JPH0499477A - Extractor for nucleic acid - Google Patents

Abstract

PURPOSE:To extract nucleic acid from a sample without requiring troublesome operation by placing the sample in a specific extraction vessel and moving the aforementioned vessel to a stirring means, a liquid ingredient discharging means and a reagent separate injecting means. CONSTITUTION:A sample is placed in a vertical cylindrical type vessel having the base formed from a separation membrane capable of permeating a liquid when a concentration difference is present between both sides without permeating the liquid under ordinary pressure and moved from a vessel fitting and inserting position (set position) (A) to a stirring position (B), reagent separate infecting positions (C1) to (C4), a nucleic acid separating position (D) with the membrane and a vessel taking out position (the vessel fitting and inserting position) (A) according to the order of treating operation.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an apparatus for extracting nucleic acids from various samples containing nucleic acids, such as biological samples.

(Prior Art) In recent years, extraction of nucleic acids from biological samples and the like has been actively performed in various fields. For example, in genetic engineering and the production of DNA probes, operations are performed to extract mRNA and DNA from cells that produce the target protein.
In addition, DNA probes can be used to detect, for example, viral DNA (
In clinical diagnosis to detect DNA (RNA), an operation is performed to extract DNA (RNA) to be detected from a biological sample.

As described above, operations for extracting nucleic acids are extremely important in various fields. Conventionally known methods for extracting nucleic acids include, for example, adding a caustic reagent to a sample, then performing phenol or chloroform/phenol extraction 1 to 3 times, and finally ethanol precipitation; There is a known method in which a surfactant and proteinase K are applied to the protein, followed by phenol extraction, and further ethanol precipitation.

In addition, methods for extracting RNA include adding thiocyanine guanidine to cells and performing ultracentrifugation in a centrifugal solution prepared to a certain density, and methods using ion exchange columns, gel filtration, or electrophoresis. It is being

Kits and extraction devices for extracting nucleic acids from biological samples have also been proposed. For example, proteinase K is used to lyse cells and simultaneously decompose proteins, and the resulting solution is passed through an ion exchange column to extract nucleic acids. There are known devices or devices that treat a sample with brotainase, then perform phenol extraction, and then perform alcohol precipitation.

Furthermore, there are proposals for devices that automate nucleic acid extraction.
For example, a device that extracts nucleic acids using a solvent such as phenol and later collects it using a dialysis cartridge, and
As described in Japanese Patent No. 3-22194, an apparatus is known that processes a sample with proteinase K, then performs phenol extraction, and then collects the ethanol-precipitated precipitate using a column or the like.

(Problems with the Prior Art) However, extraction of nucleic acids generally requires complicated operations, takes time, and, like the above-mentioned conventional method, has the problem of requiring treatment with a dangerous solvent. In addition, there is a problem in terms of yield, which is that the amount of nucleic acid obtained is small.
One of the challenges is realizing rapid and large-scale processing.

To explain this in the context of each of the conventional methods mentioned above, the conventional method of adding a caustic reagent to the sample, then performing phenol or phenol chloroform extraction 1 to 3 times, and performing ethanol precipitation in a flu-like manner requires complicated extraction. Problems include the fact that the process is time-consuming because it requires sweeping, and that it requires the use of dangerous caustic reagents and organic solvents such as chloroform. In addition, in devices that use phenol as a reagent, the metal parts are subject to the strong corrosive effects of phenol, so it is necessary to maintain and inspect the device periodically over relatively short cycles, and at this time, human body There are many problems that need to be improved, considering the risk that phenol, which is harmful to workers, may come into contact with workers.

Furthermore, in the conventional method in which a surfactant and proteinase K are allowed to interact, followed by phenol extraction and further ethanol precipitation, there is a problem in that it takes time for protein degradation by proteinase K to occur. In addition to this,
During long-term processing, the enzymatic action of proteinases is not affected by temperature changes. It is necessary to provide a temperature control means inside the device or to install the device in an environment under certain conditions, which causes the device structure to become larger and more complicated, and has the disadvantage that it tends to be expensive.

Furthermore, the conventional method of adding guanidine thiocyanate to cells and performing ultracentrifugation in a centrifugation solution prepared to a certain density has the same problem as above because the centrifugation operation takes about one day.

Conventional methods using ion exchange columns, gel filtration, or electrophoresis have the same problem of being complicated and time-consuming, and it is extremely difficult to construct an automated device. .

As explained above, there are many difficulties in the various conventional methods mentioned above, and for this reason, the equipment for implementing these methods requires complicated mechanisms and long reaction times to realize troublesome sweeping. It is necessary to provide auxiliary equipment to perform the process stably, and as a result, there are many difficulties in automating the system, and an efficient and inexpensive system has not yet been proposed.

(Means for Solving the Problems) The present inventors have conducted extensive research to solve the problems of the prior art as described above, and have found a method that enables processing with simple operations and eliminates the need for complicated operations. We have developed and proposed a novel nucleic acid extraction method5.

The present invention has been made for the purpose of providing an apparatus that can be effectively used to carry out such a novel method, particularly an apparatus suitable for mechanization and automation. A device suitable for carrying out a nucleic acid extraction method that involves adding a denaturing agent, adding alcohol, and then recovering the precipitate, especially a novel device that uses guanidine salt or urea as a protein denaturing agent. The object of the present invention is to provide a device suitable for carrying out a nucleic acid extraction method.

