a cross-sectional view showing one embodiment of a nucleic acid purification column of the present invention. [Diagram 3] FIG. 2 is a cross-sectional view showing another embodiment of the nucleic acid purification column of the present invention. [Figure 4]FIG. 2 is a cross-sectional view showing the state in which the nucleic acid purification column of the present invention is combined with a container. [Diagram 5] FIG. 2 is a perspective view showing another embodiment of the nucleic acid purification column of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The sample to be subjected to the nucleic acid purification column of the present invention is not particularly limited as long as it may contain nucleic acid. For example, samples derived from animals, samples derived from plants, samples derived from microorganisms such as fungi and bacteria, and other samples that may contain some nucleic acid can be included. Examples of samples derived from animals include, but are not limited to, body fluid samples such as blood, serum, plasma, urine, feces, cerebrospinal fluid, saliva, swabs, and various tissue fluids, as well as various tissues and formalin-fixed paraffin-embedded specimens (FFPE).

[0016] The target nucleic acid is not particularly limited. For example, DNA or RNA contained in the above sample can be used, and these nucleic acids may be nucleic acids obtained by chemical or enzymatic reaction in a test tube. Examples of DNA include chromosomal DNA, viral DNA, bacterial or fungal DNA, cDNA obtained by reverse transcription of RNA, and fragments of these. When the nucleic acid is DNA, it may be either double-stranded DNA or single-stranded DNA. Examples of RNA include messenger RNA, ribosomal RNA, small RNA, microRNA, and fragments of these. Chemically synthesized nucleic acids such as DNA or RNA can also be used as the target nucleic acid of the present invention.

[0017] The nucleic acid-binding solid phase carrier (hereinafter, simply referred to as "solid phase carrier" or "carrier") is not particularly limited as long as it is capable of adsorbing nucleic acid. Here, "nucleic acid-binding" refers to a substance that can form a hydrophobic bond with nucleic acid in the presence of a chaotropic salt.

[0018] As the material of the solid phase carrier, for example, inorganic materials such as silica, glass, alumina, zeolite, clay minerals, etc., and polymer materials such as polystyrene, etc., are preferably used. Specifically, silica-based materials such as those described in U.S. Pat. No. 5,234,809, latex and polymer materials including polystyrene-based materials such as those described in WO 96 / 18731, etc., can be used. The shape of the solid phase carrier can be particles, beads, sheets (membranes), woven fabrics, fibers, etc. Silica membranes are particularly preferred as the solid phase carrier. Silica membranes are membranes made of silica, glass, or quartz fibers, or thin films made of silica gel, and are disclosed in Japanese Patent No. 3,329,813 or Published Japanese Translation of PCT International Publication No. 8-501,321.

[0019] These solid phase carriers can be used by being packed in a column and passing a sample solution or the like therethrough, or by being directly immersed in the sample solution or the like and used in a suspended state.

[0020] When the solid carrier is used in the above-mentioned suspended state, the carrier can be easily separated and recovered from the suspension by utilizing a magnetic field, so magnetic particles are also preferably used as the solid carrier. Common materials used in magnetic particles include magnetic metal oxides, especially iron oxides. Useful magnetic oxides include iron oxides, optionally with all or part of the ferrous iron substituted with divalent transition metals such as calcium, chromium, cobalt, copper, magnesium, manganese, nickel, vanadium and / or zinc. Silica-based magnetic particles include, for example, the particles described in U.S. Pat. Nos. 6,027,945 and 5,945,525.

[0021] In general, a nucleic acid purification column is a column packed with a solid phase carrier, and is used for the purpose of passing a sample solution through it to separate nucleic acids in the solution.

