JP2005278436A - Automatic nucleic acid isolating/refining system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an automatic nucleic acid isolating/refining system at reduced cost, compactly and easily housing an anti-dropping means intended for causing neither staining nozzle surroundings nor contamination on samples as a result of e.g. scattering of droplets left on the tip of a nozzle when a liquid is injected from the dispensing injection nozzle into a nucleic acid isolating/refining cartridge. <P>SOLUTION: The system has the following construction and mechanism: The dispensing injection nozzle 51 is connected via a changeover valve 55 to pressure-generating units 52 and 53, which, in turn, are connected via the changeover valve 55 to a liquid tank 56. A prescribed amount of a liquid in the liquid tank 56 is extruded from the nozzle 51 by the pressure-generating units 52 and 53, thereafter, a prescribed minute amount of the liquid is sucked from the nozzle 51 by the identical pressure-generating units 52 and 53. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an automatic nucleic acid separation and purification apparatus for separating and purifying nucleic acid, and more particularly to a technique for preventing dripping from a nozzle after a predetermined amount of liquid is discharged from a dispensing nozzle using a pressurizing apparatus.

  A simple and efficient method for separating and purifying nucleic acids is a solid phase consisting of an organic polymer having a hydroxyl group on the surface, using a solution for adsorbing nucleic acid to the solid phase and a solution for desorbing nucleic acid from the solid phase. There is known a method for separating and purifying nucleic acid by adsorbing and desorbing the nucleic acid (for example, Patent Document 1). According to this document, it is described that the nucleic acid purification is simple, rapid, suitable for automation and downsizing, industrial mass production of nucleic acid adsorption media having the same performance is easy, and the like.

On the other hand, as a conventional liquid discharge device, for example, in a liquid passage between a pressure generation mechanism in a liquid discharge device that pressurizes and supplies liquid by a pressure generation mechanism and discharges it from a dispensing nozzle, and a discharge port of the dispensing nozzle. A one-way valve that opens only in the outflow direction by the pressurized supply of liquid by the pressurized supply means, a bypass passage that bypasses the one-way valve and is provided in the liquid passage, and a pressure generation mechanism provided in the bypass passage Patent Document 2 discloses a liquid supply apparatus provided with a liquid dripping prevention means for sucking a dispensing nozzle when a liquid pressure supply is stopped and a liquid pressure supply is stopped due to a pressure change due to the liquid being stopped.
JP 2003-128691 A JP 2003-19447 A

However, when a nucleic acid extraction apparatus that automatically performs the nucleic acid purification method described in Patent Document 1 was actually produced, dripping occurred from the liquid supply nozzle, resulting in problems such as contamination to other samples and surrounding contamination. .
Further, in the technique for preventing dripping described in Patent Document 2, the means for discharging liquid by pressurization and the means for sucking by negative pressure are separate from each other, and the structure is complicated, and the nozzle head However, there is a concern that it is difficult to fit into an automatic nucleic acid separation and purification apparatus in a compact manner, leading to an increase in production cost.

  The present invention has been made in view of the above situation, and when separating and purifying nucleic acid from a sample containing nucleic acid using a nucleic acid separation and purification cartridge and a pressure generation mechanism, the liquid is supplied from the dispensing nozzle to the nucleic acid separation and purification cartridge. A liquid dripping prevention means that prevents droplets remaining at the nozzle tip from splashing and causing contamination around the nozzle and contamination of the sample when injecting It is an object of the present invention to provide an automatic nucleic acid separation and purification apparatus that can be accommodated and that has a configuration with reduced manufacturing costs.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have ejected a certain amount of liquid from the dispensing nozzle by the pressure generating mechanism, and then from the dispensing nozzle by the same pressure generating mechanism as used for discharging. By aspirating a minute and constant amount by a suction operation, a nucleic acid generating device having a compact and simple structure and not costly and having a dripping prevention means has been solved, and the present invention has been completed. is there.

That is, the present invention has the following configuration.
1. A nucleic acid separation / purification cartridge containing a nucleic acid-adsorbing solid phase in a container having at least two openings, and a pressure generation mechanism for sending a liquid to the nucleic acid separation / purification cartridge, and a liquid pressurized by the pressure generation mechanism Is discharged from the dispensing nozzle onto the nucleic acid separation and purification cartridge, and the nucleic acid is separated and purified from the sample containing the nucleic acid injected into the nucleic acid separation and purification cartridge. The pressure generating mechanism is connected to the liquid tank via the switching valve, and a predetermined amount of liquid in the liquid tank is discharged from the dispensing nozzle by the pressure generating mechanism. Later, a dripping prevention means for sucking a minute and constant amount of liquid from the dispensing nozzle was provided by the same pressure generating mechanism as that used for the discharge. Automated nucleic acid separation and purification apparatus according to claim and.
2. 2. The automatic nucleic acid separation and purification apparatus according to claim 1, wherein the minute fixed amount sucked from the dispensing nozzle is 0.1 μl to 50 μl.
3. 3. The automatic nucleic acid separation according to claim 1 or 2, wherein the nucleic acid-adsorbing solid phase is a porous body made of an organic polymer on which nucleic acid is adsorbed by a weak interaction not involving ionic bonds. Purification equipment.
4). 4. The automatic nucleic acid separation and purification apparatus according to claim 3, wherein the porous body made of the organic polymer is made of an organic polymer having a hydroxyl group.
5). 5. The automatic nucleic acid separation and purification apparatus according to any one of claims 1 to 4, wherein the nucleic acid-adsorbing solid phase is made of an organic material obtained by saponifying a mixture of acetyl cellulose having different acetyl values.
6). 6. The automatic nucleic acid separation and purification apparatus according to claim 5, wherein the saponification rate of the mixture of acetylcelluloses having different acetyl values is 5% or more.
7). 7. The automatic nucleic acid separation and purification apparatus according to claim 5 or 6, wherein the organic material obtained by saponifying the mixture of acetylcellulose having different acetyl values is a saponified product of a mixture of triacetylcellulose and diacetylcellulose. .
8). 9. The automatic nucleic acid separation and purification apparatus according to any one of Items 1 to 8, wherein the nucleic acid-adsorbing solid phase is a front and back asymmetric porous membrane.