The device of the present invention that achieves this object is characterized by having the following configuration.

In other words, the bottom of a container used for different processes for nucleic acid extraction is formed with a separation membrane that does not allow liquid to pass through at normal pressure, but allows liquid to pass from the high-pressure side to the low-pressure side when there is a pressure difference on both sides. A vertical cylindrical extraction container, a stirring means for mixing the liquid in the extraction container to promote extraction reactions and processing, and a stirring means for solidifying and solidifying the precipitate containing nucleic acids precipitated in the liquid.
In order to separate the liquid and trap it in the separation membrane, a liquid component discharge means is provided for discharging the liquid in the container through the separation membrane, and a protein denaturant and alcohol are typically used for nucleic acid extraction in the extraction container. A reagent dispensing means for sequentially supplying into the extraction container, and a stirring means, a liquid component discharging means, and a reagent dispensing means for holding (suspending) the extraction container for continuous processing of samples. The present invention is characterized in that it includes extraction container moving means for sequentially moving the extraction containers to each position, each of which is installed in a predetermined arrangement relationship.

The present invention will be explained in more detail below.

The present invention relates to an apparatus for carrying out the method for extracting nucleic acids such as DNA and RNA from a sample as described above. Nucleic acids of interest refer to DNA and/or RNA, which may be heavy or double-stranded, and are not dependent on their genetic properties. For example, DNA includes genomic DNA and other types of DNA.

It may be DNA contained in mitochondria or chloroplasts, or may be RNA or transfer RNA.

Samples containing such nucleic acids include tissues, cells,
Examples include biological samples such as blood, bile, pus, feces, saliva, and sputum.

If the proteins or nucleic acids contained in these samples cannot come into contact with the protein denaturant or alcohol used as a reagent in the device of the present invention, that is, if the sample has cell walls or membranes but is in the form of a lump, it is necessary to For example, homogenization, surfactant treatment, or ultrasonic treatment may be carried out depending on the situation.

An overview of the process of extracting nucleic acids from the sample described above using the apparatus of the present invention will be described. The process of nucleic acid extraction performed by the apparatus of the present invention is typically performed in the following steps.

That is, the first step is to add a protein denaturant to the sample in the extraction container in order to denature and dissolve the proteins in the sample, and the sample and protein denaturant are mixed and reacted to dissolve the proteins contained in the sample. a second step in which the liquid in the extraction container is stirred in order to dissolve the liquid; The fourth step is to stir the liquid in the extraction container in order to mix and react the lysate and alcohol.The nucleic acid deposited in the extraction container is trapped on the separation membrane that also serves as the bottom of the extraction container, and the liquid components are The fifth step is to perform membrane separation treatment to remove
In this process, an alcohol aqueous solution is dispensed to wash the nucleic acid containing the denaturant trapped on the separation membrane, and the liquid in the extraction container is further stirred for mixing, and the liquid components in the extraction container are further stirred. A sixth step is to perform a membrane separation process to remove the nucleic acids, and a seventh step is to dispense pure water or a buffer solution to the nucleic acids trapped on the separation membrane in the extraction container to obtain an aqueous nucleic acid solution. and an eighth step of stirring the liquid in the extraction container in order to dissolve the nucleic acid trapped on the separation membrane.

There are no particular restrictions on the protein denaturing agent used in the first step, as long as it can denature proteins (although it can be used, guanidine salts or urea are preferred because they have a remarkable effect). As the guanidine salt, organic guanidine salts such as guanidine thiocyanate, guanidine hydrochloride, and guanidine carbonate can be used. It is added to a concentration that can denature the protein in the protein and cause nucleic acid to precipitate with the subsequent addition of alcohol.This concentration ρ is, for example, when using guanidine thiocyanate as a protein. , below 1.5M, protein denaturation does not occur sufficiently;
If the concentration is less than 6.5M, nucleic acid precipitation will not occur sufficiently when alcohol is added, so the concentration is about 2.0M to 6M.
Preferably, it is added to the sample at a concentration of 2.5M to 5.5M.

Examples of alcohols for precipitating nucleic acids include ethanol, n-propatol, 1so-propanol, n-butanol, 5ec-butanol, 1so-amyl alcohol, tert-amyl alcohol, and the like.

The alcohol used in the third step may be added at a concentration that causes nucleic acid precipitation without precipitating proteins that have been denatured by a protein denaturant and exists in a solubilized state. The amount added depends on the type of alcohol used, and alcohols with long alkyl chains, that is, alcohols with high hydrophobicity, can precipitate nucleic acids with a small amount added. Furthermore, the aqueous alcohol solution added when washing the nucleic acid after precipitation needs to have the above-mentioned concentration. Therefore, when carrying out the present invention, it is preferable to conduct a test using the alcohol to be used and sample the appropriate amount, that is, the concentration, in advance.

The outline of the apparatus of the present invention used to finally obtain nucleic acids in the form of a precipitate or an aqueous nucleic acid solution by the above nucleic acid extraction method is typically explained as follows.

As already mentioned above, one of the features of the nucleic acid extraction device of the present invention is the use of an extraction container whose bottom surface of the vertical cylinder is made of a material having the above-mentioned properties.