[0022] Nucleic acid purification columns that use silica membranes as solid phase carriers are commercially available from a number of manufacturers. Representative products include Qiagen's "miRNeasy serum / plasma kit" (model number 217184), "miRNeasy 96 kit" (model number 217061, *), "miRNeasy Mini Kit" (model number 217004), "QIAamp DNA mini kit" (model number 51304), "RNeasy micro kit" (model number 74004), "RNeasy UCP micro kit" (model number 73934), and "RNeasy MinElute Cleanup Kit" (model number 74204), as well as MACHEREY-NAGEL's "NucleoSpin miRNA Plasma" (model number 740981.1), "NucleoSpin RNA" (model number 740955.1), "NucleoSpin 96 RNA" (model number 740709.24, *), "NucleoSpin 8 RNA" (model number 740698), and "NucleoSpin RNA Plus (model number 740984.1), Zymo Research's RNA Clean & Concentrator Kit (model number R1080,*), RNA Clean & Concentrator-5 (model number R1013), RNA Clean & Concentrator-25 (model number R1017), UNIFILTER Microplate, 96-well, 800 μl, DNA binding, clear polystyrene (model number 7700-2810,*), UNIFILTER Microplate, 96-well, 800 μl, GF / C, clear polystyrene, filter bottom with long drip director (model number 7700-2801*), Thermo Fisher's mirVana miRNA Isolation Kit, with phenol (model number AM1560), PureLink miRNA Isolation Kit (model number K157001), PureLink RNA Mini Examples of such nucleic acid purification columns include those included in nucleic acid purification kits such as the "Nucleic Acid Purification Kit" (product number 12183018A).The method for isolating nucleic acids of the present invention can be applied to the isolation of target nucleic acids using these commercially available nucleic acid purification columns. Kits including nucleic acid purification columns in the 96-well plate format described below are marked with an asterisk after the model number.

[0023] Nucleic acid purification columns using the magnetic particles as the solid phase carrier are commercially available from a number of manufacturers. Representative examples include nucleic acid purification columns contained in nucleic acid purification kits such as "Maxwell RSC miRNA from Tissue and Plasma or Serum" (model number AS1680) and "Maxwell RSC Whole Blood DNA Kit" (model number AS1520) manufactured by Promega, "RNAdvance Blood Kit" (model number A35604) and "RNAdvance Cell v2 Kit" (model number A47942) manufactured by Beckman, "MagMAX mirVana Total RNA Isolation Kit" (model number A27827) and "Dynameads mRNA Purification Kit" (model number 61006) manufactured by Thermo Fisher.

[0024] Step (1) in the method for separating nucleic acid of the present invention is a step of contacting a sample containing a target nucleic acid with a solid phase carrier and adsorbing the nucleic acid in the sample to the solid phase carrier. When the solid phase carrier is used in a state where it is packed in a column, the sample solution is passed through the column, and the sample solution in the column is separated and removed by centrifugation or suction as necessary. Specifically, a sample solution containing a target nucleic acid is loaded onto a column packed with a solid phase carrier. The sample solution can be passed through the solid phase carrier by its own weight, but if it takes time, the column loaded with the sample solution can be centrifuged to pass through the solid phase carrier. The centrifugation conditions are appropriately set depending on the shape of the column, but in the case of a column packed with a commercially available solid phase carrier, the centrifugation can be performed at 8000xg to 10000xg.

[0025] Alternatively, the solid phase carrier may be immersed in the sample solution without being packed in a column, and then the solution may be separated and removed by centrifugation, suction, or the like. Specifically, an arbitrary amount of the solid phase carrier is added to the sample containing nucleic acid, and the sample is immersed. In this method, it is preferable to use a particle or bead-shaped solid phase carrier. After immersion, the solid phase carrier bound to the nucleic acid can be removed by sinking the solid phase carrier to the bottom layer of the solution by centrifugation and removing the supernatant. The centrifugation conditions at this time can be appropriately set based on the specific gravity of the solid phase carrier. If the specific gravity of the solid phase carrier is sufficiently large, the solid phase carrier sinks to the bottom layer without centrifugation, and the solid phase carrier can be removed by removing the supernatant.

[0026] Step (2) in the method for separating nucleic acid of the present invention is a step of eluting the nucleic acid adsorbed to the solid phase carrier in step (1) from the solid phase carrier. The elution of the nucleic acid can be carried out by using water or a solvent with low polarity such as TE buffer as an eluent. When the solid phase carrier is used in a state where it is packed in a column, the eluent is passed through the column, and the eluent in the column is separated by centrifugation or suction, as necessary, to obtain an eluate containing the nucleic acid. When the solid phase carrier is used without being packed in a column, the solid phase carrier is immersed in the eluent as it is, and then the solid phase carrier is separated and removed by centrifugation, suction, or the like, to obtain an eluate containing the eluted nucleic acid.

[0027] The above steps (1) and (2) themselves are well known and can be carried out by conventional methods.

[0028] The method for separating nucleic acids of the present invention is characterized in that, prior to carrying out the above-mentioned step (1) of contacting a sample containing a target nucleic acid with a solid phase carrier and adsorbing the nucleic acid in the sample to the solid phase carrier, the solid phase carrier is contacted with a solution containing a chaotropic agent.