  According to the present invention, automatic nucleic acid separation for separating and purifying nucleic acid from a sample containing nucleic acid using a nucleic acid separation and purification cartridge containing a nucleic acid-adsorbing solid phase in a container having at least two openings and a pressure generation mechanism. In the refining device, after a certain amount of liquid in the liquid tank is discharged from the dispensing nozzle by the pressure generating mechanism, a minute constant amount of liquid is discharged from the dispensing nozzle by the same pressure generating mechanism as used for this discharge via the switching valve. By sucking the liquid, dripping from the dispensing nozzle can be surely prevented. Therefore, droplets remaining at the tip of the nozzle do not scatter and cause contamination around the nozzle and contamination of the sample, and with good separation performance, simple, rapid, and automatable, from samples containing nucleic acids. Nucleic acids can be separated and purified with high reliability. Moreover, it can be set as the automatic nucleic acid separation refinement | purification apparatus of a small and cheap structure.

Hereinafter, preferred embodiments of an automatic nucleic acid separation and purification apparatus according to the present invention will be described in detail.
The present invention uses a nucleic acid separation and purification cartridge (hereinafter simply referred to as a cartridge) in which a nucleic acid-adsorbing porous body (nucleic acid-adsorbing porous membrane) is contained in a container having at least two openings, and a pressure generation mechanism. In an automatic nucleic acid separation and purification apparatus for separating and purifying nucleic acid from a sample containing nucleic acid, (1) a step of allowing a sample solution containing nucleic acid to pass through a nucleic acid-adsorbing porous membrane and adsorbing the nucleic acid in the porous membrane; (2) a step of washing the nucleic acid-adsorbing porous membrane in a state where the nucleic acid is adsorbed; and (3) passing the recovered liquid through the nucleic acid-adsorbing porous membrane to remove nucleic acid from the porous membrane. For each step of the desorption process, after a certain amount of liquid is discharged from the dispensing nozzle to the cartridge by the pressure generating mechanism, a minute constant amount of liquid is sucked by the same pressure generating mechanism to prevent dripping. Is.

Hereinafter, the dripping prevention means mounted on the automatic nucleic acid extraction apparatus of the present invention will be described with reference to the schematic diagram of FIG.
The dispensing device 5 includes a liquid dispensing nozzle 51 installed on the nozzle moving table 50, a supply pump 52 that feeds the liquid W stored in the liquid bottle 56 to the liquid dispensing nozzle 51, and the like. The cleaning liquid dispensing nozzle 51 is bent toward the lower cartridge 11, and the dispensing nozzle 51 is connected to the liquid supply pump 52 via the switching valve 55, and the liquid supply pump 52 is connected to the switching valve 55. Via the liquid bottle 56. The liquid supply pump 52 is constituted by a syringe pump, and its piston member is driven and controlled to dispense a predetermined amount of liquid W based on the position detection of the sensor 54 by a pump motor 53 (pulse motor). The liquid supply pump 53 and the pump motor 53 constitute a pressure generating mechanism.

  That is, when dispensing the liquid W into the cartridge 11, the switching valve 55 is switched to the liquid bottle 56 side, the pump motor 53 is driven, the piston member of the liquid supply pump 52 is moved backward, and the liquid W is supplied to the cleaning liquid. The pump 52 is accommodated in the suction. Subsequently, the switching valve 55 is switched to the dispensing nozzle 51 side, the pump motor 53 is driven to push the piston member of the liquid supply pump 52, and the liquid is discharged from the dispensing nozzle 51 until the air in the passage is discharged. After that, the drive of the supply pump 52 is stopped. Thereafter, the driving amount of the cleaning liquid supply pump 52 is controlled to dispense a predetermined amount of liquid W.

  Then, after the dispensing of the liquid is completed, the pump motor 53 is driven to move the piston member of the liquid supply pump 52 backward while keeping the switching valve 55 on the dispensing nozzle 51 side, and the liquid is sucked into a minute fixed amount. . Thereby, the dripping after the liquid discharge from the dispensing nozzle 51 is reliably prevented. In addition, it is preferable that said fine fixed amount is 0.1 microliter-50 microliters. If it is less than 0.1 μl, it is difficult to obtain the effect of dripping, and if it exceeds 50 μl, air accumulates in the conduit to the liquid supply pump 52, and it takes extra time for the air discharge process at the next discharge. Cause a malfunction.

Hereinafter, a nucleic acid extraction apparatus as an automatic nucleic acid separation and purification apparatus equipped with a dripping prevention means according to the present invention will be described in detail with reference to the drawings.
2 is a perspective view showing a state in which the cover is removed according to an embodiment of the nucleic acid extraction apparatus, FIG. 3 is a schematic block diagram of the nucleic acid extraction apparatus, FIG. 4 is a perspective view of a rack in the mounting mechanism, and FIG. FIG. 6 is a process diagram of the extraction operation, and FIG. 7 is a perspective view of the nucleic acid separation / purification cartridge.

Before describing the mechanism of the nucleic acid extraction apparatus 1 of the present embodiment, a nucleic acid extraction and purification process by the nucleic acid extraction apparatus will be described.
The nucleic acid extraction apparatus 1 uses a nucleic acid separation and purification cartridge 11 (cartridge) as shown in FIG. 7 to extract nucleic acids in a sample solution. In this cartridge 11, a nucleic acid-adsorbing porous membrane 11b is held at the bottom of a cylindrical main body 11a whose upper end is open, and a portion below the nucleic acid-adsorbing porous membrane 11b of the cylindrical main body 11a is formed in a funnel shape. A narrow tube nozzle-like discharge portion 11c is formed to protrude to a predetermined length, and vertical protrusions 11d are formed on both sides of the cylindrical main body 11a. After sample liquid, cleaning liquid, and recovery liquid, which will be described later, are dispensed from the upper opening 11e of the cartridge 11, pressurized air is introduced from the upper opening 11e, and each liquid is discharged from the discharge part 11c through the nucleic acid-adsorbing porous film 11b. It flows down to the waste liquid container 12 or the collection container 13 and is discharged. In the illustrated case, the cylindrical main body 11a is divided into an upper part and a lower part and is fitted.