This extraction container is specially constructed in order to carry out all or a part of the process described in step 5 of the nucleic acid extraction process in it, and the shape of the body is suitable for, for example, reagent dispensing. It has a vertical cylindrical shape that is open in the F direction, and the bottom is made of a separation membrane to form a reaction chamber in the body for dissolving the sample with a protein denaturant and precipitation of nucleic acid by adding alcohol. I'm letting you do it. In addition, in order to trap precipitated nucleic acids with a separation membrane, a liquid discharge port is provided below the separation membrane in the body of the container to discharge liquid components other than the precipitated nucleic acids to the outside of the container. In addition to an example of a configuration having a joint or the like, it is also possible to configure a combination type in which an extraction container having the separation membrane as a bottom is inserted into a cylindrical container holder having a liquid component outlet.

The separation membrane is used to trap the precipitated nucleic acids as described above, and at the same time forms the bottom surface that provides a reaction chamber within the extraction container, so that the inside of the extraction container is not exposed to the outside. Under normal pressure conditions (ordinary pressure), the internal liquid can be retained to prevent it from leaking under the separation membrane, and the inside of the container must be pressurized (or under the separation membrane). It has the property of allowing the liquid to permeate downward when the side is set to negative pressure (negative pressure), and in order to ultimately dissolve the nucleic acids, which are precipitates trapped on the separation M membrane, in the form of an aqueous nucleic acid solution, the liquid It is preferable to use a material that is resistant to penetration and adsorption of nucleic acids, such as a water-repellent material. Good resistance 1.) Products made of basic resin are exemplified;
χ-Medium (trade name: manufactured by Norton) can be mentioned.

Further, the apparatus of the present invention is characterized in that it has a stirring means for accelerating the aggregation and extraction of the liquid held in the extraction container during the extraction process.

This stirring means is used to accelerate the mixing reaction between the sample and the denaturing agent at each stage of the processing process for nucleic acid extraction, although the structure is not particularly limited. A device configured to sustain stirring in a state of contact, specifically, an extraction container holding means for suspending the extraction container so that the container can perform rotational pendulum motion using the vicinity of its upper part as a fulcrum, and this extraction container holding means. A preferable example is one having an agitating motion imparting means for causing the lower part of the container held by the container to rotate around a vertical axis, such as in a circular motion or an elliptical motion.

When constituting such a non-contact stirring means, the extraction container holding means that enables rotational pendulum movement of the container is, for example, capable of rotating the extraction container around specific or arbitrary two orthogonal horizontal axes. An example of a suspension mechanism for suspending the device is a so-called gimbal type mechanism. For example, this includes a ring member that surrounds the upper part of the extraction container in an annular shape with a gap,
A fixing member disposed on the outer periphery of the ring member is provided, and a shaft provided on either the identification member or the ring member, and a bearing provided on the other, on one diameter line of the ring member. A shaft provided on either the ring member or the container, and a shaft provided on the other of these on a diametrical line orthogonal to the above-mentioned diameter line. By forming the second rotary shaft support mechanism by engagement of the bearings, the gimbal-type suspension mechanism described above can be constructed.

Such a suspension mechanism is usually attached to the upper part of the extraction container in order to sufficiently stir the liquid inside the extraction container.

The stirring motion imparting means is for forcing the extraction container to perform stirring by giving a rotating pendulum motion to the liquid without bringing a stirring blade or the like into contact with the liquid. Since it is suspended so that it can freely rotate around two orthogonal axes, it is sufficient to allow the lower part of the extraction container to move in a circular or elliptical manner.

Specifically, means for imparting such a stirring motion include (eccentric) circular motion or (eccentric) elliptical motion of a member that applies magnetic attraction force directly or indirectly to the bottom of the extraction container around a vertical axis. Examples include means for causing rotational movement, and means for causing rotational movement in the same manner as described above for a member that is mechanically fitted into convexes and convexes directly or indirectly with the bottom of the extraction container.

The rotational motion given to the lower part of the extraction container may be a uniform motion or a non-uniform motion. In particular, traps 1. In the process of dissolving the extracted nucleic acids in pure water or buffer solution, etc., in order to shorten the dissolution time, it is recommended to reverse the direction of rotation of the extraction container at relatively short intervals, for example, at intervals of 1 to 2 seconds. This method can be recommended.

The above-described non-contact stirring means has the advantage that a minute amount of nucleic acid to be recovered can be processed with little risk of contamination of the sample liquid with foreign matter.

The apparatus of the present invention is also characterized by having a liquid component discharge means as a means for removing the liquid component from inside the extraction container in order to trap the nucleic acid precipitate deposited in the extraction container on the separation membrane.

This liquid component discharge means may be a pressurizing means that pressurizes the inside of the container by sealing the upper opening of the extraction container with a lid, etc., and pushes out the liquid component from the discharge port at the bottom of the extraction container, or For example, a negative pressure means that connects a liquid component suction member of a closed fitting type to suck and suck out the liquid component can be exemplified. Note that a suction method using negative pressure, which does not require consideration of problems such as a mechanism for sealing the inside of the container and scattering of waste liquid discharged from an outlet, is more preferable for automated equipment.

The apparatus of the present invention is also characterized by having a reagent dispensing means for supplying various reagents for nucleic acid extraction into the extraction container.