[0029] Chaotropic agents are generally substances that destabilize the structure of nucleic acids in solution by reducing the interactions between water molecules. Nucleic acids form secondary structures due to hydrogen bonds between bases, but the addition of chaotropic agents dissolves the secondary structures.

[0030] As the chaotropic agent, guanidinium salt, urea, iodide salt, chlorate, perchlorate, thiocyanate, isothiocyanate, etc. can be used. As the preferred chaotropic agent, guanidinium thiocyanate and guanidinium hydrochloride can be mentioned. The concentration of the solution containing the chaotropic agent is preferably in the range of 1M to 8M.

[0031] The solution containing the chaotropic agent can be used as an aqueous solution, but may also be an aqueous solution containing alcohol. As the alcohol, ethanol, which is generally used in molecular biology experiments, is preferable. The concentration of alcohol in the solution containing the chaotropic agent can be in the range of 10 to 80% by volume.

[0032] As a method for contacting the solid phase carrier with a solution containing a chaotropic agent, when the solid phase carrier is used in a state where it is packed in a column, the solution containing the chaotropic agent can be passed through the column, and the solution in the column can be separated by centrifugation or suction, if necessary. When the solid phase carrier is used in a state where it is not packed in a column, the solid phase carrier can be immersed in the solution containing the chaotropic agent as it is, and then the solution can be separated and removed by centrifugation, suction, or the like. In this case, the amount of the solution containing the chaotropic agent used is about 0.1 mL to 1.5 mL, preferably about 0.6 mL to 1.0 mL, per 10 mg of the solid phase carrier. The temperature is not particularly limited, and is usually about 4°C to 60°C, but it is convenient and preferable to carry out the reaction at room temperature. Furthermore, after contacting the solid phase carrier with the chaotropic agent, it is preferable to wash the solid phase carrier with water or an aqueous alcohol solution such as 10 to 80% by volume ethanol. The amount of water or aqueous alcohol solution used in this case is not particularly limited, but is usually about 0.1 mL to 1.5 mL per 10 mg of the solid phase carrier.

[0033] An example of the process of contacting a solid carrier with a solution containing a chaotropic agent is outlined in FIG. 1. First, a solution containing a chaotropic agent is passed through the solid carrier, and the solution is removed by a method such as centrifugation or suction (step 1). Next, if necessary, water is passed through the solid carrier, and the solution is removed by a method such as centrifugation or suction (step 2). Here, an alcohol aqueous solution such as 10 to 80% by volume ethanol may be used instead of water. The solid carrier prepared in this manner can be used to separate a sample containing nucleic acid.

[0034] The method for detecting nucleic acids of the present invention includes a step (3) of detecting the target nucleic acid obtained by contacting the solid phase carrier with a solution containing a chaotropic agent as described above and then carrying out the steps (1) and (2) described above.

[0035] Nucleic acids can be detected by labeling with a labeling substance. When the nucleic acid is DNA, the labeling substance can be incorporated into the nucleic acid using an enzyme such as DNA polymerase or terminal deoxidil transferase. When the nucleic acid is detected by electrophoresis, the nucleic acid can be labeled by incorporating an intercalator such as ethidium bromide.

[0036] When the nucleic acid is RNA, a method of binding a labeling substance to the 3' end of RNA, a method of binding a labeling substance to the 5' end, or a method of binding a labeling substance to a nucleoside may be used, or these methods may be used in combination. In the method of binding a labeling substance to the 3' end or the method of binding a labeling substance to the 5' end, an enzyme reaction may be used. For the enzyme reaction, T4 RNA Ligase, Terminal Deoxididyl Transferase, Poly A polymerase, etc. may be used. For both labeling methods, the method described in "miRNA Experimental Protocol" (Yodosha, 2008) may be used as a reference. In addition, the above-mentioned DNA labeling method may be used after replacing RNA with a complementary strand of DNA using reverse transcriptase.

[0037] The labeling substance may be a nucleic acid having one or more labels, or may contain a linker sequence that is a polymer of at least two or more nucleic acids.

[0038] In the present invention, the label that can be used includes known substances used for labeling, such as fluorescent dyes, phosphorescent dyes, and radioisotopes. Preferred are fluorescent dyes that are easy to measure and whose signals are easy to detect. Specific examples include known fluorescent dyes such as cyanine (cyanine 2), aminomethylcoumarin, fluorescein, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine red, Texas red, indocarbocyanine (cyanine 5), cyanine 5.5, cyanine 7, and oyster.

[0039] A fluorescent microscope, a fluorescent scanner, or the like can be used to detect the fluorescent dye.

