JP2004215676A - Method of purification and separation of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the yield of nucleic acid recovery in the method of purification and separation of nucleic acid. <P>SOLUTION: The nucleic acid is adsorbed on an inorganic carrier from a mixture containing nucleic acids, salts and organic solvents, and subsequently is collected by desorption of the nucleic acid from the carrier using an eluent. The organic solvents are compounds with 2-10 carbon atoms selected from aliphatic ethers, aliphatic esters and aliphatic ketones. The method of purification and separation of nucleic acid has an improved yield of nucleic acid recovery, an excellent operability and a little influence in pollution. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a nucleic acid purification / separation method for purifying and separating a nucleic acid by eluting the nucleic acid from a sample containing the nucleic acid such as a biological sample.

  Effective methods for purifying and separating nucleic acids in the prior art are based on the principle of recovering nucleic acids by adsorbing the nucleic acids to glass or silica gel particles in the presence of chaotropic salts (Vogelstein, B. and Gillespie, D. (1979). ); "Preparative and degradative purification of DNA from agarose", Proc. Natl. Acad. Sci. USA 76: 615-619). According to this method, using a high concentration of chaotropic salts such as sodium iodide, sodium perchlorate, or guanidine thiocyanate, DNA can be isolated and purified from agarose, or RNA or DNA can be mixed in various mixtures. (Boom, R. (1990); a rapid and simple method for purifying nucleic acids, J. Clin. Microbiol. 28: 495-503).

  After purification, the nucleic acid is often used in a polymerase chain reaction (PCR). According to the PCR reaction, nucleic acids can be amplified in a sequence-specific manner, and thus are widely applied to gene diagnosis and the like. There are several problems in routine clinical use of this PCR method. Among them, it is known that a PCR reaction is inhibited by the influence of an inhibitor that cannot be removed during nucleic acid purification. Known inhibitors include hemoglobin, surfactants used in the nucleic acid extraction step, and the like. Against this background, it has been pointed out that nucleic acid extraction and purification steps are important (Oshima et al., JJCL A, 22 (2), 145-150 (1997)).

  Further, the extraction step has conventionally been performed manually, but since the operation is complicated and requires skill, automation by an apparatus is desired. Development of an extraction method suitable for automation, including the reagents to be used, is required. As a method for extracting a nucleic acid suitable for automation, there are a method described in JP-A-11-127854 and a method using an apparatus described in JP-A-11-266864. Further, as a reagent suitable for extraction by an automated apparatus, a reagent described in JP-T-8-501321 is known. In the method using the reagent described in JP-A-8-501321, a nucleic acid to be separated and purified from a solution containing a high concentration of salt (ionic strength) and a high concentration of alcohol is brought into contact with an adsorption carrier in an extraction chip. Then, the target nucleic acid can be obtained by desorbing from the adsorption carrier with a solution containing a lower concentration of salt (ionic strength).

  The method for purifying and separating nucleic acids using the reagents described in JP-T 8-501321 has a problem that the yield of nucleic acid recovery is low. Further, in the method using an extraction chip described in JP-A-11-127854, a nucleic acid to be separated and purified from a solution containing a high concentration of salt (ionic strength) and a high concentration of alcohol is adsorbed on the extraction chip. In the step of contacting with and adsorbing the solution, there is a problem that the operation requires a long time because the viscosity of the solution is high.

  An object of the present invention is to provide a method for purifying and separating a nucleic acid which improves the recovery yield, shortens the time required for nucleic acid recovery, has excellent operability, and is less affected by contamination.

  In the method for purifying and separating a nucleic acid of the present invention, the nucleic acid is adsorbed on an inorganic carrier from a mixed solution containing a nucleic acid-containing sample, a high-concentration salt, and an organic solvent. Remove and recover from the carrier. Before recovery, the carrier is washed with a washing solution for removing components other than nucleic acids, if necessary. The nucleic acid is derived, for example, from whole blood leukocytes.

  As a salt, a chaotropic salt is used. For example, any of guanidine hydrochloride, guanidine isothiocyanate, and potassium iodide is used in a concentration range of 1 M to 8 M.

  The organic solvent (organic compound) is one or more compounds selected from aliphatic ethers, aliphatic esters, and aliphatic ketones, and is a compound having 2 to 10 carbon atoms. The concentration of the organic solvent in the mixture is 5% by volume to 50% by volume.