Although the mechanism of the reagent dispensing means is not particularly limited,
In order to shorten the stirring process time to finally obtain an aqueous nucleic acid solution dissolved in a solvent such as pure water or a buffer solution, it is recommended to use a heated solvent, and therefore a heating device should be installed. is also preferable.

The apparatus of the present invention furthermore holds a plurality of extraction containers, and places these plurality of containers at each position where the above-mentioned stirring means, liquid component discharge means such as gas pressure applying means, and reagent dispensing means are installed. It is characterized by having an extraction container moving means for moving the extraction container in a sequential manner.

A typical example of such means for moving an extraction container is a turntable in which a number of suspension mechanisms, which constitute part of the stirring means described above, are provided at equal intervals on the same circumference, and a turntable that is used intermittently. An example is a combination with a motor that rotates in the circumferential direction.

This extraction container moving means can be configured such that the extraction container is inseparably connected to the extraction container moving means represented by a turntable and the sample is injected into the extraction container. It is also possible to construct a removable device by assembling the extracted extraction container on a turntable and making it removable.

[Example] The present invention will be explained in more detail below based on examples.

Example 1 A method for extracting nucleic acids from Ni562 cells, which are human leukocyte cells (these cells are famous cells and can be purchased, for example, from Dainippon Pharmaceutical Co., Ltd.), as a material to be extracted. An automatic nucleic acid extraction apparatus configured as a specific example for use in the following will be described below with reference to FIGS. 1 to 5.

The outline of this extraction operation is as follows.

First, 562 cells were added to PRM-16 containing 1% beef tallow serum.
40 culture medium, and when the culture suspension reached 500,000 cells per mβ, 1m! The V suspension was collected in a Zumbling tube, centrifuged at 5000 rpm for 5 minutes, and the cells were collected in the precipitate, followed by the above treatments (1) to (5).
Nucleic acids are extracted from the sample.

+1+ The sample in the processing container contains 5M guanidine thiocyanate, 1 * klEDT^, which is a protein denaturant.
0 mM Tris-HCl buffer (pH 7,21 to 0.4
After dispensing m4, stir at room temperature for 30 seconds.

(2) Pour 1 mβ of 99.5% ethanol into the sample solution containing the protein denaturant and stir to precipitate the nucleic acid.

(3) Suction filtration is performed from below the separation membrane forming the inner bottom surface of the container, and the nucleic acid is trapped on the separation membrane as a precipitate.

(4) To remove the protein denaturant remaining in the nucleic acid precipitate, 7r% ethanol is dispensed, and the mixture is washed by repeated stirring and suction filtration.

(5) In order to dissolve the nucleic acid precipitate obtained on the separation membrane, a buffer solution heated to 60 degrees is dispensed and further stirred for 1 minute to finally obtain an aqueous nucleic acid solution.

The automatic nucleic acid extraction device of this example that performs the above processing operations is
A plurality of extraction containers filled with samples containing nucleic acids are suspended from respective openings provided at intervals on the turntable l'T, and automatic processing is performed by intermittent simultaneous movement of the turntable. The system is configured so that the nucleic acids can be extracted in the same container in the form of an aqueous nucleic acid solution.

In Figures 1 to 5, reference numeral 3 designates an upwardly open vertical cylindrical extraction vessel, and its bottom surface is covered with a separation membrane 4 that does not allow liquid to pass through at normal pressure, but allows liquid to pass through when a pressure difference is applied. At the same time, on the underside of this separation membrane,
A discharge port 5 for connecting a connecting pipe from a negative pressure supply source (not shown) for discharging the liquid component on the separation membrane to the outside, and a circular recess 6 for stirring operation are provided.

The extraction container of this example is formed, for example, as (trifluoroethylene resin, inner diameter 18 mm x depth 90 mm), and is used to perform all processes in this apparatus within this container. For example, the portion 1!11 on the bottom of the container is made of tetrafluoroethylene membrane (manufactured by NoRTON, ZITEX).
(trade name); pore size = fine).

Further, the outlet 5 of the extraction container 3 in this example is located in the circular recess 6 at the bottom.
It is formed so as to form a wide conical part extending downwardly at the upper part of the cone, and can be tightly fitted with a suction nozzle 15 shown in FIG.

2 is a turntable, and a plurality of openings 2a for suspending the extraction container 3 are formed at equal intervals along the circumferential direction near the edge of the disc.

Reference numeral 1 designates a container holding ring as a ring member provided in the opening 2a, and a pair of shafts 9a extending radially outward on a straight line is provided on the outer periphery of the ring.

9b, and container holding shafts 8a, 8b extending inward from the inner periphery of the ring are provided on a diameter line perpendicular to the shafts 9a, 9b.

Bearings 10a and 10b are provided at the inner edge of the opening 2a of the turntable 2 to support the shafts 9a and 9b of the container holding ring 1 so as to be rotatable about the shafts. A plurality of openings 2a are provided along the circumferential direction of the turntable 2 as shown in FIG. , turntable drive motor 2
9, by intermittent rotation, from the container insertion position (arrow A position in Figure 2), to the stirring position (arrow B position in Figure 2), to the reagent dispensing position (
01 to C4 positions in the same figure), the nucleic acid membrane separation position (arrowed position in the same figure), and further to the container extraction position (in this device, the same position as the container insertion position A) in accordance with the order of processing operations. It is set in.