  The nucleic acid extraction apparatus 1 basically performs nucleic acid extraction and purification by an extraction process as shown in FIGS. 6 (a) to 6 (g). First, in step (a) of FIG. 6, the sample solution S containing the dissolved nucleic acid is injected into the cartridge 11 located on the waste liquid container 12. Next, in step (b) of FIG. 6, pressurized air is introduced into the cartridge 11 to pressurize it, the sample solution S is passed through the nucleic acid adsorbing porous membrane 11b, and the nucleic acid is adsorbed to the nucleic acid adsorbing porous membrane 11b. The liquid component that has passed is discharged to the waste liquid container 12.

  Next, the cleaning liquid W is automatically dispensed into the cartridge 11 in the step of FIG. 6 (c), and pressurized air is introduced into the cartridge 11 and pressurized in the step (d), and the nucleic acid is retained in the nucleic acid adsorbing porous membrane 11b. Other impurities are removed by washing, and the cleaning liquid W that has passed is discharged to the waste liquid container 12. Steps (c) and (d) may be repeated a plurality of times.

  Thereafter, the waste liquid container 12 below the cartridge 11 is replaced with the recovery container 13 in the step (e), and the recovery liquid R is automatically dispensed into the cartridge 11 in the step (f). Pressurized air is introduced and pressurized to weaken the binding force between the nucleic acid-adsorptive porous membrane 11b and the nucleic acid, the adsorbed nucleic acid is released, and the recovery liquid R containing the nucleic acid is discharged into the recovery container 13 and recovered.

  The nucleic acid-adsorptive porous membrane 11b in the cartridge 11 is basically a porous body through which nucleic acid can pass, and its surface has a characteristic of adsorbing nucleic acid in the sample liquid with a chemical binding force, and is based on a cleaning liquid. The adsorption is held during washing, and the nucleic acid adsorption force is weakened and separated during collection by the collection liquid. The specific configuration of the example is described in detail in the method for separating and purifying nucleic acid in JP-A No. 2003-128691, for example, the nucleic acid-adsorbing porous membrane 11b has a weak interaction that does not involve ionic bonds, A porous film made of an adsorbing organic polymer is preferable, and the porous film made of an organic polymer is preferably made of an organic polymer having a hydroxyl group. Further, the porous membrane is more preferably a porous membrane made of an organic material obtained by saponifying a mixture of acetyl cellulose having different acetyl values, and the saponification rate of the mixture of acetyl cellulose having different acetyl values is 5% or more. It is preferable. The organic material obtained by saponifying a mixture of acetyl celluloses having different acetyl values is preferably a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose, and the mixing ratio (weight ratio) of triacetyl cellulose and diacetyl cellulose is: It is preferable that it is 99: 1-50: 50. The nucleic acid-adsorptive porous membrane preferably has an average pore size of 0.1 to 10.0 μm and a thickness of 50 to 500 μm.

  As shown in FIGS. 2 and 3, the nucleic acid extraction apparatus 1 pressurizes the apparatus main body 2, a mounting mechanism 3 that holds a plurality of cartridges 11, a waste liquid container 12 and a recovery container 13, and a nucleic acid separation and purification cartridge 11. A pressurized air supply mechanism 4 for introducing air, a dispensing mechanism 5 for dispensing the cleaning liquid W and the recovery liquid R to the nucleic acid separation and purification cartridge 11, and the like are provided. Next, each mechanism 3-5 is demonstrated concretely.

<Mounting mechanism>
The mounting mechanism 3 includes a mounting base 21 at the lower front portion of the apparatus main body 2, and a rack 6 holding a plurality of nucleic acid separation and purification cartridges 11, waste liquid containers 12 and recovery containers 13 is placed on the mounting base 21. As shown in FIG. 4, the rack 6 includes a stand 61, a cartridge holder 62, and a container holder 63.
The stand 61 holds the cartridge holder 62 on the columnar parts 61a on both sides so as to be movable up and down, and holds the container holder 63 on the bottom plate 61b between the columnar parts 61a so as to be movable back and forth.

  The cartridge holder 62 is configured in a two-part structure by joining front and rear plate materials, and includes support legs 62b extending in the vertical direction at both ends of a holding portion 62a extending in the horizontal direction. The support leg 62b is inserted into the vertical sliding groove 61c of the columnar portion 61a of the stand 61 so as to be vertically movable, and the support leg 62b is biased upward by a biasing member (not shown) built in the stand 61. Has been. A plurality of holding holes 62c are arranged in the holding portion 62a, and the nucleic acid separation / purification cartridge 11 is inserted from above, and protrusions 11d (see FIG. 7) formed on both sides of the cylindrical body 11a of the nucleic acid separation / purification cartridge 11. ) Is engaged and held by an engagement member (not shown) in the cartridge holder 62. The engaging member is movable, and at the time of movement, the engagement with the protrusion 11d is released, and the nucleic acid separation / purification cartridge 11 is dropped and discarded all at the same time.

  The cartridge holder 62 has pin holes 62d on both sides of the upper surface, and in use, a tip 49a of a presser pin 49 (see FIG. 2) as a positioning means described later is engaged and pushed down. As shown in FIG. 4, at the position where the cartridge holder 62 is raised, the lower end of the discharge part 11 c of the nucleic acid separation / purification cartridge 11 held by the cartridge holder 62 is located above the waste liquid container 12 and the collection container 13 set in the container holder 63. As shown in FIG. 5, when the cartridge holder 62 is lowered, the discharge part 11c of the nucleic acid separation / purification cartridge 11 is set to be inserted into the waste liquid container 12 or the collection container 13 by a predetermined amount. Has been.

