JP2003144150A - Method and apparatus for refining/isolating nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for refining/isolating a nucleic acid stably in high yield. SOLUTION: This method comprises the steps of (1) adding an organic solvent at -5 to 55 deg.C to a solution containing the nucleic acid and a high-concentration salt and (2) adsorbing the nucleic acid to a carrier, thus improving the collection yield of the nucleic acid with slight variation in its collection.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for purifying and separating nucleic acid, in which nucleic acid is eluted from a biological sample containing nucleic acid to purify and separate the nucleic acid.

[0002]

2. Description of the Related Art An effective method for purifying and separating nucleic acids in the prior art is based on the principle of adsorbing nucleic acids to glass or silica gel particles in the presence of a chaotropic salt to recover the nucleic acids (Prior Art-1: Vogelstein). , B. And Gillespie, D.
(1979); "Preparative and Degradative Purification of DNA from Agarose", Proc. Natl. Acad. Sci. USA 76: 615-619, Prior Art-2: Boom, R. (1990); And simple purification method ”, J. Clin. Microbiol. 28: 495-503). Conventional technology −
1, 2, using high concentrations of chaotropic salts such as sodium iodide, sodium perchlorate, or guanidine thiocyanate, DNA was isolated from agarose,
Purified, or RNA or DNA is isolated and purified from various mixtures.

Nucleic acids are often used in a polymerase chain reaction (PCR) after purification. In PCR, a nucleic acid can be specifically amplified in a sequence, and thus is widely applied to gene diagnosis and the like. PC
There are some problems in routine clinical use of the R method. It is known that PCR is inhibited by the influence of an inhibitor that could not be removed during the purification of nucleic acid. Known inhibitory substances include hemoglobin and surfactants used in the nucleic acid extraction step. Therefore, it has been pointed out that the steps of extracting and purifying nucleic acids are important (prior art-
3: Oshima et al., JJCL A, 22 (2), 145-150 (1997)).

Conventionally, the extraction process has been performed manually, but since the operation is complicated and requires skill, automation by an apparatus is desired. Further, development of an extraction method (including reagents used) suitable for automation is required.

JP-A-11-127854 (Prior Art-4) and JP-A-11-266864 (Prior Art-5) describe nucleic acid extraction methods and nucleic acid-capturing chips suitable for automation. . In addition, the special table 8-501321
Japanese Unexamined Patent Publication (Prior Art-6) describes a reagent suitable for extraction of nucleic acid by an automated device, and separates and purifies a reagent containing a high-concentration salt (high ionic strength) and a high-concentration alcohol. The nucleic acid to be adsorbed by contacting it with the adsorption carrier in the extraction chip, and then using a solution containing a lower concentration of salt (small ionic strength) to release the nucleic acid from the adsorption carrier, Is getting

Extraction of nucleic acid from whole blood is performed as follows. A proteolytic enzyme and a buffer for hemolysis are added to whole blood, stirred, and then incubated at 56 ° C or 70 ° C for 10 minutes. After incubation, add ethanol and pass the solution containing nucleic acid through the adsorption column. After washing the column with a buffer containing ethanol, the nucleic acid adsorbed on the column is recovered with a nucleic acid recovery buffer (Prior Art-7: QIAamp DNA Mini Kit an
d QIAamp DNA Blood Mini Kit Handbook and QIAamp
DNA Blood Midi / Maxi Kit Handbook, QIAGEN).

[0007]

The method for purifying and separating nucleic acid using the reagent described in Prior Art-6 has a problem that the yield of nucleic acid recovery is low. Further, the method using the extraction chip described in Related Art-4 has a problem that the amount of nucleic acid recovered varies greatly.

An object of the present invention is to provide a method and a device for purifying and separating nucleic acids, in which variations in the amount of recovered nucleic acids are small and a high amount of recovered nucleic acids can be stably obtained.

[0009]

In the method for purifying and separating a nucleic acid according to the present invention, (step 1) an organic solvent is added to a solution containing a nucleic acid and a high-concentration salt (hereinafter referred to as a nucleic acid releasing solution),
(Step 2) Nucleic acid is adsorbed on an organic or inorganic carrier, and (Step 3) nucleic acid is desorbed from the organic or inorganic carrier and recovered using a recovery solution composed of an aqueous solution. If necessary, before step (step 3), a step of washing the carrier with a washing solution to remove components other than the nucleic acid is performed. The temperature of the nucleic acid releasing solution just before the addition of the organic solvent is made substantially the same as the temperature of the organic solvent, and the temperature of the nucleic acid releasing solution immediately before the addition of the organic solvent at the temperature T (° C) is T ± (2 to 3) ° C. And In the following description, the temperature that is almost the same as the temperature T (° C) means T ± (2
3) It means a temperature of ° C. Further, the temperature of the nucleic acid-releasing solution may be different from the temperature of the organic solvent. Nucleic acid is, for example,
Derived from white blood cells of whole blood.

Nucleic acid yields are compared by relative nucleic acid yield with a maximum nucleic acid yield of 100%.

In the present invention, in (step 1), -5 ° C to 5 ° C
By adding any of ethanol, 1-propanol, 2-propanol, diethylene glycol dimethyl ether, and ethyl lactate, which were kept at a temperature in the range of 5 ° C, a relative nucleic acid yield of 80% or more was obtained, and
In (Step 1), the relative nucleic acid yield of 80% or more is obtained by adding diethylene glycol dimethyl ether or ethyl lactate, which is kept at a temperature in the range of 0 ° C to 50 ° C.

More preferably, in (step 1), the temperature is from 0 ° C to
Ethanol, 1-, kept at a temperature in the range of 50 ° C
By adding any of propanol, 2-propanol and ethyl lactate, a relative nucleic acid yield of 90% or more was obtained, and in (step 1), the temperature was kept in the range of 0 ° C to 40 ° C. Moreover, by adding diethylene glycol dimethyl ether or ethyl lactate, a relative nucleic acid yield of 90% or more is obtained.

More preferably, in (step 1), 10 ° C.
Ethanol, 1 kept at a temperature in the range of ~ 35 ° C
-By adding any of propanol, 2-propanol, diethylene glycol dimethyl ether and ethyl lactate, a relative nucleic acid yield of 95% or more is obtained.

In the method for purifying and separating a nucleic acid of the present invention (step 1), a solution containing a nucleic acid and a high-concentration salt and kept at a temperature T in the range of 0 ° C. to 50 ° C. An organic solvent kept at the same temperature is added (step 2) to adsorb the nucleic acid on the carrier. By using any of ethanol, 1-propanol, 2-propanol and ethyl lactate as the organic solvent, a relative nucleic acid yield of 90% or more can be obtained.

In the method for purifying and separating nucleic acid according to the present invention (step 1), a solution containing nucleic acid and a high-concentration salt and kept at a temperature T in the range of 0 ° C. to 40 ° C. An organic solvent kept at the same temperature is added (step 2) to adsorb the nucleic acid on the carrier. By using diethylene glycol dimethyl ether or ethyl lactate as the organic solvent, a relative nucleic acid yield of 90% or more can be obtained.