In order to fit the extraction container 3 into the opening 2a of the turntable, the extraction container 3 is lowered from above the container holding ring 1 by an operating mechanism (not shown) so that the extraction container 3 is inserted into the opening 2a of the turntable. This can be done by fitting the vertical grooves 7a and 7b into the container holders 8a and 8b from the lower end groove openings, thereby allowing the container holding ring 1 to freely rotate around two horizontal orthogonal axes. This will allow you to hang the container. Removal of the extraction container from the opening 2a
The same operating mechanism can be operated in reverse.

Reference numeral 11 denotes a stirring drive motor as a stirring motion imparting means, which is provided at one or more (one location in the figure) stirring position (arrow B position shown in Figure 2) in the circumferential direction of the turntable. A cylindrical eccentric rotation protrusion 12 that can be loosely fitted into the circular recess 6 at the bottom of the extraction container 3 is provided at the upper end of the rotation shaft 11a (see FIG. 1(a)) extending above the extraction container 3. There is. The stirring drive motor 11 is moved upwardly from the waiting position shown in FIG. 3 by means of a vertical movement means (not shown) to the upward position shown in FIG. It is designed to be moved up and down at the appropriate time. Note that the fixing plate 14 disposed above the stirring position is moved downward by the solenoid 13 to hold the upper end of the container 3 and fit the cylindrical eccentric rotary protrusion 12 into the circular recess 6 at the bottom of the container. The posture of the container 3 can be corrected when the container 3 is moved.

Next, the operation of the stirring mechanism in this embodiment will be explained.

First, the fixing plate 14 is moved downward by the solenoid 13 to hold down the upper part of the container 3, and its posture is corrected and stabilized to a substantially upright state so that the central axes of the stirring drive motor 11 and the container 3 coincide.

Next, the lower stirring drive motor 11 is moved upward by a vertical movement means (not shown), so that the eccentric rotation protrusion 12 is fitted into the circular recess 6 at the bottom of the container. In this case, if the inner diameter of the circular recess 6 is drawn by the outer circumference of the eccentric rotation protrusion 12 when the stirring drive motor 11 is rotated (larger than the diameter of the locus),
No matter what rotational position the eccentric rotating protrusion 12 is in with respect to the rotating shaft of the stirring drive motor 11, the fitting can be performed without any problem. However, if the configuration is such that the rotational position of the stirring drive motor can be determined, the circular recess 6
By making the inner diameter of the eccentric rotation projection 12 larger than the outer diameter of the circular recess 6, the same fitting as described above becomes possible. This has the advantage of eliminating the need for special operations.

After this fitting is completed, the fixing plate 14 is moved upward to allow the container 3 to swing freely again, and if necessary, to securely engage the eccentric rotation protrusion 12 with the inner wall of the circular recess 6. The turntable 2 is slightly rotated, and then the rotation of the stirring drive motor 11 is started. As a result, the eccentric rotation movement of the eccentric rotation protrusion 12 is transmitted to the container 3, and the bottom portion thereof performs a circular movement. Since the upper part of the container is suspended so that it can swing freely around the two orthogonal horizontal axes as described above, the container 3 as a whole performs a conical pendulum motion with the intersection of the two horizontal axes as the fulcrum. Become. As a result, the liquid in the sample container is forcibly rotated in a swirling manner, providing an efficient stirring action. In addition, Figure 1 (
Figure a) shows the state of the liquid during stirring.

After the stirring is completed, the stirring driving motor 11 is stopped and then moved downward to release the fitting between the eccentric rotation projection 12 and the circular recess 6. Furthermore, if there is a container waiting for its turn to be stirred, the turntable 2 is rotated and the stirring process is performed in the same manner.

Note that when stirring is performed using the above configuration, it is often preferable that the liquid level height in the container is approximately 1/2 or less from the bottom of the container 2 to the hanging position.

In addition, when dissolving nucleic acids, which are precipitates that have adhered to the separation membrane, in a buffer solution in the final step of this device, the direction of rotation of the container must be adjusted in a relatively short cycle (
For example, a stirring method in which the mixture is reversed at a cycle of about 1 second is recommended. Note that the dimensions, rotational speed, amplitude, etc. of the container may be determined as appropriate depending on the properties and amount of the target liquid and the degree of stirring.

Figure 4 shows the liquid component discharge provided at the nucleic acid membrane separation position (the arrowed position shown in Figure 2) set at one or multiple locations (one location in the figure) in the circumferential direction of the turntable. This figure shows a suction nozzle 15 as an air pressure applying means. An O-ring 16 is attached to the tip of the suction nozzle 15 so as to tightly fit into a downwardly wide conical discharge port 5 provided above the circular recess 6 at the bottom of the container. .

In addition, the suction nozzle 15 is connected to a waste liquid trap bottle which is kept under negative pressure by a vacuum pump via a tube made of, for example, Teflon (not shown), and the liquid sucked up by the suction nozzle 15 is guided here. . In addition,
Waste liquid)・Pressure inside the wrap bottle (e.g. 20 e+nH
g), it is preferable to control the drive time of the vacuum pump using a pressure regulator or the like to keep it within a certain range.

In this example, the suction nozzle 15 is moved up and down from the downward waiting position shown in FIG.
After the membrane is tightly fitted (see Fig. 3), suction filtration for membrane separation is performed.