  The container holder 63 is provided with waste liquid container holding holes 63a and recovery container holding holes 63b extending in the horizontal direction in two parallel rows, and a plurality of waste liquid containers 12 are arranged in the rear waste liquid container holding holes 63a. A plurality of collection containers 13 are respectively held in a row by 63b. The waste liquid container holding hole 63a and the recovery container holding hole 63b are disposed at the same position as the holding holes 62c of the cartridge holder 62 at the same pitch, and the waste liquid container 12 and the recovery container 13 are respectively disposed below the held nucleic acid separation and purification cartridges 11. Is set to be located. The waste liquid container 12 and the recovery container 13 are preferably different in size, shape, etc. to prevent confusion.

  The container holder 63 is biased forward by a biasing member (not shown) built in the stand 61. In the container exchange movement (back and forth movement) of the container holder 63, the operating member 31 (see FIG. 3) installed on the mounting base 21 is engaged with the bottom of the container holder 63 through the opening formed in the bottom plate 61b of the stand 61. It is performed by engaging with a hole (not shown). The container holder 63 is moved backward in accordance with the movement operation of the operation member 31 according to the drive of the container replacement motor 32 (DC motor), and the collection container 13 is operated below the cartridge holder 62. When not operating, the waste liquid container 12 is biased by a biasing member (not shown) so as to be positioned below the cartridge holder 62. The operation of the container replacement motor 32 is controlled according to the detection of the position sensors 33a and 33b.

<Pressurized air supply mechanism>
As shown in FIGS. 2 and 3, the pressurized air supply mechanism 4 is installed in a line along the pressure head 40 that moves up and down with respect to the rack 6 of the mounting mechanism 3. A plurality (eight in the figure) of air nozzles 41, an air pump 43 that generates pressurized air, a relief valve 44, an open / close valve 45 that is installed in each air nozzle 41 and opens and closes individually, and is installed in each air nozzle 41. The pressure sensor 46 is provided, and pressurized air is sequentially fed to the nucleic acid separation / purification cartridge 11.

  The pressure head 40 is held by a guide rod 24 installed in the vertical direction between the intermediate frame 22 and the upper frame 23 of the apparatus main body 2 so as to be vertically movable. Similarly, the ball nut 40a installed on the pressure head 40 is screwed to the ball screw 25 installed in the vertical direction, and the ball screw 25 is rotated through a timing belt and a pulley accompanying the driving of the elevating motor 47 (pulse motor). The pressure head 40 is moved up and down by the control accompanying the detection of the photosensors 48a to 48c. There are presser pins 49 on both sides of the pressure head 40. The presser pins 49 are urged downward by a spring 49b and can move up and down, and the tip 49a engages with a pin hole 62d on the upper surface of the cartridge holder 62. The position is restricted and can be pressed.

  The pressing pin 49 presses the front position of the cartridge holder 62 so as not to interfere with the lateral movement of a cleaning liquid dispensing nozzle 51w and a collected liquid dispensing nozzle 51r, which will be described later, while the cartridge holder 62 is being pressed. It is arranged like this.

  Each of the air nozzles 41 is installed on the pressure head 40 so as to be vertically movable and urged downward. A sheet-like sealing material 42 having a communication hole 42a (see FIG. 3) corresponding to the air nozzle 41 is provided below the air nozzle 41. When the pressurizing head 40 moves downward, the upper end opening of the nucleic acid separation / purification cartridge 11 set in the cartridge holder 62 is pressed and sealed through the sealing material 42 at the tip of the air nozzle 41 to communicate with each other. The pressurized air can be fed into the nucleic acid separation / purification cartridge 11 through the hole 42a.

  The relief valve 44 is opened to the atmosphere when the air in the passage between the air pump 43 and the opening / closing valve 45 is discharged. The open / close valve 45 is selectively opened, and an air circuit is configured to introduce pressurized air from the air pump 43 into the nucleic acid separation / purification cartridge 11 through the corresponding air nozzle 41.

<Dispensing mechanism>
The dispensing mechanism 5 divides the cleaning liquid W contained in the cleaning liquid dispensing nozzle 51w and the recovered liquid dispensing nozzle 51r installed in the nozzle moving table 50 that can move horizontally on the rack 6 and the cleaning liquid W contained in the cleaning liquid bottle 56w. The cleaning liquid supply pump 52w for feeding to the injection nozzle 51w, the recovery liquid supply pump 52r for supplying the recovery liquid R accommodated in the recovery liquid bottle 56r to the recovery liquid dispensing nozzle 51r, and the mounting base 21 are mounted. A waste bottle 57 is provided.

  The nozzle moving table 50 is held by a guide rail 27 installed in the horizontal direction on the vertical wall 26 of the apparatus main body 2 and can move in the horizontal direction, and the movement is performed by a nozzle moving motor (pulse motor) (not shown). Drive control is performed so that the cartridge is sequentially stopped on the nucleic acid separation and purification cartridge 11 and stopped on the waste liquid bottle 57 in the return state. The cleaning liquid dispensing nozzle 51w and the recovered liquid dispensing nozzle 51r are bent at the tips downward, the cleaning liquid dispensing nozzle 51w is connected to the cleaning liquid supply pump 52w via the switching valve 55w, and the cleaning liquid supply pump 52w is switched to the switching valve 55w. The recovery liquid dispensing nozzle 51r is connected to the recovery liquid supply pump 52r via the switching valve 55r, and the recovery liquid supply pump 52r is connected to the recovery liquid bottle 56r via the switching valve 55r. Has been. The cleaning liquid bottle 56w and the recovery liquid bottle 56r are attached to the side portions of the apparatus main body 2, respectively. The cleaning liquid supply pump 52w and the recovery liquid supply pump 52r are constituted by syringe pumps, and piston members of the cleaning liquid W and the recovery liquid of a predetermined amount based on detection of the positions of the sensors 54w and 54r by pump motors 53w and 53r (pulse motors), respectively. The drive is controlled to dispense R. The pump motors 53w and 53r and the switching valves 55w and 55r operate based on a command from the control unit 70.