The method for purifying and separating nucleic acid of the present invention has the following features. (A) (1) Containing nucleic acid and high-concentration salt, 10 ° C to 4
The temperature T in the range of 0 ° C
A method for separating and purifying nucleic acid, comprising: a step of adding an organic solvent maintained at about the same temperature as that of (1); and (2) a step of adsorbing the nucleic acid on a carrier. (B) The method for separating and purifying nucleic acid as described in (A), wherein the organic solvent is any one of ethanol, diethylene glycol dimethyl ether and ethyl lactate. The methods (A) and (B) give a relative nucleic acid yield of 95% or more. (C) (1) Containing nucleic acid and high concentration of salt, 20 ° C to 3
The solution maintained at a temperature T in the range of 6 ° C was
And a step of (2) causing the carrier to adsorb the nucleic acid at a temperature substantially the same as the temperature T. (D) The method for separating and purifying nucleic acid as described in (C), wherein the temperature T is set to a temperature in the range of 22 ° C to 32 ° C. The method (C) gives a relative nucleic acid yield of 80% or more, and the method (D) gives a relative nucleic acid yield of 95% or more.

The nucleic acid purifying / separating apparatus for automatically executing the purifying / separating method of the present invention has the following features. (1) In a nucleic acid separating and purifying apparatus in which an organic solvent is added to a solution containing a nucleic acid and a high-concentration salt, the carrier is adsorbed on the carrier in a temperature range of 0 ° C to 40 ° C. An apparatus for separating and purifying nucleic acid, characterized by having a means for maintaining the temperature. (2) The apparatus for separating and purifying nucleic acid as described in (1), wherein the organic solvent is maintained at a temperature in the range of 0 ° C to 40 ° C. (3) The apparatus for separating and purifying nucleic acid as described in (1), wherein the temperature of the solution and the temperature of the organic solvent are substantially the same. (4) The apparatus for separating and purifying nucleic acid as described in (1), wherein the temperature of the solution is different from the temperature of the organic solvent. With the devices (1) to (4), a relative nucleic acid yield of 90% or more can be obtained. (5) In the apparatus for separating and purifying nucleic acid, which comprises adsorbing the nucleic acid on a carrier after adding the organic solvent to a solution containing the nucleic acid and a high-concentration salt, the temperature of the organic solvent and the solution is adjusted to 20
A means for holding the temperature T in the range of ° C to 36 ° C,
An apparatus for separating and purifying nucleic acid, comprising adding the organic solvent at the temperature T to the solution at the temperature T and adsorbing the nucleic acid on the carrier at about the same temperature as the temperature T. (6) The apparatus for separating and purifying nucleic acid as described in (5), wherein the temperature T is set to a temperature in the range of 22 ° C to 32 ° C. The device of (5) gives a relative nucleic acid yield of 80% or more, and the device of (6) gives a relative nucleic acid yield of 95% or more.

In the apparatus of the present invention, T is set to -20 ° C to 8 ° C.
A common heating / cooling temperature control circuit as a means for holding the nucleic acid-releasing solution at a temperature T as an arbitrary temperature between 0 ° C. and a means for holding an organic or inorganic carrier at a temperature substantially the same as the temperature T. To use. The heating / cooling temperature control circuit is configured by combining a temperature sensor, a Peltier element, and a Peltier element control circuit, and holds the temperature control target at a constant temperature. With such a configuration, temperature control can be performed at high speed. Further, the heating / cooling temperature control circuit may be configured by combining a temperature sensor, a circulating water cooling device, and a controlling circuit for the circulating water cooling device.

Further, in the apparatus of the present invention in which the thermal cycler section for carrying out PCR is incorporated, after purification and separation of nucleic acid,
Since the PCR can be immediately and automatically executed, for example, genetic testing can be performed at high speed.

The reagents and constituents preferably used in the method and apparatus for purifying and separating nucleic acid of the present invention will be described below.

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

As the organic solvent, a compound having a carbon number in the range of 2 to 10 is used. The compound is selected from aliphatic alcohols, aliphatic ethers, aliphatic esters, aliphatic ketones. One compound or a mixture of compounds is used as the organic solvent. The organic solvent is preferably added so that the concentration thereof is 5% by volume to 50% by volume.

Typical examples of the aliphatic alcohol include ethanol (EtOH) and 1-propanol (PrO).
H), 2-propanol (i-PrOH).

As typical examples of the aliphatic ethers, ethylene glycol dimethyl ether, ethylene glycol diethyl ether (EGDE), propylene glycol dimethyl ether (PGDME), propylene glycol dimethyl ether, propylene glycol diethyl ether (PGDEE), diethylene glycol dimethyl ether (DIGLYME), There are diethylene glycol diethyl ether (DGDEE), tetrahydrofuran (THF), 1,4-dioxane (DX).

As a typical example of the aliphatic ester, propylene glycol monomethyl acetate (PGME
A) and ethyl lactate (EL).

Representative examples of aliphatic ketones are hydroxyacetone (HAC), acetone (AC), and methyl ethyl ketone (MEK).

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

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

The recovery liquid is an aqueous solution containing a low concentration of salt, and the nucleic acid adsorbed on the organic or inorganic carrier is eluted by contacting with the organic or inorganic carrier.

In the present invention, 0.1% by volume to 50% by volume of a surfactant is added to a solution containing nucleic acid and a high concentration of salt. Preferably, the surfactant is 0.1% by volume to 5%.
It is advisable to use a volume% defoamer.

[0031]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a diagram for explaining a nucleic acid extraction process according to an embodiment of the present invention. FIG. 2 is a diagram for explaining the constitution of whole blood used in the examples of the present invention. 3 to 5 explain the procedure of nucleic acid extraction from whole blood using a nucleic acid extraction reagent in the method for purifying and separating nucleic acid of the present invention based on the method of FIG. 1 described in Prior Art-4. FIG. In the following description, nucleic acid extraction from 100 μL of whole human blood is taken as an example.

The nucleic acid extraction process shown in FIG. 1 is composed of six steps described in Prior Art-4. The nucleic acid extraction procedure promotes the binding of the nucleic acid to the solid phase in the nucleic acid releasing solution containing the released nucleic acid obtained in the first step 501 and the first step 501 for releasing the nucleic acid from the nucleic acid-containing material (sample). Second step 50 of adding and mixing an additive liquid (alcohol-based, ester-based, ether-based organic solvent, etc.)
2. Third step 50 of bringing the mixed solution obtained in the second step 502 into contact with the nucleic acid-binding solid phase (carrier for adsorbing nucleic acid)
3, Fourth step 5 for separating a solid phase bound with nucleic acid and a liquid
04, a fifth step of washing the nucleic acid-bonded solid phase with a solution containing a salt, and a sixth step of releasing the nucleic acid from the nucleic acid-bonded solid phase. Through the above steps, the purified nucleic acid can be obtained from the material (sample) containing the nucleic acid.

Hereinafter, the first step and the second step are collectively called a decomposition step, and the third step and the fourth step are collectively called an adsorption step.

As shown in FIG. 2, in human whole blood 1,
Components such as red blood cells 2 and white blood cells 3 are included, and nucleic acids are mainly present in the nucleus 4 of the white blood cells 3.