  Representative examples of the aliphatic ether that can be used as an organic solvent (organic compound) in the mixture include ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, and tetrahydrofuran. And 1,4-dioxane.

  Representative examples of the aliphatic ester that can be used as the organic solvent (organic compound) in the mixture include propylene glycol monomethyl ether acetate and ethyl lactate.

  Representative examples of aliphatic ketones that can be used as an organic solvent (organic compound) in the mixed solution include hydroxyacetone, acetone, and methyl ethyl ketone.

  In the present invention, 0.1% to 50% by volume of a surfactant is contained in the mixed solution, and more preferably, 0.1% to 5% by volume of an antifoaming agent is used as the surfactant. good.

  As the inorganic carrier, a porous or non-porous material made of silica, alumina, zeolite, titanium dioxide, or the like is used.

  The washing liquid contains an organic solvent such as alcohol at a high concentration, and the inorganic carrier is washed a plurality of times by suction or centrifugation to remove components other than nucleic acids.

  According to the method for purifying and separating a nucleic acid of the present invention, the viscosity of a solution containing a nucleic acid is reduced, the generation of bubbles is suppressed, and the defoaming is promoted, so that the operability is improved and the influence of contamination is reduced. As a result, the amount of recovered nucleic acid increases. Further, the operation time required for nucleic acid recovery can be reduced.

  ADVANTAGE OF THE INVENTION According to the method of this invention, while improving the recovery yield, shortening the time required for nucleic acid recovery, it is possible to provide a method for purifying and separating nucleic acids that is excellent in operability and less affected by contamination. .

  Purification and separation of the nucleic acid of the present invention are performed based on the method shown in FIG. Here, the same items as those in the four steps described in JP-A-11-127854 are described. The details of each step will be described below.

  FIGS. 2 to 5 are diagrams illustrating the procedure for extracting nucleic acids from whole blood using the nucleic acid extraction reagent of the present invention with respect to the items shown in FIG. In the following description, nucleic acid extraction from 100 μL of human whole blood is taken as an example. As shown in FIG. 2, human whole blood 1 contains components such as red blood cells 2 and white blood cells 3, and nucleic acids mainly exist in nuclei 4 in white blood cells 3.

  As shown in FIG. 3, first, 10 μL of Proteinase K (6) is injected into the tube 5. Next, 100 μL of whole blood 1 is added to tube 5. This step is 101. Next, 100 μL of the reaction solution is added and stirred. This step is 102. The reaction contains 1M to 8M chaotropic salt and up to 50% by volume of surfactant. By incubating the solution in the tube 5 at 56 ° C. for 10 minutes, leukocytes are destroyed, and the nucleic acid in the nucleus 4 comes out of the solution. This step is 103. Thereafter, 100 μL of an additive liquid (for example, an organic solvent such as EGDE (ethylene glycol diethyl ether) or DIGLYME (diethylene glycol dimethyl ether)) is added and stirred, whereby a mixed liquid 7 is obtained. This step is 104. The operation up to this point is called a decomposition step.

  After this decomposition step, there are several types of nucleic acid recovery methods. Each method includes an adsorption step, a washing step, and a recovery step. Typical examples include a spin column method and a suction / discharge method.

  FIG. 4 shows a spin column method (hereinafter, referred to as a first method). The mixed solution 7 in the tube 5 to which the additive solution has been added is transferred into the spin column 8. The bottom of the spin column 8 is filled with a filter paper-shaped adsorption carrier 9 made of very fine silica or glass fiber. The mixed solution 7 containing the additive liquid is caused to pass through the adsorption carrier 9 by suction, and the nucleic acid is adsorbed on the adsorption carrier 9 (adsorption step). This step is 105. Thereafter, 500 μL of the washing liquid 11 containing 20% to 80% of an organic solvent such as alcohol is injected into the spin column 8. This step is 106. Next, in step 107, the adsorption carrier 9 is washed by suction to remove components other than nucleic acids (washing step). This washing step is repeated twice, but the adsorbed nucleic acid is not eluted. Thereafter, the eluate 12 having a low salt concentration is added, and the mixture is allowed to stand for 2 to 5 minutes. This step is 108. The step 109 is to elute the nucleic acid from the adsorption carrier 9 and finally collect it in the tube 10 by suction (elution step). The composition of a typical eluate is 50 mmol Tris / HCl (pH 8.5), 0.1 mmol EDTA · 2Na. In the first method, a centrifugation operation can be used in each step for simple and quick processing.