When dispensing the cleaning liquid W or the recovery liquid R, the switching valve 55w or 55r is switched to the cleaning liquid bottle 56w or the recovery liquid bottle 56r, and the pump motor 53w or 53r is driven to supply the cleaning liquid supply pump 52w or the recovery liquid supply pump. The piston member 52r is moved backward. Thereby, the cleaning liquid W or the recovery liquid R is sucked and accommodated in the cleaning liquid supply pump 52w or the recovery liquid supply pump 52r. Subsequently, the switching valve 55w or 55r is switched to the cleaning liquid dispensing nozzle 51w or the recovered liquid dispensing nozzle 51r, and the pump motor 53w or 53r is driven to push the piston member of the cleaning liquid supply pump 52w or the recovered liquid supply pump 52r. . After the cleaning liquid or the recovery liquid is discharged from the cleaning liquid dispensing nozzle 51w or the recovery liquid dispensing nozzle 51r until the air in the passage is discharged to the waste liquid bottle 57, the cleaning liquid supply pump 52w or the recovery liquid supply pump 52r Stop driving. Thereafter, the cleaning liquid dispensing nozzle 51w or the recovery liquid dispensing nozzle 51r is moved onto the nucleic acid separation and purification cartridge 11, and then the driving amount of the cleaning liquid supply pump 52w or the recovery liquid supply pump 52r is controlled to control a predetermined amount of the cleaning liquid W or The recovered liquid R is dispensed into the nucleic acid separation and purification cartridge 11.
When the dispensing is completed, the switching valve 55w or 55r is set to the cleaning liquid dispensing nozzle 51w or the recovered liquid dispensing nozzle 51r, and the cleaning liquid supply pump 52w or the recovered liquid that is the same pressure generating mechanism as that used for discharge is used. The piston member of the supply pump 52r is moved backward. Thus, a minute and constant amount of liquid is sucked from the dispensing nozzles 51w and 51r to prevent dripping from the dispensing nozzles 51w and 51r into the cartridge 11.

  The cleaning liquid bottle 56w and the recovery liquid bottle 56r are composed of container bodies 56wb and 56rb and caps 56wu and 56ru. The caps 56wu and 56ru are respectively provided with suction pipes 58w and 58r in the form of thin pipes. The lower end of 58r opens near the bottom of the container bodies 56wb and 56rb, and the cleaning liquid W and the recovery liquid R are sucked up in accordance with the operation of the cleaning liquid supply pump 52w or the recovery liquid supply pump 52r.

Next, each of the mechanisms 3 to 5 described above is based on a program set in advance by the linked control unit 70 in response to an input operation of the operation panel 7 installed on the upper part of the apparatus body 2. Drive control is automatically performed.
Here, the extraction operation by the nucleic acid extraction apparatus 1 will be specifically described. First, the nucleic acid separation / purification cartridge 11 is set in the cartridge holder 62 of the rack 6 of the mounting mechanism 3, the waste liquid container 12 and the recovery container 13 are set in the container holder 63, and the rack 6 is mounted on the mounting base 21 of the apparatus body 2. To prepare. Next, the dissolved sample solution S is sequentially injected into each nucleic acid separation / purification cartridge 11 by a pipette or the like. Note that the sample solution S may be first injected into the nucleic acid separation / purification cartridge 11 after being set in the rack 6 before being mounted on the apparatus 1 or before being set.
Thereafter, when the apparatus is operated by operating the operation panel 7, the pressure head 40 is moved downward by driving of the lifting motor 47 of the pressure air supply mechanism 4, and the tip 49 a of the presser pin 49 is moved to the pin hole 62 d of the cartridge holder 62. The cartridge holder 62 is lowered to regulate the position, and the lower end discharge part 11c of the nucleic acid separation / purification cartridge 11 is inserted into the waste liquid container 12 as shown in FIG. Do not let the liquid leak out due to splashing etc. and cause contamination. Further, the pressure head 40 moves downward, and the lower end portion of each air nozzle 41 is pressed against the upper end opening of the nucleic acid separation / purification cartridge 11 through the sealing material 42 to be sealed. At this time, since the presser pins 49 regulate the position of the cartridge holder 62, each air nozzle 41 can be accurately pressed against each nucleic acid separation and purification cartridge 11 to ensure a reliable sealing.

  Thereafter, pressurized air is supplied. In response to a command from the control unit 70, the air pump 43 is driven with all the open / close valves 45 closed, and the first open / close valve 45 is first opened. A predetermined amount of pressurized air from the air pump 43 is supplied to the first nucleic acid separation and purification cartridge 11 through the first air nozzle 41.

  Next, after the first opening / closing valve 45 is closed, the second opening / closing valve 45 is opened and pressurized air is supplied to the second nucleic acid separation / purification cartridge 11 through the second air nozzle 41. . This operation is repeated in order to apply pressure to all the nucleic acid separation and purification cartridges 11. The sample solution S on which the pressure is applied is adsorbed and held by the nucleic acid through the nucleic acid adsorbing porous membrane 11b, and the other liquid components are discharged from the discharge portion 11c at the lower end to the waste liquid container 12. When all the sample liquid S passes through the nucleic acid adsorbing porous membrane 11b, the pressure drops below the liquid discharge completion pressure, and when the completion of extraction is detected by all the nucleic acid separation and purification cartridges 11 by the pressure sensors 46, the pressure head 40 Is activated.