[0040] Methods for detecting nucleic acids include PCR, sequencing, hybridization, etc., when the nucleic acid is DNA. When the nucleic acid is RNA, it can be converted into DNA fragments by reverse transcriptase and then detected by PCR, sequencing, hybridization, etc. When the nucleic acid is RNA, it can be hydrolyzed into monomers and then detected by LC / MS / MS.

[0041] The detection step (3) itself is well known and can be carried out by a conventional method.

[0042] The variation of measurement data can be expressed as an index using the coefficient of variation. The coefficient of variation is obtained by dividing the standard deviation of values ​​obtained from multiple measurements by the average value and then multiplying the result by 100. A small coefficient of variation indicates low variation in the measurement data. In general, a coefficient of variation of less than 5% indicates low variation in the measurement data.

[0043] The nucleic acid purification column of the present invention has a hollow body (tube) with an inlet opening and an outlet opening, and a nucleic acid-binding solid phase carrier, and is manufactured by a method including step A, which is a step of "passing a solution containing a chaotropic agent through the nucleic acid-binding solid phase carrier, and then drying the nucleic acid-binding solid phase carrier."

[0044] One embodiment of the nucleic acid purification column of the present invention is shown in Figure 2. The hollow body 1 has a cylindrical structure and has an inlet opening 2 for injecting a sample solution and an outlet opening 6 for discharging the solution that has passed through the solid phase carrier 5. Materials that can be used for the hollow body 1 include resins such as polypropylene, polyethylene, polymethyl methacrylate, polytetrafluoroethylene, polyethyl terephthalate, and polyacrylonitrile, and glass, which are similar to those used for existing nucleic acid purification columns.

[0045] The inside of the hollow body 1 is filled with a nucleic acid-binding solid phase carrier 5. A fixing device 4 for fixing the solid phase carrier 5 may be attached to the inlet opening side. The fixing device 4 can be made of the same resin material as the hollow body 1. The fixing device 4 preferably has an outer diameter that is the same as the inner diameter of the hollow body 1 and has an O-ring structure with a hollowed-out center.

[0046] The hollow body 1 may have a rim 3. By providing the rim 3, when the present device is used with a centrifuge, it can be attached to a container 8 as shown in FIG.

[0047] Another embodiment of the nucleic acid purification column of the present invention is shown in Figure 3. A support 7 for holding the solid phase carrier 5 can be provided so that the solid phase carrier 5 does not deform or fall off during, for example, centrifugation. The support 7 has a microporous structure of a size that does not allow the solid phase carrier 5 to pass through, but allows the solution to pass through. Membranes made of plastics such as sintered glass, polypropylene, glass, ceramics, and nylon, or nonwoven fabrics made of polypropylene, polyethylene, nylon, and the like can be used.

[0048] FIG. 5 shows a 96-well plate format, which is another embodiment of the nucleic acid purification column of the present invention. This structure is formed by arranging 8×12 of the nucleic acid purification columns shown in FIG. 2 and FIG. 3 and connecting them at the outer periphery of a hollow body 1. Each column has an inlet opening 2 and an outlet opening 6 for discharging the solution that has passed through a solid phase carrier 5. The solution can be separated and removed by centrifugation or suction to discharge the solution. Note that, although columns in a 96-well plate format are commercially available as a combination of multiple columns arranged in a plate, the number and arrangement of columns are not limited to 8×12, and other numbers and arrangements (different numbers of rows and columns) can also be adopted.

[0049] The nucleic acid purification column having the above-mentioned hollow body provided with an inlet opening and an outlet opening and a nucleic acid-binding solid phase carrier can be produced by the following procedure.

[0050] First, the hollow body 1 is filled with the solid phase carrier 5. In this step, when the solid phase carrier 5 is in the form of particles or beads, it may be filled as it is using a medicine spoon or the like, or it may be dispersed in a solvent such as water or a buffer solution and then poured in using a pipette or the like. When the solid phase carrier 5 is in the form of a sheet or woven fabric, the solid phase carrier 5 is cut out to fit the inner diameter of the hollow body 1, filled into the hollow body 1, and if necessary, the above-mentioned O-ring structured fixing device 4 is fitted thereon to fix the solid phase carrier 5.

[0051] The nucleic acid purification column of the present invention is produced by a method comprising a step A of passing a solution containing a chaotropic agent through the nucleic acid-binding solid phase carrier and then drying the nucleic acid-binding solid phase carrier.