FIG. 3 is a diagram for explaining the procedure of nucleic acid extraction from whole blood in the embodiment of the present invention. First, tube 5
Inject 10 μL of Proteinase K (6) into the tube. Next, add 100 μL of whole blood 1 to the tube 5 (addition of whole blood 1
01). Next, 100 μL of a reaction solution (containing 1M to 8M chaotropic salt and 50% or less of a surfactant) is added and stirred (addition of reaction solution and stirring 102). Incubating the solution in tube 5 at 70 ° C for 10 minutes (incubation 103) destroys leukocytes,
The nucleic acid in the nucleus 4 comes out in the solution (a nucleic acid releasing solution before addition of the additive solution is obtained). Thereafter, 100 μL of an additive solution (organic solvent such as alcohol, ester or ether) that promotes the binding of the nucleic acid to the solid phase is added to the nucleic acid releasing solution and stirred (addition solution and stirring 104). A mixed solution 7 is obtained. The process until the mixed liquid 7 is obtained is a decomposition process.

There are several types of nucleic acid recovery methods performed after this decomposition step. Both methods include an adsorption process, a cleaning process, and a recovery process. Typical examples are a spin column method and a suction discharge method.

FIG. 4 is a diagram for explaining the procedure of the first method for recovering nucleic acid from whole blood (spin column method) in the example of the present invention. First, the mixed solution 7 in the tube 5 is transferred into the spin column 8. The bottom of the spin column 8 is filled with a filter paper-like adsorption carrier 9 made of very fine silica or glass fiber.

The mixed solution 7 containing the additive is passed through the adsorption carrier 9 by suction to adsorb the nucleic acid to the adsorption carrier 9 (adsorption step 7: nucleic acid adsorption step 105 by suction). After that, 50% containing 20% to 80% of an organic solvent such as alcohol.
0 μL of the washing liquid 11 is injected into the spin column 8 (addition of washing liquid to column 106).

Next, the adsorption carrier 9 is washed by suction to remove components other than nucleic acid (washing step 107). This washing step is repeated twice, but the nucleic acid adsorbed on the adsorption carrier is not eluted by the washing step. Then, the low salt concentration eluate 12
Is added and the mixture is allowed to stand for 2 to 5 minutes (addition of eluate and standing 108). The nucleic acid is eluted from the adsorption carrier and finally
It is collected in the tube 10 by suction (elution step: nucleic acid collection 109). The composition of the typical eluate 12 is 50 m
mol / L Tris / HCl (pH 8.5) +0.1
It is EDTA.2Na of mmol / L.

FIG. 5 is a diagram for explaining the procedure of the second method for recovering nucleic acid from whole blood (suction / ejection method) in the embodiment of the present invention. In the suction discharge method, glass is used as the adsorption carrier 13,
Cylindrical hollow column 14 in which fibers such as quartz are packed at low density
To use. Using the column 14, suction and discharge of the mixed liquid 7 in the tube 5 are repeated a plurality of times to adsorb the nucleic acid to the adsorption carrier 13 (adsorption step: nucleic acid adsorption 110 by suction and ejection). Then, suction and discharge are performed using another tube 16 containing 500 μL of the washing liquid 15 containing 20% to 80% of an organic solvent such as alcohol to remove components other than nucleic acid (washing step). This cleaning process is performed twice or more. Then, the eluate 16 (100 μL) in the tube 17 heated in advance to 70 ° C. is sucked up to a position where it comes into contact with the adsorption carrier 13 in the column 14 and then rested for about 2 minutes (absorption and suction of the eluent). 111). By this operation, the nucleic acid is eluted from the adsorption carrier 13 into the eluate 18. The eluate 18 is discharged to collect the nucleic acid in the tube 17 (nucleic acid recovery 112). A typical eluent composition is 50
mmol / L Tris / HCl (pH 8.5) +0.
It is 1 mmol / L of EDTA.2Na.

Materials, chemicals and samples used in the examples will be described in detail below. 1. Nucleic acid adsorbing material and column 1.1 Spin column: For example, a three-layer glass fiber filter paper (manufactured by Whatman) was fixed to a centrifugal chromatography column (spin column). The spin column is
It was used in the spin column protocol shown in FIGS. 1.2 Nucleic acid capture column: the nucleic acid capture chip (column) described in Related Art-5 has a diameter of 0.5 μm to 30 μm.
Of quartz fiber (manufactured by Tosoh Quartz Co., Ltd. or Toshiba Ceramics Co., Ltd.). The nucleic acid-capturing chip was used in the suction / ejection protocol shown in FIGS. 2. Sample containing nucleic acid 2.1 Blood (human): Blood immediately after blood collection or -20
Blood cryopreserved at ° C was used. 2.2 Tissues: Fresh tissues or frozen tissues were used. 2.3 Plants: The plants were ground in liquid mortar in a mortar and used directly or after frozen storage. 2.4 Cells: The isolated or isolated / cultured cells were used directly, or the cells that had been cryopreserved were used. 3. Reagent 3.1 Reaction liquid: The reaction liquid contains the following chaotropic salt, surfactant, antifoaming agent and other salt components. 3.1.1 Chaotropic salt: potassium iodide (K
I), guanidine hydrochloride (GuHCl), or guanidine thiocyanate (GuHSCN) was used. 3.1.2 Surfactant: polyoxyethylene (20)
Sorbitan monolaurate (Tween 20), polyoxyethylene (20) sorbitan monopalmilate (T
Either ween 40) or polyoxyethylene (10) isooctyl phenyl ether (Triton X-100) was used. 3.1.3 Defoaming agent: CE-457 (defoaming agent home made by NOF CORPORATION, polyalkylene glycol derivative) 3.1.4 Other salts: ethylenediaminetetraacetic acid / disodium salt (EDTA / 2Na) ), Tris (hydroxymethyl) aminomethane (Tris) 3.2 Additives: PGDME, PGDEE, DIGLY
ME, DGDEE, THF, DX, PGMEA, EL,
HAC, AC, MEK, EtOH, PrOH, i-Pr
Any organic solvent of OH was used. 3.3 Washing solution: 25 mmol / L potassium acetate +7
0% by volume ethanol (W1), 25 mmol / L potassium acetate + 50% by volume ethanol (W2), 10 mmo
1 / L Tris / HCl (pH 8.5) + 0.1 mm
Either composition of ol / L EDTA.2Na + 50% by volume ethanol (W3) was used. 3.4 Eluent: water (100%, pH 8.0) (E
1), 10 mmol / L Tris / HCl (pH 8.
5) +0.1 mmol of EDTA.2Na (E2), 5
0 mmol / L Tris / HCl (pH 8.5) +
Any composition of 0.1 mmol / L EDTA.2Na (E3) was used. 3.5. Enzyme: Proteolytic enzyme Protease (prot
ease), alkaline proteinase K
Either enzyme was used. Example 1 Nucleic Acid Extraction from Whole Blood (Part 1) According to the method shown in FIGS. 3 and 5, 100 μL of human whole blood 1 was subjected to nucleic acid extraction. In the disassembly process, 10μ
Tube 100 containing L proteinase K (6)
1 μL of whole blood and 100 μL of reaction solution (containing guanidine hydrochloride having a concentration of 3M and 5% by volume of Tween 40) were added and stirred. The solution in tube 5 was incubated at 70 ° C for 10 minutes to obtain a nucleic acid releasing solution. EL, DIGLYME, EtOH, PrOH, i
An additive solution (100 μL) selected from —PrOH was added and stirred to obtain a mixture solution 7.

In the adsorption step, the quartz wool adsorption carrier 13
Column 14 filled with (Toshiba Ceramics Co., Ltd.)
The operation of sucking and discharging the mixed liquid 7 is performed at a temperature of 25
Repeated 10 times at ° C.