  Next, the process proceeds to a cleaning process. The pressure head 40 is lifted after the pressure air is supplied to a position where the air nozzle 41 is moved away from the nucleic acid separation / purification cartridge 11 and moved to a height at which the movement of the nozzle moving table 50 is allowed. The holding pin 49 presses the cartridge holder 62, and the lower end of the nucleic acid separation / purification cartridge 11 is inserted into the waste container 12 while maintaining the state shown in FIG. Then, the nozzle moving table 50 is moved to stop the washing liquid dispensing nozzle 51 w on the first nucleic acid separation and purification cartridge 11 to dispense a predetermined amount of the washing liquid W, and the nozzle moving table 50 is moved to the next nucleic acid separation and purification cartridge 11. The cleaning liquid W is sequentially dispensed. When dispensing of the cleaning liquid W to all the nucleic acid separation and purification cartridges 11 is completed, the cleaning liquid supply pump 52w is driven to suck a small amount of the cleaning liquid W from the cleaning liquid dispensing nozzle 51w to prevent dripping. Next, after the pressure head 40 is moved downward and the lower end of each air nozzle 41 is pressed against the upper end opening of the nucleic acid separation / purification cartridge 11 via the sealing material 42 and sealed, the open / close valve 45 is sequentially moved in the same manner as described above. Opening operation is performed, and pressurized air is supplied to each nucleic acid separation and purification cartridge 11 in the same manner as described above. The cleaning liquid W on which the pressure is applied passes through the nucleic acid adsorbing porous membrane 11b to clean and remove impurities other than nucleic acids, and the cleaning liquid W is discharged to the waste liquid container 12 from the discharge portion 11c at the lower end. When all the cleaning liquid W in all the nucleic acid separation and purification cartridges 11 is discharged through the nucleic acid adsorbing porous membrane 11b, the pressure head 40 is raised to the initial position. The above operation is repeated when the cleaning process is performed a plurality of times.

  Next, the process proceeds to collection processing. First, as the pressure head 40 rises after the cleaning process, the presser pin 49 rises and the cartridge holder 62 of the rack 6 also moves upward, and the lower end discharge part 11c of the nucleic acid separation and purification cartridge 11 moves upward from the waste liquid container 12. After that, the operation member 31 of the mounting mechanism 3 is operated to move the container holder 63 backward, and the container is changed so that the collection container 13 is positioned below the nucleic acid separation / purification cartridge 11.

  Subsequently, the pressure head 40 is moved downward, the tip of the presser pin 49 engages and presses against the pin hole 62d of the cartridge holder 62, and the lower end of the nucleic acid separation and purification cartridge 11 is inserted into the collection container 13. Hold. Then, the nozzle moving base 50 is moved to stop the recovery liquid dispensing nozzle 51r on the first nucleic acid separation and purification cartridge 11 to dispense a predetermined amount of the recovery liquid R, and the nozzle movement base 50 is subjected to the next nucleic acid separation and purification. The collected liquid R is sequentially dispensed by moving to the cartridge 11. When dispensing of the recovery liquid R to all of the nucleic acid separation and purification cartridges 11 is completed, the recovery liquid supply pump 52r is driven to suck a small and constant amount of the recovery liquid R from the recovery liquid dispensing nozzle 51r, thereby dripping the liquid. To prevent. Next, the pressure head 40 is further lowered in the same manner as described above, and the lower end portion of each air nozzle 41 is pressed against the upper end opening of the nucleic acid separation / purification cartridge 11 via the sealing material 42 and sealed, and then the open / close valve 45 is sequentially opened. Opening operation is performed, and pressurized air is supplied to each nucleic acid separation and purification cartridge 11 in the same manner as described above. The recovery liquid R on which the pressure is applied passes through the nucleic acid adsorbing porous membrane 11b to release the nucleic acid adsorbed thereto, and the nucleic acid is discharged together with the recovery liquid R from the lower end discharge portion 11c to the recovery container 13. When all the recovery liquid R in all the nucleic acid separation and purification cartridges 11 is discharged to the recovery container 13, the pressurizing head 40 is lifted and the series of operations is completed.

  The rack 6 for which the extraction operation has been completed is lowered from the mounting table 21, and the nucleic acid separation and purification cartridge 11 and the waste liquid container 12 are taken out from the cartridge holder 62 and the container holder 63 and discarded, while the collection container 13 is taken out from the container holder 63. Then, the lid is covered as necessary, and the next nucleic acid analysis process or the like is performed.

In the present embodiment, a plurality of the nucleic acid separation / purification cartridges 11 are mounted. However, the present invention is not limited to this, and the present invention can be applied to a single nucleic acid separation / purification cartridge 11.
The air supplied from the air pump 43 to the nucleic acid separation / purification cartridge 11 may be any other gas as long as it does not affect the properties of the liquid 16.

Next, the nucleic acid adsorbing porous membrane (nucleic acid adsorbing porous body) 11b included in the nucleic acid separation and purification cartridge 11 will be described in detail.
The nucleic acid adsorbing porous membrane 11b in the nucleic acid separation and purification cartridge 11 is basically porous so that the nucleic acid can pass through, and its surface has a characteristic of adsorbing the nucleic acid in the sample solution with a chemical binding force. In addition, the adsorption is maintained during washing with the washing liquid, and the adsorption force of the nucleic acid is weakened and separated during the collection with the collecting liquid.

  The nucleic acid-adsorptive porous membrane 11b in the nucleic acid separation and purification cartridge 11 is a porous membrane that adsorbs nucleic acids by an interaction that does not substantially involve ionic bonds. This means that it is not “ionized” under the usage conditions on the porous membrane side, and it is presumed that the nucleic acid and the porous membrane come to attract each other by changing the polarity of the environment. As a result, the nucleic acid can be isolated and purified with excellent separation performance and good washing efficiency. Preferably, the nucleic acid-adsorptive porous membrane is a porous membrane having a hydrophilic group, and it is presumed that the hydrophilic group of the nucleic acid and the porous membrane are attracted to each other by changing the polarity of the environment.