Note that the solenoid 17 and fixing plate 18 placed at the top of the container at the membrane separation position are used to tightly fit the suction nozzle JL15 into the outlet 5 at the bottom of the container, similar to the solenoid 13 and fixing plate 14 at the stirring position. This is for correcting the posture of the container 3 when the container 3 is moved.

At the top of the turntable 2, there are reagent dispensing positions set at multiple locations in the circumferential direction (arrows C1 to C1 shown in FIG. 2).
A dispensing nozzle (not shown) is provided corresponding to position C4), and its tip is directed toward the opening 2a on the turntable. Reagents discharged from these dispensing nozzles are provided so as to be supplied by a plunger type dispensing pump from a tank storing a corresponding type of reagent. Note that C1 in the figure indicates the dispensing position of the protein denaturant, C2 and C indicate the dispensing position of alcohol, and C4 indicates the dispensing position of pure water (or buffer solution).

FIGS. 5(a) to (e) show specific examples of the dispensing means used in this example. Of these, FIG. 5(a) shows a Koshida dispensing device that dispenses alcohol or pure water. It is for dispensing, and there is a pipe between the reagent tank 32 and the dispensing nozzle 30.
A device similar to that used for known reagent dispensing, which is composed of a plunger pump 31 and a pair of aMi valves 33 and 34 provided upstream of the plunger pump 31, can be used as is.

In contrast, the dispensing device shown in FIGS. 5(b) and (c)! is used to dispense protein denaturant,
It has the following contrived configuration. In other words, this example! The protein denaturing agent used in the 0-ring is often used in high concentrations, so if a boat-type plunger pump is used, crystals of the denaturing agent will precipitate on the surface of the plunger, causing damage to the plunger and O-ring. There is a risk of seal failure between the Therefore, in this example, a main pump chamber 366 and a main plunger 361 for pumping up and feeding the protein denaturant are provided on the way from the protein denaturant tank 37 to the dispensing nozzle 35, as shown in FIG. 5(c). At the same time, a cleaning chamber 367 is provided to clean the outer periphery of the main plunger 361, which is removed from the O-ring 364 provided to prevent liquid leakage from the main pump chamber 366 and comes out of the main pump chamber. The cleaning liquid from the cleaning liquid tank 38 is provided here to pass through.This eliminates the problem of seal leakage caused by protein denaturant depositing on the outer periphery of the main plunger 361. The diameter section 363 is a drive shaft connected to a drive device (not shown), 365 is an O-ring that seals the cleaning chamber from the outside, and is upstream of the main pump chamber 366 and the cleaning chamber 367.

A pair of solenoid valves 39, 40 and 41, 42 are provided downstream. 43 is a drain tank connected to a drain receiver.

In addition, in FIG. 2 shown in the above explanation, for the sake of simplicity, A, B, and D are shown at each location, whereas the dispensing positions are shown at four locations, C1 to C4. However, in this case, mechanisms corresponding to the first to eighth steps described above are arranged in one direction (for example, clockwise in FIG. 2) along the circumferential direction of the turntable, and the turntable is rotated intermittently. Of course, it is also possible to provide a system in which each mechanism sequentially performs processing while the other mechanisms are being controlled. Such a provision is convenient when configuring a continuous automation device that attempts to process a large number of extraction vessels in a continuous manner.

Figure 6 conceptually shows the above processing steps in a block diagram, and the steps A to D are shown in the lower part of the figure.
- Nucleic acid extraction is performed in the order indicated by ■ and (execution position).

Note that EtOH in this Figure 6 is ethanol, Gu
SCN stands for protein denaturant.

Next, an example of actual processing performed by the nucleic acid extraction apparatus having the above configuration will be described in accordance with FIG. 7 above.

A plurality of extraction containers 3 holding samples containing 562 cells were set in the opening 2a of the turntable 2, and the series of processing operations described above were performed.

The mechanism that performs each processing operation is controlled by a controller (not shown), and the turntable is activated at appropriate times according to pre-programmed processing procedures and various parameters depending on the type, form, and amount of the sample. The upper extraction containers were automatically transferred to each processing section one after another to perform processing for nucleic acid extraction. Number 2 in Figure 3
Reference numeral 8 indicates a sensor of a known position detection device used to control the rotational operation and stop position of the turntable.

First, the container containing the sample was moved to the protein denaturant dispensing nozzle position (arrow C1 position in Figure 2) by rotating the turntable 20 times, and 5M guanidine thiocyanate (
GuSCN), 0.4 mR of 10 mM hydrochloric acid buffer pH 7.2 containing 1 mM EDTA was dispensed. Next, in order to mix and react the sample in the container with the dispensed denaturant, the container was moved to the stirring position (B), and the stirring process was carried out at a rotation speed of 600.
It was performed for 30 seconds with rpc+. Next, position the alcohol dispensing nozzle (arrow C2 in Figure 2) to precipitate the nucleic acid in the container.
position) and 99.5% ethanol (EtOH)
After dispensing IllI2, move to which stirring position and stir to mix the ethanol.Next, only the precipitated nucleic acid is trapped on the separation membrane,
In order to remove the liquid component containing dissolved protein from under the separation membrane by suction, move the container to the membrane separation position ■ (position indicated by the arrow in Figure 2).
The suction nozzle 15 was connected to the discharge port 5 to remove the liquid component by suction. Next, washing with 70% ethanol was performed to further remove the denaturant remaining in the nucleic acid. In other words, first dispense 70% ethanol! (Fig. 2 arrow C
The container was moved to position (s) and β was dispensed into two wells, and then moved to the stirring position (B) and stirred for 10 seconds. Furthermore, it was moved to the membrane separation position (D) and the liquid component was removed by suction.