In the cleaning step, the operation of sucking and discharging the cleaning liquid (W1) 15 contained in another tube 16 was repeated three times.

In the collecting step, the eluate 18 contained in another tube 17 was sucked up to a position where the entire adsorption carrier 13 was immersed, and was left still for 2 minutes. Then, the eluate 18 was discharged and the solution containing nucleic acid was recovered. The recovered nucleic acid has a good purity and is subjected to the next PCR without any steps such as precipitation and purification.
Etc. could be used for reaction and analysis.

FIG. 6 shows the temperature T when the additive solution at the temperature T (° C.) was added to the nucleic acid releasing solution at the same temperature T using EL as the additive solution in Example 1 of the present invention. It is a figure which shows the relationship between a relative nucleic acid yield.

In the following description, when the relationship between the temperature T and the relative nucleic acid yield is obtained, the nucleic acid yield when the temperature T is changed under the same chemical reaction conditions except the temperature T, and the maximum nucleic acid yield is 100 Comparison is made by relative nucleic acid yield in%.

6, FIG. 7, FIG. 8 and FIG. 11, the temperature on the horizontal axis is the temperature T when the additive solution at temperature T (° C.) is added to the nucleic acid releasing solution at substantially the same temperature T. The vertical axis represents the relative nucleic acid yield obtained when the temperature T was changed.

As shown in FIG. 6, the temperature T is -5 ° C to 5 ° C.
At 5 ° C, the target relative nucleic acid yield of 80% or more was obtained. When the temperature T is 0 ° C to 50 ° C, 90%
The above preferable relative nucleic acid yields were obtained. Furthermore, when the temperature I was 10 ° C to 40 ° C, a more preferable relative nucleic acid yield of 95% or more was obtained.

PGMEA is an ester organic solvent that can be used in the same manner as EL.

FIG. 7 shows the temperature T when the additive solution at the temperature T (° C) was added to the nucleic acid releasing solution at the same temperature T using DIGLYME as the additive solution in Example 1 of the present invention. It is a figure which shows the relationship between a relative nucleic acid yield. As shown in FIG. 7, when the temperature T is −5 ° C. to 55 ° C.
A relative nucleic acid yield of 0% or more was obtained. Also, the temperature T is 0
Suitable relative nucleic acid yields of 90% or higher were obtained between ° C and 45 ° C. Furthermore, when the temperature T is 10 ° C to 40 ° C,
A better relative nucleic acid yield of 95% or higher was obtained.

EDGME, EGDEE, PGD can be used as an ether type organic solvent which can be used similarly to DIGLYME.
ME, PGDEE, DGDEE, THF, DX.

FIG. 8 shows the temperature T when the additive solution at temperature T (° C) was added to the nucleic acid releasing solution at the same temperature T using EtOH as the additive solution in Example 1 of the present invention. It is a figure which shows the relationship between a relative nucleic acid yield. As shown in FIG.
When the temperature T was -5 ° C to 55 ° C, the target relative nucleic acid yield of 80% or more was obtained. Further, the temperature T is 0 ° C to
At 50 ° C, a good relative nucleic acid yield of 90% or more was obtained. Further, when the temperature T is 10 ° C to 40 ° C, 95%
The more preferable relative nucleic acid yield was obtained.

As the alcoholic organic solvent which can be used similarly to EtOH, there are PrOH and i-PrOH. Using either PrOH or i-PrOH as the additive liquid,
As a result of investigating the relationship between the temperature T and the relative nucleic acid yield when the additive solution at the temperature T (° C) was added to the nucleic acid releasing solution at the same temperature T, when the temperature T was -5 ° C to 55 ° C, Targeted 80
A relative nucleic acid yield of>% was obtained. Also, the temperature T is 0 °
Suitable relative nucleic acid yields of 90% or higher were obtained at C-50 ° C.

When an alcoholic organic solvent is used,
Higher relative nucleic acid yields were obtained over a slightly wider temperature range than when using ether organic solvents. Example 2: Extraction of nucleic acid from whole blood (Part 2) According to the method shown in FIGS. 3 and 5, nucleic acid was extracted from 1 mL of human whole blood 1. 100 μL in the decomposition process
1 ml of whole blood 1 and 1 ml of reaction solution (concentration of 3M guanidine hydrochloride,
5% by volume of Triton X-100 was included) and stirred. The solution in tube 5 was incubated at 70 ° C for 10 minutes and then cooled to 20 ° C to obtain a nucleic acid-releasing solution. EGDME, EGDEE, PGDME, PGDE
E, DIGLYME, DGDEE, THF, DX, PG
MEA, EL, HAC, AC, MEK, EtOH, Pr
Additive liquid 8 (1 mL) selected from OH and i-PrOH was added to tube 5 and stirred to obtain mixed liquid 7.

In the adsorption step, the mixed solution 7 was poured into the spin column 8 at 20 ° C. and a centrifugal process (rotation speed 6000 rpm) was performed for 1 minute using a tabletop centrifuge.

In the washing process, the spin column 8 is set to 500 μm.
L of the cleaning liquid (W2) 11 was poured, and the centrifugal treatment (rotation speed 6000 rpm) was performed for 1 minute.

In the recovery step, 100 μ was applied to the spin column 8.
Pour L eluate (E2) 12 and let it stand for 2 minutes at room temperature (25 °
After leaving it in C), centrifuge for 1 minute (rotation speed 6000
rpm) was performed to recover the nucleic acid, and quantification and purity evaluation were performed. The recovered nucleic acid could be used for PCR.

FIG. 9 is a diagram showing the nucleic acid yields obtained when different types of additive solutions were added in Example 2 of the present invention. The temperature of the nucleic acid-releasing solution was set to 20 ° C., and the additive solution at 20 ° C. was added to the nucleic acid-releasing solution. 20 adsorption steps
It was performed at ° C. The target nucleic acid yield was 15 μg, but a value higher than the target nucleic acid yield was obtained regardless of which type of additive solution was used. Example 3: Extraction of nucleic acid from whole blood (3) Extraction of nucleic acid from 100 μL of human whole blood 1 was performed.

In the decomposition step, 10 μL of proteinase K was used.
In a tube 5 containing (6), 100 μL of whole blood 1 and 100 μL of reaction liquid (concentration of 4 M guanidine hydrochloride, 5
Volume% Tween 20 was included) was dispensed and stirred. The solution in tube 5 was incubated at 70 ° C for 10 minutes.

FIG. 10 is a diagram showing the structure of the reaction tank used for incubation in Example 3 of the present invention. The sample tube 251 containing the whole blood 1 and the reaction solution is heated on the heat block 254. The temperature control signal for the heating / cooling substrate 256, which is generated by the temperature control circuit 255 based on the temperature measured by the temperature sensor 259, is applied to the heating / cooling substrate 256 through the control line 260, and the temperature of the heat block 254 is changed. ， Adjusted. A heat radiating fan 257 is attached to the bottom of the heating / cooling substrate 256 to accelerate heating and cooling. The operation of the heat radiation fan 257 is controlled by the temperature control circuit 255 through the control line 261. Heat block 254
The outside of the heat insulating material 2 is used for stable temperature control.
Covered with 53. The heat insulating material 253 is covered with a cover 252. The entire reaction tank is supported by columns 258.