  The hydrophilic group refers to a polar group (atomic group) capable of interacting with water, and all groups (atomic groups) involved in nucleic acid adsorption are applicable. As the hydrophilic group, one having a moderate interaction with water is good (see Chemical Encyclopedia, Kyoritsu Shuppan Co., Ltd., “Hydrophilic group”, “Group that is not very hydrophilic”), Examples thereof include a hydroxyl group, a carboxyl group, a cyano group, and an oxyethylene group. Preferably it is a hydroxyl group.

  Here, the porous film having a hydrophilic group means that the material forming the porous film itself is formed by treating or coating the porous film having the hydrophilic group or the material forming the porous film. It means the introduced porous membrane. The material for forming the porous film may be either organic or inorganic. For example, a porous film in which the material forming the porous film itself is an organic material having a hydrophilic group, a porous film in which a hydrophilic group is introduced by treating a porous film of an organic material having no hydrophilic group, A porous film in which a hydrophilic group is introduced by coating a porous film made of an organic material that does not have a hydrophilic group, a porous film in which the material forming the porous film itself is an inorganic material having a hydrophilic group, hydrophilic Porous membranes with hydrophilic groups introduced by treating porous membranes of inorganic materials that do not have groups, and inorganic groups without inorganic groups coated with materials having hydrophilic groups to introduce hydrophilic groups A porous film or the like can be used, but an organic material such as an organic polymer is preferably used as a material for forming the porous film from the viewpoint of ease of processing.

  Examples of the porous film made of a material having a hydrophilic group include a porous film made of an organic material having a hydroxyl group. As porous membranes of organic materials having hydroxyl groups, polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid, polyoxyethylene, acetylcellulose, different acetyl values Examples of the porous film formed include a mixture of acetylcellulose, and a porous film of an organic material having a polysaccharide structure can be preferably used.

As the porous film made of an organic material having a hydroxyl group, an organic polymer porous film made of a mixture of acetylcelluloses having different acetyl values can be preferably used. As a mixture of acetyl cellulose having different acetyl values, a mixture of triacetyl cellulose and diacetyl cellulose, a mixture of triacetyl cellulose and monoacetyl cellulose, a mixture of triacetyl cellulose, diacetyl cellulose and monoacetyl cellulose, a mixture of diacetyl cellulose and monoacetyl cellulose Can be preferably used.
In particular, a mixture of triacetyl cellulose and diacetyl cellulose can be preferably used. The mixing ratio (mass ratio) of triacetyl cellulose and diacetyl cellulose is preferably 99: 1 to 1:99, and more preferably 90:10 to 50:50.

  More preferable organic materials having a hydroxyl group include surface saponified products of acetyl cellulose described in JP-A No. 2003-128691. The surface saponified product of acetyl cellulose is a mixture of acetyl cellulose having different acetyl values, saponified product of triacetyl cellulose and diacetyl cellulose, saponified product of triacetyl cellulose and monoacetyl cellulose, triacetyl A saponified product of a mixture of cellulose, diacetylcellulose and monoacetylcellulose, and a saponified product of a mixture of diacetylcellulose and monoacetylcellulose can also be preferably used. More preferably, a saponified product of a mixture of triacetyl cellulose and diacetyl cellulose is used. The mixing ratio (mass ratio) of the triacetyl cellulose and diacetyl cellulose mixture is preferably 99: 1 to 1:99. More preferably, the mixing ratio of the mixture of triacetyl cellulose and diacetyl cellulose is 90:10 to 50:50. In this case, the amount (density) of hydroxyl groups on the solid surface can be controlled by the degree of saponification treatment (saponification rate). In order to increase the nucleic acid separation efficiency, it is preferable that the amount (density) of hydroxyl groups is large. For example, in the case of acetylcellulose such as triacetylcellulose, the saponification rate (surface saponification rate) is preferably about 5% or more, and more preferably 10% or more. In order to increase the surface area of the organic polymer having a hydroxyl group, it is preferable to saponify the porous membrane of acetylcellulose. In this case, the porous membrane may be a front-back symmetrical porous membrane, but a back-asymmetric porous membrane can be preferably used.

The saponification treatment refers to bringing acetyl cellulose into contact with a saponification treatment solution (for example, sodium hydroxide aqueous solution). Thereby, it becomes a regenerated cellulose and a hydroxyl group is introduced into the portion of acetylcellulose that has come into contact with the saponification solution. The regenerated cellulose thus prepared is different from the original cellulose in terms of crystal state and the like.
In order to change the saponification rate, the saponification treatment may be performed by changing the concentration of sodium hydroxide. The saponification rate can be easily measured by NMR, IR, or XPS (for example, it can be determined by the degree of peak reduction of the carbonyl group).

As a method for introducing a hydrophilic group into a porous film made of an organic material having no hydrophilic group, a graft polymer chain having a hydrophilic group in a polymer chain or in a side chain can be bonded to the porous film.
As a method of bonding the graft polymer chain to the porous film of the organic material, a method of chemically bonding the porous film and the graft polymer chain, and a compound having a double bond that can be polymerized starting from the porous film are polymerized. There are two ways to make the graft polymer chain.

  First, in the method of attaching the porous membrane and the graft polymer chain by chemical bonding, a polymer having a functional group that reacts with the porous membrane is used at the terminal or side chain of the polymer, and this functional group and the porous It can be grafted by chemically reacting with a functional group of the membrane. The functional group that reacts with the porous membrane is not particularly limited as long as it can react with the functional group of the porous membrane. For example, a silane coupling group such as alkoxysilane, an isocyanate group, an amino group, and a hydroxyl group. And carboxyl group, sulfonic acid group, phosphoric acid group, epoxy group, allyl group, methacryloyl group, acryloyl group and the like.