  FIG. 5 shows a suction-discharge method (hereinafter, referred to as a second method). In the second method, a cylindrical hollow column 14 filled with fibers such as glass or quartz at a low density is used as the adsorption carrier 13. Step 110 is to repeatedly suction and discharge the mixed solution 7 to which the additive liquid has been added in the column 14 a plurality of times, and to adsorb the nucleic acid to the adsorption carrier 13 (adsorption step). Subsequently, suction and discharge are performed using another tube 16 containing a cleaning solution 15 containing 20 to 80% of an organic solvent such as 500 μL of an alcohol to remove components other than nucleic acids. This washing step is performed twice or more. Thereafter, the eluate 18 (100 μL) in the tube 17 preheated to 70 ° C. is sucked up to a position where the eluate 18 comes into contact with the adsorption carrier 13 in the column 14, and is stopped for about 2 minutes. This step is 111. By this operation, the nucleic acid is eluted from the adsorption carrier 13 into the eluate 18. The eluate 18 is discharged to recover the nucleic acid. This step is 112. The composition of a typical eluate is 50 mmol Tris / HCl (pH 8.5), 0.1 mmol EDTA · 2Na.

  Hereinafter, materials, chemicals, and samples used in the examples will be described in detail.

1. Nucleic Acid Adsorbing Material and Column 1.1 Spin Column For example, three layers of Whatman glass fiber filter paper were fixed on a centrifugal separation chromatography column (spin column). This was used in the protocol of the first method shown in FIG.
1.2 Column for Nucleic Acid Capture A quartz fiber (manufactured by Tosoh Quartz Co., Ltd. or Toshiba Ceramics Co., Ltd.) having a diameter of 0.5 μm to 30 μm used for the nucleic acid capture chip described in JP-A-11-266864 was packed. This was used in the protocol of the second method shown in FIG.

2. Sample containing nucleic acid 2.1 Blood (human)
Blood immediately after blood collection or blood frozen at -20 ° C was used.
2.2 Tissue Fresh tissue or cryopreserved tissue was used.
2.3 Plants Plants were ground in a mortar under liquid nitrogen and used directly or frozen and stored.
2.4 Cells Isolated or isolated / cultured cells were used directly, or cells that had been cryopreserved were used.

3. Reagent 3.1 Reaction solution The reaction solution is composed of the following chaotropic salts, surfactants, defoamers, and other salt components.
3.1.1 Chaotropic salt As a chaotropic salt, any of the following was used.
KI: Potassium iodide GuHCl: Guanidine hydrochloride GuHSCN: Guanidine thiocyanate

3.1.2 Surfactant As a surfactant, any of the following was used.
Tween20: polyoxyethylene (20) sorbitan monolaurate
Tween40: polyoxyethylene (20) sorbitan monopalmitate
Tween60: polyoxyethylene (20) sorbitan monostearate
Tween80: polyoxyethylene (20) sorbitan monooleate
Tween85: polyoxyethylene (20) sorbitan triolate
Triton X-100: polyoxyethylene (10) isooctyl phenyl ether 3.1.3 defoamer CE-457: defoamer disperse manufactured by NOF CORPORATION, polyalkylene glycol derivative 3.1.4 other salts EDTA 2Na: Ethylenediaminetetraacetic acid disodium salt Tris: Tris (hydroxymethyl) aminomethane

3.2. Additive liquid Any of the following organic solvents was used for the additive liquid.
EGDME: ethylene glycol dimethyl ether EGDEE: ethylene glycol diethyl ether PGDME: propylene glycol dimethyl ether PGDEE: propylene glycol diethyl ether DIGLYME: diethylene glycol dimethyl ether DGDEE: diethylene glycol diethyl ether THF: tetrahydrofuran DX: 1,4-dioxane PGMEA: propylene glycol monomethyl ether acetate EL: Ethyl lactate HAC: hydroxyacetone AC: acetone MEK: methyl ethyl ketone