After incubation, sample tube 25
The temperature of the solution of 1 was cooled to 20 ° C. to obtain a nucleic acid releasing solution. EL, DIGLY on the sample tube 251
An additive solution (100 μL) selected from ME, EtOH, PrOH, and i-PrOH was added, and the mixture was shaken and stirred to obtain a mixed solution 7.

In the adsorption step, the spin column 8 was used to suction-filter the mixed solution 7 with a vacuum pump.

In the washing process, the spin column 8 is set to 500 μm.
The washing liquid (W3) 11 of L was poured and suction filtration was performed with a vacuum pump.

In the recovery step, the eluate (E3) 18 was poured into the spin column 8 and left standing for 2 minutes. Then, suction filtration was performed using a vacuum pump to collect a solution containing nucleic acid. The recovered nucleic acid has sufficient yield and good purity,
It was possible to use it for PCR.

FIG. 11 shows that, in Example 3 of the present invention, DIGLYME was used as the additive solution and the additive solution at the temperature T (° C) was added to the nucleic acid releasing solution at the same temperature T. It is a figure which shows the relationship between a relative nucleic acid yield. As shown in FIG. 11, when the temperature T was −5 ° C. to 45 ° C., the target relative nucleic acid yield of 80% or more was obtained. Also, the temperature T
A suitable relative nucleic acid yield of 90% or more was obtained when the temperature was from 0 ° C to 40 ° C. Furthermore, when the temperature T was 10 ° C to 35 ° C, a more preferable relative nucleic acid yield of 95% or more was obtained.

Further, using EL as an additive liquid, the temperature T
As a result of investigating the relationship between the temperature T and the relative nucleic acid yield when the addition solution of (° C) was added to the nucleic acid releasing solution of the same temperature T, when the temperature T was -5 ° C to 45 ° C, A relative nucleic acid yield of 80% or more was obtained. Further, the temperature T is 0 ° C to 4
At 0 ° C, a good relative nucleic acid yield of over 90% was obtained.

Further, as an additive liquid, EtOH, PrOH,
The temperature T when the additive solution at the temperature T (° C) is added to the nucleic acid releasing solution at the same temperature T using any of i-PrOH
As a result of investigating the relationship between the relative nucleic acid yield and the
At ˜55 ° C., the targeted relative nucleic acid yield of 80% or more was obtained. When the temperature T is 0 ° C to 50 ° C, 9
Suitable relative nucleic acid yields of 0% and above were obtained.

When an alcoholic organic solvent is used,
Higher relative nucleic acid yields were obtained over a slightly wider temperature range than when ester-based organic solvents or ether-based organic solvents were used. Example 4 Nucleic Acid Extraction from Whole Blood by Automated Apparatus (Part 1) FIG. 12 is a block diagram showing the configuration of the automated nucleic acid purification / separation apparatus 206 in Example 4 of the present invention. Figure 1
The device 206 shown in FIG. 2 automatically executes the method described in the first embodiment. The controller 201 controls the operation of the device. The device 206 includes an extraction column, a separation unit 202 that separates nucleic acids, and a dispensing unit 20 that dispenses a reagent.
4, a construction unit 203 in which a sample, a reaction container, a storage container, a nucleic acid-capturing chip, a reagent container, and the like are arranged, and a thermal cycler unit 20 that controls the temperature of the liquid in the reaction container to perform PCR
Have five. The procedure of nucleic acid extraction is automatically performed according to FIGS. 3 and 5.

FIG. 13 is a plan view showing the arrangement of the components of the automated nucleic acid purification / separation device 206 according to the fourth embodiment of the present invention. A sample such as blood is placed in the sample erection unit 223. A reaction container is placed in the reaction container installation parts 224 and 225. Storage container installation parts 226, 227, 2
28 and 229 are provided with storage containers for storing the solution after the reaction. The chemicals used in the device 206 are placed in the reagent installation section 218. Thermal cycler section 22
In 2,230, the temperature of the liquid in the reaction vessel is controlled to control the PC.
Perform R. The cleaning liquid such as ethanol is used for the waste liquid outlet 212,
It is discharged from 221 to the waste liquid container. Chip installation part 21
Chips or columns are placed at 3,214, 215, 216, 217, and 219. The used chips or columns are discarded from the chip disposal ports 211 and 220 into a disposal container.

The sample blood is placed in the sample erection section 223. In the device 206, the decomposition process and the adsorption process are performed at the same temperature. Nucleic acid was extracted from 100 μL of human whole blood 1 using the device 206 shown in FIGS. 12 and 13. The reagents used for nucleic acid extraction are the same as the reagents described in Example 1.

The disassembling process was performed in the reaction container erection section 224. 100 μL of proteinase K (6) in the dispensing unit 204
1 mL of whole blood 1 and 1 mL of the reaction solution were added to the tube 5 into which Tube 5 solution at 30 ° C
Incubation was carried out for 10 minutes to obtain a nucleic acid releasing solution.
An additive solution (1 mL) selected from EL, EtOH, and DIGLYME was added to the tube 5 and stirred to obtain a mixed solution 7.

The adsorption process was carried out in the reaction container installation section 224. The separation unit 202 includes an adsorption carrier 13 made of quartz wool.
Column 14 filled with (Toshiba Ceramics Co., Ltd.)
Is installed. The operation of sucking and discharging the mixed liquid 7 by the drive pump was repeated 10 times at 30 ° C.

The cleaning step was carried out by moving the separating section 202 to the reaction container erection section 225. Another tube 16 is installed in the reaction container installation section 225. The operation of sucking and discharging 1 mL of the cleaning liquid (W1) 15 contained in the tube 16 was repeated three times.

The recovery step was carried out by moving the separation section 202 to the storage container installation section 227. A heater is arranged in the storage container installation section 227. 1 mL of the eluate 18 was dispensed into the tube 17 installed in the storage container erection section 227 and preheated to 70 ° C. The eluate 18 contained in the tube 17 was sucked up to a position where the entire adsorption carrier 13 was dipped, and was left still for 2 minutes. Then, the eluate 18 was discharged and the solution containing nucleic acid was recovered.

Next, the eluate 18 containing the nucleic acid is dispensed in the storage container erection section 228, and the dispensed eluate containing the nucleic acid is mixed with dNTP, magnesium chloride, DNA polymerase, and a buffer solution for PCR. After that, the reaction container was transferred to the thermal cycler unit 230, and PCR could be performed.

FIG. 14 shows that in Example 4 of the present invention, DIGLYME was used as the additive solution, and the additive solution at the temperature T (° C.) was added to the nucleic acid releasing solution at the same temperature T, followed by the same temperature T. It is a figure which shows the relationship between temperature T and the relative nucleic acid yield at the time of performing an adsorption process by. The decomposition process and adsorption process are
The reaction container installation unit 224 executes the reaction container installation unit 22.
The temperature of 4 is maintained at the temperature T. FIG. 14 shows the temperature T
Relative nucleic acid yield when changing (horizontal axis in FIG. 14) (FIG. 1)
4 shows the results of examining (4 vertical axis). The target relative nucleic acid yield of 80% or more was obtained when the temperature of the reaction vessel installation portion in the decomposition step and the adsorption step was 20 ° C to 35 ° C.