The above washing operation is repeated 5 times, and finally, in order to dissolve the nucleic acid attached to the separation membrane in the buffer solution, the buffer solution is moved to the buffer dispensing nozzle position (C4) and the necessary amount of buffer solution is dispensed. Move it to the stirring position for stirring and set the rotation speed to 600 rpm.
Nucleic acids were extracted by stirring for 1 minute while reversing the rotational direction every 1 second.

Nucleic acids extracted from Ni562 cells, which are human leukocytes, were treated with restriction enzymes (old Bam, Ec.

As a result of the action of R1, Alu I), RNase, and DNase, the reaction by these enzymes was not inhibited. Furthermore, the replication reaction by DNA polymerase using the extracted nucleic acid as a template was not inhibited. These results indicate that restriction enzyme inhibitors such as histones are removed.

The present processing apparatus cited as an example has been described as one that automatically extracts nucleic acids after setting an extraction container containing a sample in the opening 28 having a container holding ring of the turntable 2. In addition to this first analysis device, for example D
Viral DNA (
Applications such as incorporating a detection device for detecting (RNA) or combining an independent nucleic acid analyzer to use it as an automatic analyzer that includes sample pretreatment are also possible. In this case, in order to remove the extraction container containing the aqueous nucleic acid solution after extraction from the container holding ring of the processing device and transfer it to the analysis device, for example, the container must be lifted using a chuck attached to the tip of a robot arm, etc. mechanism can be used.

Example 2 This example shown in FIG. 7 is a simple example in which the bottom surface of the vertical cylindrical body 23 is formed with a membrane 231 instead of the extraction container in the automatic nucleic acid extraction apparatus described in Example 1. The structure is
Another example is shown in which this is changed to a type in which the container is fitted into a cylindrical container holder 24 having a liquid discharge port 24 at the bottom, and five magnets are used as means for providing stirring motion.

Specifically, the vertical direction 1117a7b of the extraction container 3 in Example 1
A cylindrical sample container 23 with a simple structure omitting the outlet 6 at the bottom, and a container holder 24 into which the extraction container 23 is inserted and held, and this container holder 24 is connected to the container holding ring 27 of the turntable. It is characterized by being provided in a structure in which it is inseparably suspended.

Further, as the stirring motion imparting means, an example is shown in which a first magnet 25 provided at the bottom of the container holder 24 and a second magnet 26 that exerts a magnetic attraction force are eccentrically rotated. That is, in Figure 7,
The container holding ring 27 is provided with a pair of shafts 20a and 20b extending radially outward on the diameter line of its outer periphery.
Further, a pair of shafts 19a are provided radially outward of the container holder 24 on a diametrical line perpendicular to the shafts 20a and 20b.
.

19b, and a bearing 2+ that rotatably supports these shafts.
a, 21b, 22a, and 22b are provided.

The turntable 2 of this example has a pair of shafts 20a, 20b provided in the container holder 24 inseparable by bearings 22a, 22b that rotatably support each of the plurality of openings 2a provided along the circumferential direction. Similar to the first embodiment, the container holders are suspended from the container holders and are provided so that they can be moved to each processing operation position by intermittent rotation of the turntable 2.

It should be noted that fitting the extraction container 23 downward into the container holder 24 or lifting it out can easily be done manually, but in consideration of the danger to the worker, this is done automatically using a chuck or the like of a robot arm. It is also preferable to do so.

In this example, the container holder 24 is always suspended inseparably from the opening 2a of the turntable 2,
Since the extraction container 23 is inserted into and removed from the inner cylindrical portion, when the container 3 is set on the turntable as in the first embodiment, the vertical grooves 7a and 7b of the container and the container holding shaft 8a are used. .

There is no need for fitting 8b.

Next, the motion imparting means in this example will be explained. In this example, a first magnet 25 is attached to the bottom of the container holder 24.
embedded, and the output shaft 11 of the stirring drive motor 11 on the other hand.
The above-mentioned first
A second magnet 26 that exerts a magnetic attraction force between the second magnet 25 and the second magnet 25 is embedded, thereby causing the bottom of the container holder 24 to move circularly as the motor rotates.

With this configuration, as in the first embodiment, it is possible to forcibly give a rotating pendulum motion to the extraction container 23 fitted and held inside the container holder 24, resulting in highly efficient and rapid stirring. It has the effect of

As in Example 1, the dimensions, rotational speed, amplitude, etc. of the container may be determined as appropriate depending on the properties, volume, and degree of agitation of the target liquid. Depending on the rotational speed of the magnet 26, etc., the centrifugal force of the container may exceed the attraction force between the magnets, causing disturbances in the rotational movement of the container, so this point should also be considered depending on the desired degree of agitation. In some cases, it may be preferable to take measures such as, for example, limiting the range in which the container can swing.

(Effects of the Invention) According to the present invention, nucleic acids can be extracted without using dangerous organic solvents by adding a protein denaturant and alcohol to a sample in an extraction container, and then separating the precipitate with a membrane. This has the effect of providing a simple device.