3.3 Cleaning solution Any of the following compositions was used as the cleaning solution.
W1: 25 mmol / l potassium acetate, 70 vol% ethanol W2: 25 mmol / l potassium acetate, 50 vol% ethanol W3: 25 mmol / l Tris / HCl, 50 vol% ethanol W4: 10 mmol / l Tris / HCl (pH 8.5) , 0.1 mmol / l EDTA · 2Na, 50% by volume ethanol

3.4 Eluate Any of the following compositions was used for the eluate.
E1: Water (100%), pH 8.0
E2: 10 mmol / l Tris / HCl (pH 8.5), 0.1 mmol / l EDTA · 2Na
E3: 50 mmol / l Tris / HCl (pH 8.5), 0.1 mmol / l EDTA · 2Na

3.5. Enzymes Any of the following enzymes were used.
protease: Proteolytic enzyme protease
proteinase K: alkaline proteinase K

Nucleic acid extraction from whole blood 1
Nucleic acid was extracted from human whole blood 1 (100 μL) according to the second method shown in FIGS. 3 and 5. In the decomposition step, whole blood 1 (100 μL) and the reaction solution (100 μL) were added to a tube 5 containing proteinase K (6) (10 μL) and stirred to obtain a mixed solution. The reaction solution used here contains 3M guanidine hydrochloride and 5% by volume of Triton X-100. The mixture was incubated at 70 ° C. for 10 minutes. Thereafter, an additive liquid (100 μL) selected from EGDME, EGDEE, PGDME, PGDEE, DIGLYME, DGDEE, THF, DX, PGMEA, EL, HAC, AC and MEK was added and stirred.

  In the adsorption step, the operation of suctioning and discharging the mixed solution 7 was repeated 10 times using the column 14 filled with the adsorption carrier 13 (manufactured by Toshiba Ceramics Co., Ltd.).

  In the cleaning process, the operation of suctioning and discharging the cleaning liquid (W1) 15 placed in another tube 16 was repeated three times.

  In the recovery step, the eluate 18 placed in another tube 17 was sucked up to a position where the entire adsorption carrier 13 was immersed, and was stopped for 2 minutes. Thereafter, the eluate 18 was discharged, and the nucleic acid solution was recovered.

  The obtained nucleic acid solution could be used for the next reaction and analysis such as PCR without any steps such as precipitation purification.

  Table 1 shows nucleic acid yields when various additive solutions were used, and A260 / A280 as an index of nucleic acid purity in the extracted nucleic acid solution.

  Here, A260 represents the absorbance of an aqueous solution containing a nucleic acid at a wavelength of 260 nm. When the A260 / A280 value is 1.8, the nucleic acid is considered pure. Further, when the value of A260 / A280 is between 1.7 and 1.9, the purity is considered to be sufficient. In the series of experiments in Table 1, the same enzyme (proteinase K), reaction solution, washing solution and eluate were used. The obtained nucleic acid was quantified by fluorescence measurement using a fluorescent dye PicoGreen (Molecular Probes Inc.) for quantifying double-stranded nucleic acid. It can be seen that high-purity nucleic acids were obtained using any of the additive solutions.

  FIG. 6 shows a change in the nucleic acid yield when the amount of the addition solution DIGLYME is changed. The horizontal axis represents the DIGLYME concentration (% by volume) in the mixture 7, and the vertical axis represents the relative yield (%) when the maximum yield is 100. The target yield is obtained when the relative yield is 80% or more. It can be seen that a relative yield of 80% or more is obtained when the DIGLYME concentration is from 5% to 50%, and the maximum yield is obtained particularly when the concentration is about 43%.

  FIG. 7 shows a change in the nucleic acid yield when the amount of the addition liquid EL is changed. In a series of experiments, the same enzyme (proteinase K), reaction solution, washing solution and eluate were used. The target yield is obtained when the relative yield is 80% or more. It can be seen that a relative yield of 80% or more is obtained when the amount of EL added is 10% to 50%, and the maximum yield is obtained particularly when the amount of EL is about 33%.

  FIG. 8 shows a change in nucleic acid yield when DIGLYME and EL are mixed and used as an additive solution. Here, the addition amount of the additive liquid corresponds to 33% of the entire mixture. The horizontal axis in FIG. 8 represents the DIGLYME ratio (volume ratio) in the additive solution. The target yield is obtained when the relative yield is 80% or more. It can be seen that the desired yield can be obtained at any DIGLYME ratio.