FIG. 15 is a diagram showing the result of agarose electrophoresis of the PCR product obtained by PCR of the exon 8 site of the p53 gene using the nucleic acid obtained in Example 4 of the present invention as a template. is there. The lane-1 (301) and the lane-2 (302) were subjected to migration of PCR products in the migration direction 304. Migration of DNA molecular weight markers was performed in Lane-3 (303). As is clear from FIG. 15, a PCR product having a chain length of 200 base pairs was confirmed. Example 5: Extraction of Nucleic Acid from Whole Blood by Automated Device (Part 2) Similarly to Example 4, the device 206 shown in FIGS. 12 and 13 was used to extract nucleic acid from 100 μL of human whole blood 1. went. The reagents used for nucleic acid extraction are the same as the reagents described in Example 1. The sample blood is placed in the sample erection unit 223.

The disassembling process was performed in the reaction container erection section 224. 100 μL of proteinase K (6) in the dispensing unit 204
1 mL of whole blood 1 and 1 mL of the reaction solution were added to the tube 5 into which The solution in tube 5 was incubated at 70 ° C for 10 minutes and then cooled to 25 ° C to obtain a nucleic acid-releasing solution.

FIG. 16 is a diagram showing the structure of the reaction container installation section 224 used in the fifth embodiment of the present invention. In the reaction container installation section 224, the organic solvent is automatically supplied based on the temperature detected by the temperature sensor.

The tube 5 containing the solution 403 containing the whole blood 1 and the reaction solution is installed in the heat block 414. Measurement control unit 40 incorporating a temperature control program
7 is incorporated in the support base 420. Measurement control unit 4
Reference numeral 07 drives the Peltier element 405 incorporated in the heat block 414 based on the temperature detected by the temperature sensor 406 incorporated in the heat block 414 to control the temperature of the heat block 414.

After the incubation of the solution 403 in the tube 5 at 70 ° C. is completed, the heat block 414 is cooled by the Peltier device 405. When the temperature sensor 406 detects the time when the temperature of the heat block 414 reaches 25 ° C., a reagent supply command for starting the addition of the organic solvent (addition liquid) 409 is sent to the pump 410 via the control line 415. The pump 410, which is sent and is incorporated in the support 421, is driven. The organic solvent 409 sucked up from the nozzle 408 is added to the tube 5 from the reagent supply nozzle 402 arranged on the arm 422 via the reagent supply passage 411.

With the structure shown in FIG. 16, after the temperature of the reaction vessel erection section 224 was lowered to 25 ° C.
L of DIGLYME was automatically added to obtain a mixed solution 7.

The adsorption process was performed on the reaction container erection section 224. The separation unit 202 includes an adsorption carrier 13 made of quartz wool.
Column 14 filled with (Toshiba Ceramics Co., Ltd.)
Is installed. The operation of sucking and discharging the mixed liquid 7 by the drive pump was repeated 10 times at 25 ° C.

The cleaning step was carried out by moving the separating section 202 to the reaction container erection section 225. Another tube 16 is installed in the reaction container installation section 225. The operation of sucking and discharging 1 mL of the cleaning liquid (W2) 15 contained in the tube 16 was repeated three times.

The collecting step was performed by moving the separating section 202 to the storage container erection section 227. A heater is arranged in the storage container installation section 227. 1 mL of the eluate 18 was dispensed into the tube 17 installed in the storage container erection section 227 and preheated to 70 ° C. The eluate 18 contained in the tube 17 was sucked up to a position where the entire adsorption carrier 13 was dipped, and was left still for 2 minutes. Then, the eluate 18 was discharged and collected to obtain 25 μg of nucleic acid.

Next, the eluate 18 containing nucleic acid is dispensed in the storage container erection section 228, and dNTP, magnesium chloride, DNA polymerase, and PCR buffer solution are prepared in the dispensed eluate containing nucleic acid. After that, the reaction container was transferred to the thermal cycler unit 230, and PCR could be performed. Example 6 Nucleic Acid Extraction from Tissue 1 In a tube, 25 mg of liver tissue (rat) and 80 μm
L of PBS (phosphate buffered saline) is mixed,
Mechanically homogenized. Add 20 μL of Pr to the tube.
After adding oteinase K and stirring, the solution in the tube was
Heat to 56 ° C until the tissue has dissolved. On the tube,
200 μL of reaction mixture (concentration of 3M guanidine hydrochloride, 5
After addition of 10% by volume Tween 80) and stirring, the solution in the tube was heated at 70 ° C. for 10 minutes.
The solution in the tube was cooled to 25 ° C by water cooling to obtain a nucleic acid-releasing solution. 200 μL DIGLY in the tube
ME was added and mixed to obtain a mixed solution 7. The above is the decomposition step.

In the adsorption step, the mixed solution 7 is put on the spin column 8.
Was poured and a centrifugal process (rotation speed 6000 rpm) was performed for 1 minute using a tabletop centrifuge.

In the washing step, the spin column 8 is set to 500 μm.
The washing liquid (W3) of L was poured, and the centrifugal treatment (rotation speed 6000 rpm) was performed for 1 minute.

In the recovery step, E1,
Eluate 13 (100 μL) selected from E2 and E3 was poured, left at room temperature (25 ° C.) for 2 minutes, and then centrifuged for 1 minute (rotation speed 6000 rpm) to recover and quantify nucleic acid. The purity was evaluated. As a result, A260
A high-purity nucleic acid having a / A280 of 1.8 was obtained and could be used for PCR. A260 represents the absorbance of the solution containing nucleic acid at a wavelength of 260 nm,
280 indicates the absorbance of the solution containing the nucleic acid at a wavelength of 280 nm. Example 7: Nucleic acid extraction from tissue 2 Nucleic acid was extracted from 20 mg of bladder tissue (rat) by the same method as in Example 6. As a result, A26
High-purity nucleic acid with 0 / A280 of 1.7 was obtained, and PCR was performed.
It was possible to use Example 8: Extraction of nucleic acid from urine A tube containing 20 mL of a urine sample was subjected to a centrifugal treatment for 5 minutes (rotation speed 14000 rpm). The supernatant was removed from the tube and the urine sediment was separated. For the urine sediment obtained in the tube, 20 μL of proteinase K and 200 μL of reaction solution (concentration of 3M guanidine hydrochloride, 5% by volume of Trito) were added.
(including nX-100) was added and stirred. After heating the solution in the tube at 70 ° C for 10 minutes, the
The solution was cooled to ° C to obtain a nucleic acid-releasing solution. Tube, 20
0 μL of EtOH was added and mixed to obtain a mixed solution 7. The above is the decomposition step.

In the adsorption step, the mixed solution 7 is put on the spin column 8.
Was poured and a centrifugal process (rotation speed 6000 rpm) was performed for 1 minute using a tabletop centrifuge.

In the washing process, the spin column 8 is set to 500
μL of the washing solution (W4) 12 was poured, and centrifugation treatment (rotation speed: 6000 rpm) was performed for 1 minute.