[0052] The solution containing the chaotropic agent may be the same as that used in the method for separating nucleic acid of the present invention. Specifically, an aqueous solution or an alcohol-containing aqueous solution containing a chaotropic agent such as guanidinium salt, urea, iodide salt, chlorate, perchlorate, thiocyanate, isothiocyanate, etc. at a concentration of 1M to 8M may be preferably used.

[0053] A method for passing (passing) a solution containing a chaotropic agent through a nucleic acid-binding solid phase carrier can be carried out in the same manner as in the method for separating nucleic acid of the present invention shown in FIG. 1, by passing (passing) a solution containing a chaotropic agent through a column filled with solid phase carriers, separating the solid phase carriers by centrifugation or suction (step 1), and then passing water or an aqueous alcohol solution such as 10 to 80% by volume ethanol through the column as necessary, and removing the chaotropic agent by centrifugation or suction (step 2).

[0054] The step of passing the solution containing a chaotropic agent through the nucleic acid-binding solid phase carrier can also be carried out before the solid phase carrier is packed into a column. Specifically, the solid phase carrier is immersed in a solution containing a chaotropic agent, and then, if necessary, in water, and the solution is separated and removed by centrifugation or suction.

[0055] After passing the solution containing the chaotropic agent, the nucleic acid-binding solid carrier is preferably washed with water or an aqueous alcohol solution as described above, and then the nucleic acid-binding solid carrier is dried. When the solid carrier is filled in the hollow body, the solid carrier may be dried naturally, specifically, the water content of the solid carrier can be evaporated by leaving it to stand for several minutes to several hours. Alternatively, the water content may be evaporated by leaving it in a heated oven. The temperature in this case can be appropriately set depending on the materials of the hollow body, the fixing device, and the support, but is preferably performed in the range of 20°C to 80°C. Even when the solution containing the chaotropic agent is passed through the solid carrier before it is filled in the column, the solid carrier can be dried in the same manner, and then the solid carrier can be filled inside the hollow body. In this case, as described above, if necessary, a fixing device with an O-ring structure is placed on the solid carrier to fix it, and a nucleic acid purification column can be prepared.

[0056] As described above, nucleic acid purification columns equipped with solid phase carriers are commercially available. Therefore, as described in the Examples below, the nucleic acid purification column of the present invention can also be produced by passing a chaotropic agent through a commercially available nucleic acid purification column as described above, then preferably washing the solid phase carrier with water or an aqueous alcohol solution, and then drying the solid phase carrier. EXAMPLES

[0057] The present invention will be described in detail with reference to the following examples, although the technical scope of the present invention is not limited to these examples.

[0058] In the following Examples and Comparative Examples, the following nucleic acid purification columns having a commercially available nucleic acid-binding solid phase carrier were used: a nucleic acid purification column using an 8 mm diameter silica membrane included in the Qiagen nucleic acid purification kit "miRNeasy Mini Kit" (hereinafter referred to as "Column A"), a nucleic acid purification column using an 8 mm diameter silica membrane included in the MACHEREY-NAGEL nucleic acid purification kit "NucleoSpin miRNA Plasma" (hereinafter referred to as "Column B"), a nucleic acid purification column used in the Qiagen nucleic acid purification kit "RNeasy 96 kit" (hereinafter referred to as "Column C"), a nucleic acid purification column used in the Qiagen nucleic acid purification kit "RNeasy UCP micro kit" (hereinafter referred to as "Column D"), a nucleic acid purification column used in the Zymo Research nucleic acid purification kit "RNA Clean & Concentrator Kit" (hereinafter referred to as "Column E"), and a nucleic acid purification column used in the cytiva nucleic acid purification kit "96 Well 800 μl UNIFILTER Microplate" (hereinafter referred to as "Column F") (Table 1).

[0059] [Table 1]

[0060] <Examples 1 and 2, Comparative Examples 1 and 2> (1) A step of passing a solution containing a chaotropic agent through a nucleic acid-binding solid phase carrier As the nucleic acid-binding solid phase carriers, columns A and B were used.

[0061] The process of passing a solution containing a chaotropic agent through the nucleic acid-binding solid phase carrier of the nucleic acid purification column was carried out as follows. First, 800 μL of the solution containing a chaotropic agent was placed on the column, centrifuged at 12,000×g for 1 minute, and the solution was removed. Here, a 1.3 M guanidine hydrochloride solution (Example 1) and a 67% ethanol solution of 1.3 M guanidine hydrochloride (Example 2) were used as the solution containing a chaotropic agent. Next, 800 μL of water was placed on the column, centrifuged at 12,000×g for 1 minute, and the water was removed.