  FIGS. 9, 10 and 11 show the relationship between the surfactant concentration in the reaction solution and the nucleic acid yield. In a series of experiments shown in FIGS. 9 to 11, proteinase K, an additive solution, a washing solution, and an eluate were used as enzymes. The target yield is obtained when the relative yield is 80% or more. It is shown that a relative nucleic acid yield of 80% or more can be obtained at a surfactant content of 5% to 50%.

  FIG. 12 shows the relationship between the amount of the antifoaming agent (CE-457) added in the mixture 7 and the nucleic acid yield. In a series of experiments, the same enzyme (proteinase K), additive solution, washing solution and eluate were used. The composition of the reaction solution is the same except for the antifoaming agent. It is shown that a high nucleic acid yield of 80% or more in relative yield can be obtained when the amount of the antifoaming agent added is 0.2% to 2.5%. Therefore, it was shown that the addition of the antifoaming agent was effective not only for suppressing foaming in the mixed solution but also for improving the nucleic acid yield.

Table 2 shows that the operability in the adsorption step is improved by adopting DIGLYME as the additive solution ( ^{* "} Organic Solvents, Fourth Edition", John Wiley & Sons, Inc., 1986).

  Comparison of the viscosity of ethanol, which is a typical compound of alcohol described in JP-T-8-501321, with the additive solution DIGLYME in the method of the present invention shows that the viscosity of DIGLYME is lower. This shows that the time required for the adsorption step was reduced by 10 seconds because the viscosity was reduced and the liquid flow resistance in the column 14 was reduced. In addition, since foaming was suppressed, contamination was reduced and operability was improved.

  Table 3 shows the flash points of the various additive solutions, and their safety was examined ("Solvent Pocket Book", edited by The Society of Synthetic Organic Chemistry, Ohmsha).

  Comparing the flash points, DIGLYME, EL, EGDEE and DGDEE are higher than the flash point of ethanol. Therefore, it can be said that, among them, DIGLYME, EL, EGDEE and DGDEE are preferred from the viewpoint of safety since they have less risk of explosion and fire.

Nucleic acid extraction from whole blood 2
Nucleic acids were extracted from human whole blood 1 (1 mL) according to the second method shown in FIGS. In the decomposition step, whole blood 1 (1 mL) and the reaction solution (1 mL) were added to a tube 5 containing proteinase K (6) (100 μL) and stirred to obtain a mixed solution. The reaction solution used here contains 3M guanidine hydrochloride and 5% by volume of Triton X-100. The mixture was incubated at 70 ° C. for 10 minutes. Thereafter, an additive solution (1 mL) selected from DIGLYME and ethanol was added and stirred.

  In the adsorption step, the operation of sucking and discharging the mixed solution 7 was repeated 10 times using the column 14 filled with the adsorption carrier 13 (100 mg) (manufactured by Tosoh Quartz Co., Ltd.).

  In the cleaning step, the operation of suctioning and discharging the cleaning liquid (W1) (3 ml) 15 placed in another tube 16 was repeated three times.

In the recovery step, the eluate (1 ml) 18 placed in another tube 17 was sucked up to a position where the whole of the adsorption carrier 13 was immersed, and kept still for 2 minutes. Thereafter, the eluate 18 was discharged, and the nucleic acid solution was recovered.
The obtained nucleic acid solution could be used for the next reaction and analysis such as PCR without any steps such as precipitation purification.

  Table 4 shows the yield of nucleic acid and A260 / A280 of the obtained nucleic acid solution when ethanol and DIGLYME were used as the additive liquid.

  When ethanol was used as the additive solution, the nucleic acid yield was 0.6 μg, whereas when DIGLYME was used, the yield was 15.0 μg, indicating that DIGLYME was superior as the additive solution. .

Nucleic acid extraction from whole blood 3
Nucleic acids were extracted from 100 μL of human whole blood according to the first method shown in FIGS.

  The decomposition step was performed in the same manner as in Example 1. The enzymes, reaction solutions, additive solutions, washing solutions and eluates used were the same as in Example 1.

  In the adsorption step, the mixed solution was poured into the spin column 8 and centrifuged for 1 minute (at 6000 rpm) using a tabletop centrifuge (at 6,000 rpm).