In the recovery step, 100 is applied to the spin column 8.
Pour the eluate (E3) 13 of μL into the well and incubate it at room temperature (25 °
After being left in C), centrifugation was performed for 1 minute to recover the nucleic acid, which was quantified and the purity was evaluated. As a result, A26
High-purity nucleic acid with 0 / A280 of 1.8 was obtained, and PCR was performed.
It was possible to use Example 9: 100 μL of 10 ⁶ HeLa cells in a tube for nucleic acid extraction from cells
After dispersing in PBS, add 20 μL of prot to the tube.
Einase K and 200 μL of reaction solution (concentration of 3M guanidine hydrochloride and 5% by volume of Triton X-100 included)
Was added and stirred. Tube of solution at 70 ° C for 10
After heating for 1 minute, the solution was cooled with water to 25 ° C. to obtain a nucleic acid-releasing solution. Add 200 μL DIGLY to the tube.
ME was added and mixed to obtain a mixed solution 7. The above is the decomposition step.

In the adsorption step, the mixed solution 7 is put on the spin column 8.
Was poured and a centrifugal process (rotation speed 6000 rpm) was performed for 1 minute using a tabletop centrifuge.

In the washing process, the spin column 8 is set to 500
μL of the washing liquid (W2) 11 was poured, and the centrifugal treatment (rotation speed 6000 rpm) was performed for 1 minute.

In the recovery process, 100 is applied to the spin column 8.
Pour μL of Eluent (E1) 12 and let it stand for 2 minutes at room temperature (25 °
After leaving it in C), centrifuge for 1 minute (rotation speed 6000
rpm) was performed to recover the nucleic acid, and the nucleic acid was quantified to evaluate the purity. As a result, a high-purity nucleic acid with A260 / A280 of 1.8 was obtained and could be used for PCR. Example 10: Nucleic acid extraction from whole blood (4) Nucleic acid was extracted from 100 μL of human whole blood 1 according to the method shown in FIGS. 3 and 5. In the disassembly process, 10μ
Tube 100 containing L proteinase K (6)
1 μL of whole blood 1 and 100 μL of a reaction solution (containing guanidine hydrochloride having a concentration of 4M and 5% by volume of Tween 20) were added and stirred. The solution in tube 5 was incubated at 70 ° C for 10 minutes to obtain a nucleic acid releasing solution. Tube 5
To the above, an additive solution (100 μL) selected from EL, EtOH, and DIGLYME was added and stirred to obtain a mixed solution 7.

In the adsorption step, the quartz wool adsorption carrier 13
Column 14 filled with (Toshiba Ceramics Co., Ltd.)
The operation of sucking and discharging the mixed liquid 7 is performed at a temperature of 25
Repeated 10 times at ° C.

In the cleaning step, the operation of sucking and discharging the cleaning liquid (W1) 15 contained in another tube 16 was repeated three times.

In the collecting step, the eluate 18 contained in another tube 17 was sucked up to a position where the entire adsorption carrier 13 was immersed, and was left still for 2 minutes. Then, the eluate 18 was discharged and the solution containing nucleic acid was recovered. The recovered nucleic acid has a good purity and is subjected to the next PCR without any steps such as precipitation and purification.
Etc. could be used for reaction and analysis.

FIG. 17 shows the tenth embodiment of the present invention.
It is a figure which shows the relationship between temperature T and relative nucleic acid yield when EtOH is used as an addition liquid and the addition liquid of 20 degreeC is added to the nucleic acid free solution of temperature T (degreeC). The mixed solution 7 is prepared by adding EtOH at 20 ° C. to the nucleic acid releasing solution at the temperature T. In FIGS. 17 and 18, the horizontal axis represents the temperature T of the nucleic acid releasing solution to which the additive solution is added, and the vertical axis represents the relative nucleic acid yield with the maximum nucleic acid yield being 100%. As shown in FIG. 17, the temperature T of the nucleic acid releasing solution was −5 ° C. to 60 °
At C, a target relative nucleic acid yield of 80% or higher was obtained. Further, when the temperature T of the nucleic acid-releasing solution was 0 ° C to 50 ° C, a suitable relative nucleic acid yield of 90% or more was obtained. Furthermore, when the temperature T of the nucleic acid releasing solution is 10 ° C to 45 ° C,
A better relative nucleic acid yield of 95% or higher was obtained.

As an alcoholic organic solvent which can be used similarly to EtOH, there are PrOH and i-PrOH.

FIG. 18 shows the tenth embodiment of the present invention.
It is a figure which shows the relationship between the temperature T and the relative nucleic acid yield when DIGLYME is used as an addition liquid and the addition liquid of 20 degreeC is added to the nucleic acid free solution of temperature T (degreeC). The mixed solution 7 is prepared by adding DIGLYME at 20 ° C. to the nucleic acid releasing solution at the temperature T. As shown in FIG. 18, when the temperature T of the nucleic acid releasing solution was −5 ° C. to 50 ° C.
A relative nucleic acid yield of>% was obtained. When the temperature T of the nucleic acid-releasing solution was 10 ° C to 45 ° C, a suitable relative nucleic acid yield of 90% or more was obtained. Furthermore, the temperature T of the nucleic acid releasing solution is 95% when the temperature is 20 ° C to 40 ° C.
The more preferable relative nucleic acid yield was obtained.

In the present invention, based on the above-described experimental results, the temperature of the organic solvent such as ethanol added to the nucleic acid releasing solution containing the nucleic acid and the high-concentration salt, and the mixing of the nucleic acid releasing solution and the organic solvent. As a result of examining the influence of the temperature of the liquid on the recovery amount of nucleic acid, it was found that there is a close relationship between these temperatures and the recovery amount of nucleic acid. It was concluded that controlling these temperatures is important to reduce the variation in the amount of nucleic acid recovered.

[0103]

Industrial Applicability According to the method of the present invention, it is possible to provide a method and an apparatus for purifying and separating nucleic acid, which can improve the yield of nucleic acid recovery, have a small variation in nucleic acid recovery amount, and stably obtain a high nucleic acid recovery amount. .

[Brief description of drawings]

FIG. 1 is a diagram illustrating a nucleic acid extraction process according to an example of the present invention.

FIG. 2 is a view for explaining the constitution of whole blood used in the examples of the present invention.

FIG. 3 is a view for explaining the procedure of nucleic acid extraction from whole blood in the example of the present invention.

FIG. 4 is a view for explaining the procedure of the first method for recovering nucleic acid from whole blood in the example of the present invention.

FIG. 5 is a view for explaining the procedure of the second method for recovering nucleic acid from whole blood in the example of the present invention.

FIG. 6 shows an EL as an additive liquid in Example 1 of the present invention.
The figure which shows the relationship between temperature T and the relative nucleic acid yield when the addition liquid of temperature T (° C) is added to the nucleic acid release solution of the same temperature T using.

FIG. 7 shows DI as an additive liquid in Example 1 of the present invention.
The figure which shows the relationship between temperature T and the relative nucleic acid yield when the addition liquid of temperature T (degreeC) is added to the nucleic acid free solution of the same temperature T using GLYME.

FIG. 8 shows Et as an additive liquid in Example 1 of the present invention.
The figure which shows the relationship between temperature T and relative nucleic acid yield when the addition liquid of temperature T (° C) is added to the nucleic acid free solution of the same temperature T using OH.

FIG. 9 is a diagram showing the nucleic acid yields obtained when different types of additive solutions were added in Example 2 of the present invention.

FIG. 10 is a diagram showing the structure of a reaction tank used in Example 3 of the present invention.

FIG. 11 shows D as an additive liquid in Example 3 of the present invention.
The figure which shows the relationship between temperature T and relative nucleic acid yield when the addition liquid of temperature T (degreeC) is added to the nucleic acid free solution of the same temperature T using IGLYME.