[0062] In order to compare the effect of the present invention, a column was prepared in which 67% ethanol was passed through the column in the same manner as above, instead of the solution containing a chaotropic agent (Comparative Example 2).

[0063] In addition, an untreated column was prepared in which the solid phase carrier was not subjected to a step of passing a solution containing a chaotropic agent (Comparative Example 1).

[0064] (2) Preparation of samples containing nucleic acids and isolation of nucleic acids Using "Isogen LS" (Nippon Gene Co., Ltd.), a solution containing nucleic acids was obtained from 300 μL of human serum, and the nucleic acids were separated using the above-mentioned columns A and B. To verify reproducibility, column A was run four times and column B was run three times to obtain samples for detecting nucleic acids.

[0065] First, 1.5 times the amount of ethanol was added to the solution containing nucleic acid obtained from 300 μL of human serum described above, and the solution was placed on columns A and B, respectively. The solution containing nucleic acid was contacted with the solid phase carrier in the column by centrifuging the solution at 12,000×g for 1 minute, and the nucleic acid contained in the solution was adsorbed onto the solid phase carrier. Next, the washing solution included in each of the nucleic acid purification kits for columns A and B was placed on the column, and the column was centrifuged at 12,000×g for 1 minute to wash away components other than nucleic acid remaining in the column. Finally, 70 μL of water was placed on the column, and the column was centrifuged at 12,000×g for 1 minute to obtain an eluate containing nucleic acid.

[0066] (3) Measurement of nucleic acids using DNA microarrays The following experiment was carried out using Toray Industries' "3D-Gene" human miRNA oligo chip (compatible with miRBase release 21, capable of detecting 2556 types of miRNA).

[0067] The nucleic acids in the eluate obtained as described above were labeled using the "3D-Gene" miRNA labeling kit (manufactured by Toray Industries, Inc.). The labeled nucleic acids (RNA) were subjected to hybridization and washing according to the standard protocol for the "3D-Gene" miRNA chip (manufactured by Toray Industries, Inc.). The fluorescent signals of the reacted DNA microarray were detected using a "microarray scanner" (manufactured by Toray Industries, Inc.), and the number of miRNAs that gave signals significantly higher than the background noise was determined. The standard deviation and coefficient of variation of the values ​​between multiple measurements were calculated (Table 1).

[0068] As a result, when Column A was used, in Comparative Example 1 (untreated), an average of 1535 miRNAs were detected. The standard deviation was 130, and a large variation was observed for each measurement. In Comparative Example 2 (67% ethanol was passed through), the number of detected miRNAs also varied, similar to Comparative Example 1. The coefficient of variation was 8.5% in Comparative Example 1 and 6.6% in Comparative Example 2.

[0069] On the other hand, in Example 1 (1.3 M guanidine hydrochloride solution was passed through), the average number of detected miRNAs was 1451, the standard deviation was 34, and the coefficient of variation was 2.4%. In Example 2 (1.3 M guanidine hydrochloride in 67% ethanol solution was passed through), the average number of detected miRNAs was 1407, the standard deviation was 21, and the coefficient of variation was 1.5%, and both Examples 1 and 2 showed significantly reduced variation compared to Comparative Examples 1 and 2.

[0070] When column B was used, in Comparative Example 1 (untreated), an average of 1593 miRNAs were detected. The standard deviation was 82, and a large variation was observed for each measurement. In Comparative Example 2, the number of detected miRNAs also varied similarly to Comparative Example 1. The coefficient of variation was 5.1% in Comparative Example 1 and 5.6% in Comparative Example 2.

[0071] On the other hand, in Example 1 (1.3 M guanidine hydrochloride solution was passed through), the average number of detected miRNAs was 1548, the standard deviation was 47, and the coefficient of variation was 3.0%. In Example 2 (1.3 M guanidine hydrochloride in 67% ethanol solution was passed through), the average number of detected miRNAs was 1401, the standard deviation was 18, and the coefficient of variation was 1.3%, and both Examples 1 and 2 showed significantly reduced variation compared to Comparative Examples 1 and 2.

[0072] [Table 2]

[0073] <Example 3, Comparative Example 3> Using guanidine thiocyanate instead of guanidine hydrochloride as a chaotropic agent, and nucleic acid purification columns A to F shown in Table 1, nucleic acids were separated from a sample containing nucleic acids and the separated nucleic acids were measured using a DNA microarray in the same manner as in Examples 1 and 2 (Example 3).