  In the washing step, the washing solution (W2) 11 (500 μl) was poured into the spin column 8 and centrifuged for 1 minute (at 6,000 rpm).

In the recovery step, the eluate (E2) 12 (100 μl) was poured into the spin column 8, allowed to stand at room temperature (25 ° C.) for 2 minutes, and then centrifuged for 1 minute (at 6000 rpm) to recover the nucleic acid, and then quantified. And the purity was evaluated.
In this method, the same results as in Example 1 were obtained.

Extraction of nucleic acid from tissue 1 (first method)
25 mg of liver tissue (rat) and 80 μl of PBS (phosphate buffered saline) were mixed and homogenized mechanically. After adding Proteinase K (20 μl), the mixture was stirred and heated to 56 ° C. until the tissue was dissolved. After adding 200 μl of a reaction solution containing 3M guanidine hydrochloride and 5% by volume of Tween 80 to form a mixed solution, the mixture was stirred and heated at 70 ° C. for 10 minutes. DIGLYME (200 μl) was added and mixed. The above is the decomposition step.

  In the adsorption step, the mixed solution was poured into the spin column 8 and centrifuged at 6000 rpm for 1 minute using a desktop centrifuge.

  In the washing step, the washing solution (W3) 11 (500 μl) was poured into the spin column 8 and centrifuged (at 6,000 rpm) for 1 minute.

  In the recovery step, the eluate 12 (100 μl) selected from the above E1, E2 and E3 is poured into the spin column 8, left at room temperature (25 ° C.) for 2 minutes, and then centrifuged for 1 minute (at 6,000 rpm). The nucleic acid was recovered by the method described above, and quantification and purity were evaluated.

  As a result, a highly pure nucleic acid having A260 / A280 of 1.8 was obtained, and could be used for the PCR reaction.

Extraction of nucleic acid from tissue 2 (first method)
Nucleic acid extraction was performed from 20 mg of bladder tissue (rat) in the same manner as in Example 4. As a result, a high-purity nucleic acid having A260 / A280 of 1.7 was obtained, and could be used for the PCR reaction.

Nucleic acid extraction from urine (first method)
A 20 ml urine sample was centrifuged (rotation speed: 14000 rpm) for 5 minutes. The supernatant was removed and the urine sediment was separated. After adding 200 μl of a reaction solution containing proteinase K (20 μl) and guanidine hydrochloride at a concentration of 3 M and 5% by volume of Triton X-100 to the obtained urine sediment, the mixture was stirred and heated at 70 ° C. for 10 minutes. 200 μl of DIGLYME was added and mixed. The above is the decomposition step.

  In the adsorption step, the mixed solution was poured into the spin column 8 and centrifuged (at 6000 rpm) for 1 minute using a tabletop centrifuge.

  In the washing step, the washing solution (W4) 11 (500 μl) was poured into the spin column 8 and centrifuged (at 6,000 rpm) for 1 minute.

  In the recovery step, the eluate (E3) 12 (100 μl) was poured into the spin column 8, allowed to stand at room temperature (25 ° C.) for 2 minutes, and then centrifuged for 1 minute to recover nucleic acids, and quantitatively and purity evaluation was performed. Was.

  As a result, a highly pure nucleic acid having A260 / A280 of 1.8 was obtained, and could be used for the PCR reaction.

Nucleic acid extraction from cells (first method)
10 ⁶ HeLa cells were dispersed in PBS (100 μl), and 200 μl of a reaction solution containing proteinase K (20 μl), guanidine hydrochloride at a concentration of 3 M, and 5% by volume of Triton X-100 was added, followed by stirring and 70 ° C. For 10 minutes. 200 μl of DIGLYME was added and mixed. The above is the decomposition step.

  In the adsorption step, the mixed solution was poured into the spin column 8 and centrifuged (at 6000 rpm) for 1 minute using a tabletop centrifuge.

  In the washing step, the washing solution (W2) 11 (500 μl) was poured into the spin column 8 and centrifuged for 1 minute (at 6,000 rpm).

  In the recovery step, the eluate (E1) 12 (100 μl) was poured into the spin column 8, allowed to stand at room temperature (25 ° C.) for 2 minutes, and then centrifuged for 1 minute (at 6,000 rpm) to recover the nucleic acid. And the purity was evaluated.