Furthermore, since the apparatus of the present invention can perform a series of operations quickly using an extraction container equipped with a separation membrane, the processing time can be significantly shortened compared to conventional nucleic acid extraction operations that require half a day to two days. The advantages of the method using protein denaturants and alcohol can be effectively utilized, and the equipment can be easily configured to carry out rapid and automated processing operations.
It is particularly useful in the field of genetic engineering, etc., which requires operations to extract nucleic acids from a large number of samples.

In addition, with the device of the present invention, all operations can be performed within the same extraction container, and furthermore, once a sample is set in the device, it is easy to configure the device without requiring manual operations. Therefore, it has the extremely beneficial effect of virtually eliminating the risk of infection or contamination when, for example, the nucleic acid to be extracted is dangerous viral DNA, etc.
Its usefulness is extremely great.

[Brief explanation of the drawing]

FIG. 1(a) is a front view including a partial cross section of the configuration outline of Example 1 of the automatic nucleic acid extraction apparatus according to the present invention, and FIG. 1(b)
Figure 1 is a front view of the extraction container, Figure 1 is a sectional view to explain the suspension mechanism. Figure 2 is an overview of the entire turntable, and the insertion position of the container, stirring position, and reagent dispensing position. , a diagram showing the relationship between the nucleic acid membrane separation position and the extraction position. Figure 3 is a diagram showing the overall outline of the automatic nucleic acid extraction device, and Figure 4 is a diagram showing the overall outline of the automatic nucleic acid extraction device.
The figure shows a mechanism for removing liquid components within the container. Figures 5 (a) to (c) are diagrams showing the outline of the configuration of the dispensing device, in which (a) is the dispensing device for alcohol 1 pure water, and (b) is the dispensing device for protein denaturant. The outline of the dispensing device is shown, and FIG. 6(c) is a diagram showing the detailed structure of the plunger pump of the protein denaturant dispensing device. FIG. 6 is a conceptual block diagram showing the processing procedure in the first embodiment. FIG. 7 is a diagram showing another embodiment of the present invention, FIG. 7(a) is a longitudinal sectional view, and FIG. 7(b) is a line A-B in FIG. 7(a).
FIG. 1: (Container) Holding ring turntable 3 Extraction container 4: Separation membrane 5: Discharge port and other 4 people Figure Figure Figure ＼Δ

Claims (1)

[Claims] 1. A nucleic acid extraction device that performs nucleic acid extraction processing from a sample containing nucleic acids such as a biological sample, which does not allow liquid to pass through at normal pressure, but when there is a pressure difference on both sides, the pressure is increased from the high pressure side to the low pressure side. A vertical cylindrical extraction container whose bottom surface is formed by a separation membrane that allows liquid to pass through the sides; a stirring means for stirring the liquid held in the extraction container; and a stirring means for stirring the liquid held in the extraction container; A means for discharging liquid components in the extraction container which discharges and removes the liquid through a separation membrane by applying gas pressure to the liquid in the extraction container in order to trap it; and a reagent dispensing means for supplying a reagent into the extraction container. 1. A nucleic acid extraction apparatus comprising extraction container moving means for sequentially moving the extraction container to a position where each of the liquid component discharging means and the reagent dispensing means is installed. 2. The stirring means consists of a container holding means for holding the extraction container so as to allow rotational pendulum movement of the lower part of the extraction container, and a stirring motion imparting means for applying rotational movement to the lower part of the held extraction container. The nucleic acid extraction device according to claim 1. 3. The nucleic acid extraction apparatus according to claim 2, wherein the extraction container holding means is a means for suspending the extraction container rotatably around two orthogonal horizontal axes. 4. The stirring motion imparting means has a member that directly or indirectly applies a magnetic attraction force or mechanically engages with the bottom of the extraction vessel, and the member moves circularly or mechanically around a vertical axis. Claim 2 or 3 characterized in that it moves in an elliptical manner.
The nucleic acid extraction device described in . 5. The extraction container moving means includes a turntable having a plurality of extraction container holding means spaced apart at equal intervals in the circumferential direction for holding the extraction container so as to be capable of pendulum movement, and a drive means for intermittent rotational movement of the turntable. The nucleic acid extraction device according to any one of claims 1 to 4, characterized in that it consists of a. 6. Any one of claims 1 to 5, wherein the reagent dispensing means comprises a first dispensing means for dispensing a protein denaturant and a second dispensing means for dispensing alcohol. The nucleic acid extraction device described in . 7. It has a vertical cylinder shape with an open top, and the bottom is formed by a separation membrane that does not allow liquid to pass through at normal pressure, but allows liquid to pass from the high pressure side to the low pressure side when there is a pressure difference on both sides, and below this separation membrane. An extraction container used for nucleic acid extraction processing, characterized in that a liquid outlet is provided on the side. 8. An extraction container that is shaped like a vertical cylinder and opens upward, and whose bottom surface is formed by a separation membrane that does not allow liquid to pass through at normal pressure, but allows liquid to pass from the high pressure side to the low pressure side when there is a pressure difference between the two sides. 1. A container used for nucleic acid extraction processing, comprising a container holder having a cylindrical shape into which an extraction container is inserted and having a liquid outlet at the bottom.