[0074] Here, a 67% ethanol solution of 2.1 M guanidine thiocyanate was used as the solution containing the chaotropic agent. In addition, since columns C, E, and F were in a 96-well plate format, the centrifugal acceleration condition for removing the solution was set to 3,000×g.

[0075] For comparison, a column was prepared in which sterilized purified water was used instead of the solution containing a chaotropic agent (Comparative Example 3).

[0076] As in Examples 1 and 2, nucleic acids were separated from the prepared nucleic acid samples using each column, and then the nucleic acids were measured using a DNA microarray. The standard deviation and coefficient of variation of the number of miRNAs detected between multiple measurements were calculated from each measurement (Table 3).

[0077] As a result of the analysis, the coefficient of variation was 5.2% to 5.9% when sterilized purified water was passed through in Comparative Example 3, but was 1.2% to 1.9% when a chaotropic agent was passed through in Example 3, confirming the effect of reducing variation in all columns to which the present invention was applied.

[0078] [Table 3]

[0079] <Example 4> A nucleic acid purification column of the present invention was prepared by passing a solution containing a chaotropic agent through the nucleic acid-binding solid phase carrier and then drying the nucleic acid-binding solid phase carrier.

[0080] A silica membrane (manufactured by Advantec Toyo Co., Ltd., model number QR-100) was used as the nucleic acid-binding solid phase carrier. A microbio spin chromatography column (Bio-Rad, model number 7326204) was used as the hollow body provided with an inlet opening and an outlet opening.

[0081] A circular hole was punched out of the silica filter paper with a diameter of 7 mm, and the silica membrane was packed into a column for microbio-spin chromatography. A polypropylene O-ring with a diameter of 7 mm was attached from above to fix the silica membrane to the column (hollow body). The process of passing a solution containing a chaotropic agent through the nucleic acid-binding solid phase carrier was carried out as follows. 800 μL of a 67% ethanol solution of 2.1 M guanidine thiocyanate as a solution containing a chaotropic agent was placed on the column, centrifuged at 12,000 × g for 1 minute, and the solution was removed. Next, 800 μL of water was placed on the column, centrifuged at 12,000 × g for 1 minute, and the water was removed. The column was then left to stand in a room for 6 hours and allowed to dry naturally.

[0082] In this manner, a nucleic acid purification column of the present invention (hereinafter referred to as "Column G") was prepared.

[0083] <Example 5> A solution containing a chaotropic agent was passed through nucleic acid purification columns A to F included in the commercially available nucleic acid purification kits shown in Table 1 to prepare the nucleic acid purification columns of the present invention.

[0084] For columns A to F, the step of passing a solution containing a chaotropic agent (including a water washing step) was carried out in the same manner as in Example 3. After removing the solution by centrifugation, each column was left to stand indoors for 6 hours and allowed to dry naturally.

[0085] In this manner, nucleic acid purification columns of the present invention (hereinafter referred to as "Column A'" to "Column F'", respectively) through which a solution containing guanidine thiocyanate as a chaotropic agent was passed were prepared.

[0086] <Example 6> Using columns A' to F' prepared in Example 5 and column G prepared in Example 4, nucleic acids were separated from the prepared nucleic acid samples in the same manner as in Examples 1 and 2, and the nucleic acids were measured using a DNA microarray. The standard deviation and coefficient of variation of the number of miRNAs detected between multiple measurements were calculated from each measurement value (Table 4).

[0087] As a result of the analysis, the coefficient of variation was 1.2% to 1.7% for columns A' to F' and 1.0% for column G. Compared with the coefficient of variation of 5.2% to 5.9% in Comparative Example 3 in which sterilized purified water was passed through, it was confirmed that the nucleic acid purification column of the present invention had the effect of reducing measurement variability.

[0088] [Table 4]

[0089] <Examples 6 and 7, Comparative Example 6> In Example 3, each of the commercially available nucleic acid purification columns A to F after passing a 67% ethanol solution of 2.1 M guanidine thiocyanate through each column (Example 6), columns A' to F' prepared in Example 5, and column G (Example 7) prepared in Example 4 were examined for the presence or absence of nucleic acid contamination (residual) in each column by detecting nucleic acids contained in the eluate eluted with sterile purified water. For comparison, the same measurement was performed on each of the commercially available nucleic acid purification columns A to F after passing sterile purified water through each column in Example 3 (Comparative Example 6).