FIG. 12 is a block diagram showing the configuration of an automated nucleic acid purification / separation apparatus according to a fourth embodiment of the present invention.

FIG. 13 is a plan view showing the arrangement of components of an automated nucleic acid purification / separation device according to a fourth embodiment of the present invention.

FIG. 14 shows D as an additive liquid in Example 4 of the present invention.
FIG. 7 is a diagram showing the relationship between temperature T and relative nucleic acid yield when an adsorption step is performed at the same temperature T after adding an additive solution at temperature T (° C) to a nucleic acid releasing solution at the same temperature T using IGLYME. .

FIG. 15 is a view showing the results of agarose electrophoresis in Example 4 of the present invention.

FIG. 16 is a diagram showing a configuration of a reaction container installation section 224 used in Example 5 of the present invention.

FIG. 17 is a graph of Example 10 of the present invention, wherein EtOH is used as an additive liquid and the additive liquid at 20 ° C. is heated to a temperature T (° C.).
FIG. 6 is a graph showing the relationship between the temperature T and the relative nucleic acid yield when added to the nucleic acid releasing solution of.

FIG. 18 In Example 10 of the present invention, using DIGLYME as an additive liquid, adding the additive liquid at 20 ° C. to a temperature T
The figure which shows the relationship between temperature T and relative nucleic acid yield when it adds to the nucleic acid free solution of (degree C).

[Explanation of symbols]

1 ... Human whole blood, 2 ... Red blood cells, 3 ... White blood cells, 4 ... Nucleus, 5,
10, 16, 17 ... Tube, 6 ... Proteinase K, 7 ...
Mixed liquid, 8 ... Spin column, 9, 13 ... Adsorption carrier, 1
1, 15 ... Wash solution, 12, 18 ... Eluate, 14 ... Column, 101 ... Addition of whole blood, 102 ... Addition and stirring of reaction solution, 103 ... Incubation, 104 ... Addition and stirring of addition solution, 105 ... By suction Nucleic acid adsorption, 106 ... Addition of washing solution to column, 107 ... Washing step, 108 ... Addition and standing of eluate, 109 ... Recovery of nucleic acid, 110 ... Adsorption of nucleic acid by suction and ejection, 111 ... Suction and rest of eluate Location, 112
... recovery of nucleic acid, 201 ... control unit, 202 ... separation unit, 20
3 ... Erection part, 204 ... Dispensing part, 205 ... Thermal cycler part, 206 ... Nucleic acid purification / separation device, 211, 220 ...
Chip waste port, 212, 221 ... Waste liquid port, 213, 21
4, 215, 216, 217, 219 ... Chip installation section,
218 ... Reagent installation section, 222, 230 ... Thermal cycler section, 223 ... Sample installation section, 224, 225 ... Reaction container installation section, 226, 227, 228, 229 ... Storage container installation section, 251 ... Sample tube, 252 ... Cover , 2
53 ... Insulating material, 254 ... Heat block, 255 ... Temperature control circuit, 256 ... Heating / cooling substrate, 257 ... Radiating fan, 258 ... Post, 259 ... Temperature sensor, 260, 26
1 ... Control line, 301 ... Lane-1, 302 ... Lane-
2, 303 ... Lane-3, 304 ... Migration direction, 305 ...
Band with a chain length of 200 base pairs, 402 ... Reagent supply nozzle,
403 ... Solution containing whole blood and reaction solution, 405 ... Peltier element, 406 ... Temperature sensor, 407 ... Measurement control section, 40
8 ... Nozzle, 409 ... Organic solvent, 410 ... Pump, 41
DESCRIPTION OF SYMBOLS 1 ... Reagent supply path, 414 ... Heat block, 415 ... Control line, 420 ... Support stand, 421 ... Support post, 4
22 ... Arm, 501 ... 1st process, 502 ... 2nd process,
503 ... Third step, 504 ... Fourth step, 505 ... Fifth step, 506 ... Sixth step.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hideki Hasegawa             1-280, Higashi Koikekubo, Kokubunji, Tokyo             Central Research Laboratory, Hitachi, Ltd. (72) Inventor Shinichi Fukuzono             882 Ichimo, Hitachinaka City, Ibaraki Stock Association             Company Hitachi Ltd. measuring instrument group (72) Inventor Takayuki Kanda             882 Ichimo, Hitachinaka City, Ibaraki Stock Association             Company Hitachi Ltd. measuring instrument group F-term (reference) 4B024 AA20 CA01 HA11 HA19

Claims (15)

[Claims] 1. A solution containing a nucleic acid and a high-concentration salt,
A method for separating and purifying nucleic acid, comprising: a step of adding an organic solvent at a temperature in the range of -5 ° C to 55 ° C; and (2) a step of adsorbing the nucleic acid on a carrier. 2. The method for separating and purifying nucleic acid according to claim 1, wherein the organic solvent is ethanol, 1-propanol, 2-propanol, diethylene glycol dimethyl ether, or ethyl lactate. Method for separating and purifying nucleic acid. 3. The method for separating and purifying nucleic acid according to claim 1, wherein the organic solvent at a temperature in the range of −5 ° C. to 45 ° C. is added. 4. The method for separating and purifying nucleic acid according to claim 3, wherein the organic solvent is diethylene glycol dimethyl ether or ethyl lactate. 5. The method for separating and purifying nucleic acid according to claim 1, wherein the organic solvent at a temperature in the range of 0 ° C. to 50 ° C. is added. 6. The method for separating and purifying nucleic acid according to claim 5, wherein the organic solvent is any one of ethanol, 1-propanol, 2-propanol and ethyl lactate. Purification method. 7. The method for separating and purifying nucleic acid according to claim 1, wherein the organic solvent at a temperature in the range of 0 ° C. to 40 ° C. is added. 8. The method for separating and purifying nucleic acid according to claim 7, wherein the organic solvent is diethylene glycol dimethyl ether or ethyl lactate. 9. The method for separating and purifying nucleic acid according to claim 1, wherein the organic solvent at a temperature in the range of 10 ° C. to 35 ° C. is added. 10. The method for separating and purifying nucleic acid according to claim 1, wherein the temperature of the solution and the temperature of the organic solvent are substantially the same. 11. The method for separating and purifying nucleic acid according to claim 1, wherein the temperature of the solution is different from the temperature of the organic solvent. (1) A nucleic acid and a high-concentration salt are included, and the temperature is 0 °.
A step of adding an organic solvent maintained at a temperature substantially the same as the above temperature to a solution maintained at a temperature in the range of C to 50 ° C, and (2) a step of adsorbing the nucleic acid on a carrier. A method for separating and purifying a featured nucleic acid. 13. The method for separating and purifying nucleic acid according to claim 12, wherein the organic solvent is any one of ethanol, 1-propanol, 2-propanol and ethyl lactate. Purification method. (1) A nucleic acid and a high-concentration salt are contained, and the temperature is 0 °.
A step of adding an organic solvent maintained at a temperature substantially the same as the above temperature to a solution maintained at a temperature in the range of C to 40 ° C, and (2) a step of adsorbing the nucleic acid onto a carrier. A method for separating and purifying a featured nucleic acid. 15. The method for separating and purifying nucleic acid according to claim 14, wherein the organic solvent is diethylene glycol dimethyl ether or ethyl lactate.