  As a result, a highly pure nucleic acid having A260 / A280 of 1.8 was obtained, and could be used for the PCR reaction.

1 shows an extraction process of a nucleic acid according to an embodiment of the present invention. 1 shows the structure of whole blood in an example of the present invention. Fig. 3 shows a decomposition step in a procedure of nucleic acid extraction from whole blood in an example of the present invention. 1 shows a procedure (first method) of nucleic acid extraction from whole blood in an example of the present invention. 2 shows a procedure (second method) of nucleic acid extraction from whole blood in an example of the present invention. 3 shows the correlation between the concentration of DIGLYME in a mixed solution and the relative nucleic acid yield in Examples of the present invention. 4 shows the correlation between the EL concentration in a mixed solution and the relative nucleic acid yield in Examples of the present invention. 4 shows the DIGLYME ratio and the relative nucleic acid yield in the additive solution when using the additive solution of DIGLYME and EL in Examples of the present invention. 4 shows the correlation between the Tween 20 content in the reaction solution and the relative nucleic acid yield in Examples of the present invention. 4 shows the correlation between the content of Tween 80 in the reaction solution and the relative nucleic acid yield in Examples of the present invention. 4 shows the correlation between the content of Triton X-100 in the reaction solution and the relative nucleic acid yield in Examples of the present invention. 4 shows the correlation between the amount of the antifoaming agent CE-457 added to the reaction solution and the relative nucleic acid yield in Examples of the present invention.

Explanation of reference numerals

1: Human whole blood, 2: Red blood cell, 3: White blood cell, 4: Nuclear, 5: Tube, 6: Proteinase K, 7: Mixed solution, 8: Spin column, 9: Adsorption carrier, 10: Tube, 11: Washing liquid, 12 ... eluate, 13 ... adsorption carrier, 14 ... column, 15 ... washing solution, 16 ... tube, 17 ... tube, 18 ... eluate,
101 addition of whole blood, 102 addition and stirring of a reaction solution, 103 incubation of a mixed solution, 104 addition and stirring of an addition solution, 105 adsorption of nucleic acid by suction, 106 addition of a washing solution to a column, 107 washing steps , 108: Addition and standing of eluate, 109: Recovery of nucleic acid, 110: Adsorption of nucleic acid by suction and discharge, 111: Suction and standing of eluate, 112: Recovery of nucleic acid

Claims (22)