[0090] 70 μL of sterile purified water was dropped onto each column, and the column was centrifuged to obtain an eluate. The nucleic acids contained in the eluate were measured in the same manner as in Examples 1 and 2. The number of detected miRNAs is shown in Table 5.

[0091] In the eluates of columns A to F after passing sterile purified water without passing a solution containing a chaotropic agent, 50 to 269 kinds of miRNA were detected. On the other hand, in columns A to F after treatment with 67% ethanol solution of 2.1 M guanidine thiocyanate, and columns A' to F' and G of the present invention manufactured by passing a chaotropic agent, almost no nucleic acid was detected in the eluates. From this, it was found that all commercially available nucleic acid purification columns were contaminated with nucleic acid, and that the contaminated nucleic acid was removed by passing a chaotropic agent through the commercially available nucleic acid purification column by the method of the present invention. Furthermore, it was found that no nucleic acid contamination was observed in the nucleic acid purification column of the present invention manufactured by passing a chaotropic agent.

[0092] [Table 5] [Explanation of symbols]

[0093] 1 hollow body 2 Inlet opening 3 Rims 4 Fixtures 5. Nucleic Acid Binding Solid Phase Carriers 6 Outlet opening 7 Fixtures 8 containers

Claims

1. The following steps (1) and (2) (1) contacting a sample containing a target nucleic acid with a nucleic acid-binding solid phase carrier capable of adsorbing the nucleic acid; and (2) a step of eluting the nucleic acid from the nucleic acid-binding solid phase carrier to which the nucleic acid has been adsorbed in the step (1); A method for isolating a nucleic acid from a sample containing the nucleic acid, comprising: Prior to the step (1), the nucleic acid-binding solid phase carrier is brought into contact with a solution containing a chaotropic agent, The method further comprises the step of washing the nucleic acid-binding solid phase carrier with water or an aqueous alcohol solution after contacting the nucleic acid-binding solid phase carrier with the solution containing the chaotropic agent. method.

2. The method according to claim 1, wherein the nucleic acid-binding solid phase carrier is a silica membrane.

3. 3. The method of claim 1 or 2, wherein the chaotropic agent is any one of a guanidinium salt, urea, an iodide salt, a chlorate, a perchlorate, a thiocyanate, and an isothiocyanate.

4. The method according to any one of claims 1 to 3, wherein the solution containing a chaotropic agent contains an alcohol.

5. The method of claim 4 , wherein the alcohol is ethanol.

6. The method according to any one of claims 1 to 5, wherein the nucleic acid is DNA or RNA.

7. The following steps (1), (2) and (3) (1) contacting a sample containing a target nucleic acid with a nucleic acid-binding solid phase carrier capable of adsorbing the nucleic acid; (2) eluting the nucleic acid from the nucleic acid-binding solid phase carrier to which the nucleic acid has been adsorbed in the step (1); and (3) labeling and detecting the eluted nucleic acid A method for detecting a nucleic acid, comprising: Prior to the step (1), the nucleic acid-binding solid phase carrier is brought into contact with a solution containing a chaotropic agent, The method further comprises the step of washing the nucleic acid-binding solid phase carrier with water or an aqueous alcohol solution after contacting the nucleic acid-binding solid phase carrier with the solution containing the chaotropic agent. method.

8. A nucleic acid purification column having a hollow body provided with an inlet opening and an outlet opening, and a nucleic acid-binding solid phase carrier, A nucleic acid purification column produced by a method including a step A of passing a solution containing a chaotropic agent through a nucleic acid-binding solid phase carrier and then drying the nucleic acid-binding solid phase carrier.

9. A method for producing a nucleic acid purification column having a hollow body provided with an inlet opening and an outlet opening and a nucleic acid-binding solid phase carrier, comprising the steps of: A production method comprising a step A of passing a solution containing a chaotropic agent through a nucleic acid-binding solid phase carrier and then drying the nucleic acid-binding solid phase carrier.

10. The method according to claim 9, further comprising the step of washing the nucleic acid-binding solid phase carriers with water or an aqueous alcohol solution after passing the solution containing the chaotropic agent through the nucleic acid-binding solid phase carriers.

11. The method according to claim 9 or 10, wherein the step A is carried out in a state where the nucleic acid-binding solid phase carrier is filled in a hollow body.

12. The method according to any one of claims 9 to 11, wherein the nucleic acid-binding solid phase carrier is a silica membrane.

13. The method according to any one of claims 9 to 12, wherein the chaotropic agent is a guanidinium salt, urea, an iodide salt, a chlorate, a perchlorate, a thiocyanate or an isothiocyanate.

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