Protease, chaotropic salt, and a first mixed solution obtained by mixing the surfactant and the sample at a predetermined temperature,
After the predetermined temperature, a solution containing any compound of aliphatic ethers, aliphatic esters, and aliphatic ketones having 2 to 10 carbon atoms is added to the first mixture. As a second mixture,
The nucleic acid contained in the second mixture is adsorbed on a carrier,
Washing the carrier with a solution containing ethanol,
A nucleic acid extraction method, comprising eluting and recovering the nucleic acid adsorbed on the carrier.   The compound is ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propylene glycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, The nucleic acid extraction method according to claim 1, wherein the method is at least one of acetone and methyl ethyl ketone.   The method according to claim 1, wherein the sample is a tissue.   The method according to claim 1, wherein the sample is urine. Proteases,
A first liquid containing a chaotropic salt and a surfactant, and a second liquid containing any compound of an aliphatic ether, an aliphatic ester, and an aliphatic ketone having 2 to 10 carbon atoms,
A carrier for adsorbing nucleic acids,
A third liquid containing ethanol;
A nucleic acid extraction kit, comprising: an eluate for releasing the nucleic acid adsorbed on the carrier from the carrier.   The nucleic acid extraction kit according to claim 5, wherein the chaotropic salt is at least one of potassium iodide, guanidine hydrochloride, and guanidine thiocyanate.   Examples of the surfactant include polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, and polyoxyethylene (20) sorbitan monooleate. The nucleic acid extraction kit according to claim 5, wherein the kit is at least one of oxyethylene (20) sorbitan triolate and polyoxyethylene (10) isooctyl phenyl ether.   The compound is ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propylene glycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, The nucleic acid extraction kit according to claim 5, wherein the kit is at least one of acetone and methyl ethyl ketone.   The kit according to claim 5, wherein the carrier is made of at least one of silica, alumina, zeolite, and titanium dioxide. Protease, chaotropic salt, and a first mixture of a surfactant and blood mixed with a predetermined temperature,
After the predetermined temperature, a solution containing any compound of aliphatic ethers, aliphatic esters, and aliphatic ketones having 2 to 10 carbon atoms is added to the first mixture. As a second mixture,
The nucleic acid contained in the second mixture is adsorbed on a carrier,
Washing the carrier with a solution containing ethanol,
A method for extracting nucleic acids from blood, comprising eluting and recovering nucleic acids adsorbed on the carrier.   The compound is ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propylene glycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, The method for extracting nucleic acid from blood according to claim 10, wherein the method is at least one of acetone and methyl ethyl ketone. Proteases,
A first liquid containing a chaotropic salt and a surfactant, and a second liquid containing any compound of an aliphatic ether, an aliphatic ester, and an aliphatic ketone having 2 to 10 carbon atoms,
A carrier for adsorbing nucleic acids,
A third liquid containing ethanol;
A kit for extracting nucleic acids from blood, comprising: an eluate for releasing nucleic acids adsorbed on the carrier from the carrier.   The compound is ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propylene glycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, The kit for extracting nucleic acid from blood according to claim 12, wherein the kit is at least one of acetone and methyl ethyl ketone. Decompose the cells to obtain a first mixture,
To the first mixture, having a carbon number of 2 to 10, a liquid containing a compound of any of aliphatic ethers, aliphatic esters, and aliphatic ketones to obtain a second mixture,
Aspirating and discharging the second mixed solution into a container filled with a carrier, allowing the carrier to adsorb nucleic acids contained in the second mixed solution,
Washing the container filled with the carrier,
A nucleic acid extraction method, comprising eluting the nucleic acid adsorbed on the carrier.   15. The nucleic acid extraction method according to claim 14, wherein in at least one of the washing and the elution, a liquid is sucked and discharged into a container filled with the carrier.   The compound is ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propylene glycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, The nucleic acid extraction method according to claim 14, wherein the method is at least one of acetone and methyl ethyl ketone. Decompose the cells to obtain a first mixture,
To the first mixture, having a carbon number of 2 to 10, a liquid containing a compound of any of aliphatic ethers, aliphatic esters, and aliphatic ketones to obtain a second mixture,
Transfer the second mixture to the column containing the carrier,
Aspirating the second mixed solution from the column, allowing the nucleic acid contained in the second mixed solution to be adsorbed to the carrier,
Washing the carrier by injecting a washing solution into the column and aspirating the washing solution from the column,
A nucleic acid extraction method, comprising injecting an eluate into the column and aspirating the eluate from the column to elute the nucleic acid adsorbed on the carrier.   The compound is ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propylene glycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, 18. The method according to claim 17, wherein the method is at least one of acetone and methyl ethyl ketone. Proteolytic enzymes,
A reaction solution containing a chaotropic salt and a surfactant, supplied to the sample,
An additive liquid having a carbon number of 2 to 10, including a compound of an aliphatic ether, an aliphatic ester, and an aliphatic ketone, and added to a reaction liquid supplied to the sample;
An adsorption carrier for adsorbing nucleic acids,
A washing solution for washing the adsorption carrier,
A nucleic acid extraction kit, comprising: an eluate for eluting the nucleic acid from the adsorption carrier on which the nucleic acid has been adsorbed.   The compound is ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propylene glycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, 20. The nucleic acid extraction kit according to claim 19, wherein the kit is at least one of acetone and methyl ethyl ketone. Proteinase K,
A first liquid containing guadinine hydrochloride, polyoxyethylene (20) sorbitan monolaurate, and a polyalkylene glycol derivative; diethylene glycol dimethyl ether;
A carrier for adsorbing nucleic acids,
A second liquid comprising potassium acetate and ethanol;
A nucleic acid extraction kit comprising: a third liquid containing tris (hydroxymethyl) aminomethane and ethylenediaminetetraacetic acid disodium salt.   The first liquid is a reaction liquid supplied to the sample, the diethylene glycol dimethyl ether is an additive liquid added to the reaction liquid supplied to the sample, and the second liquid is a cleaning liquid for cleaning the carrier. 22. The nucleic acid extraction kit according to claim 21, wherein the third liquid is an eluate for eluting the nucleic acid from the carrier.

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