  Particularly useful compounds as a polymer having a reactive functional group at the end of the polymer or in the side chain are a polymer having a trialkoxysilyl group at the polymer end, a polymer having an amino group at the polymer end, and a polymer having a carboxyl group at the polymer end. , A polymer having an epoxy group at the polymer end, and a polymer having an isocyanate group at the polymer end. The polymer used at this time is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption. Specifically, polyhydroxyethylacrylic acid, polyhydroxyethylmethacrylic acid and salts thereof , Polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, and polyoxyethylene.

A method of polymerizing a compound having a polymerizable double bond based on a porous membrane to form a graft polymer chain is generally called surface graft polymerization. The surface graft polymerization method is a method of polymerizing a compound having a polymerizable double bond disposed so as to be in contact with the porous film by giving active species on the surface of the substrate by a method such as plasma irradiation, light irradiation, and heating. It refers to the method of bonding with a porous membrane.
A compound useful for forming a graft polymer chain bound to a substrate has a double bond that can be polymerized and has a hydrophilic group that participates in nucleic acid adsorption. It is necessary to be. As these compounds, any polymer, oligomer, or monomer compound having a hydrophilic group can be used as long as it has a double bond in the molecule. Particularly useful compounds are monomers having hydrophilic groups.
Specific examples of the particularly useful monomer having a hydrophilic group include the following monomers. For example, a hydroxyl group-containing monomer such as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and glycerol monomethacrylate can be particularly preferably used. Also, carboxyl group-containing monomers such as acrylic acid and methacrylic acid, or alkali metal salts and amine salts thereof can be preferably used.

  As another method for introducing a hydrophilic group into a porous film made of an organic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for the coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of easy work. Examples of polymers include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, acetylcellulose, and different acetyl values. Examples thereof include a mixture of acetylcellulose, and a polymer having a polysaccharide structure is preferable.

  In addition, after coating a porous film of an organic material having no hydrophilic group with acetyl cellulose or a mixture of acetyl celluloses having different acetyl values, the coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values is saponified. You can also. In this case, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.

  Examples of the porous film that is an inorganic material having a hydrophilic group include a porous film containing a silica compound. A glass filter can be mentioned as a porous film containing a silica compound. Further, a porous silica thin film as described in Japanese Patent Publication No. 3058342 can be given. This porous silica thin film is an amphiphilic substance by spreading a developing solution of a cationic type amphiphilic substance having a bilayer-forming ability on a substrate and then removing the solvent from the liquid film on the substrate. This multilayer bilayer thin film can be prepared by bringing the multilayer bilayer thin film into contact with a solution containing a silica compound, and then extracting and removing the multilayer bilayer thin film.

As a method for introducing a hydrophilic group into a porous membrane of an inorganic material having no hydrophilic group, a method of chemically bonding the porous membrane and a graft polymer chain, and a hydrophilic group having a double bond in the molecule are used. There are two methods for polymerizing a graft polymer chain using a monomer having a porous membrane as a starting point.
When the porous membrane and the graft polymer chain are attached by chemical bonding, a functional group that reacts with the functional group at the terminal of the graft polymer chain is introduced into the inorganic material, and the graft polymer is chemically bonded thereto. In addition, when a graft polymer chain is polymerized starting from a porous membrane using a monomer having a hydrophilic group having a double bond in the molecule, a compound having a double bond is polymerized. The starting functional group is introduced into the inorganic material.

  As a graft polymer having a hydrophilic group and a monomer having a hydrophilic group having a double bond in the molecule, the porous film of the organic material having no hydrophilic group is chemically bonded to the graft polymer chain. In the process, the described graft polymers having hydrophilic groups and monomers having a hydrophilic group having a double bond in the molecule can be preferably used.

  As another method for introducing a hydrophilic group into a porous film made of an inorganic material having no hydrophilic group, a material having a hydrophilic group can be coated. The material used for the coating is not particularly limited as long as it has a hydrophilic group involved in nucleic acid adsorption, but an organic material polymer is preferable from the viewpoint of easy work. Examples of polymers include polyhydroxyethyl acrylic acid, polyhydroxyethyl methacrylic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, polymethacrylic acid and salts thereof, polyoxyethylene, acetylcellulose, and different acetyl values. Examples thereof include a mixture of acetylcellulose.

  Also, after coating a porous membrane of an inorganic material having no hydrophilic group with acetyl cellulose or a mixture of acetyl celluloses having different acetyl values, coated acetyl cellulose or a mixture of acetyl celluloses having different acetyl values Can also be saponified. In this case, the saponification rate is preferably about 5% or more. Furthermore, the saponification rate is preferably about 10% or more.

  Examples of the porous film made of an inorganic material having no hydrophilic group include a porous film produced by processing a metal such as aluminum, ceramics such as glass, cement and ceramics, or new ceramics, silicon and activated carbon. .

  The nucleic acid-adsorptive porous membrane allows a solution to pass through and has a thickness of 10 μm to 500 μm. More preferably, the thickness is 50 μm to 250 μm. The thinner the thickness, the easier it is to clean.

  The nucleic acid-adsorbing porous membrane that allows the solution to pass through the inside has a minimum pore size of 0.22 μm or more. More preferably, the minimum pore diameter is 0.5 μm or more. Moreover, it is preferable to use a porous membrane having a ratio of the maximum pore diameter to the minimum pore diameter of 2 or more. As a result, a sufficient surface area for adsorbing nucleic acids is obtained and clogging is difficult. More preferably, the ratio of the maximum pore diameter to the minimum pore diameter is 5 or more.

The nucleic acid-adsorptive porous membrane through which the solution can pass inside has a porosity of 50 to 95%. More preferably, the porosity is 65 to 80%. Moreover, it is preferable that a bubble point is 0.1-10 kgf / cm < ² >. More preferably, a bubble point is 0.2-4 kgf / cm < ² >.

  The nucleic acid-adsorbing porous membrane through which the solution can pass is preferably 0.1 to 100 kPa in pressure loss. Thereby, a uniform pressure is obtained at the time of overpressure. More preferably, the pressure loss is 0.5 to 50 kPa. Here, the pressure loss is the minimum pressure required to pass water per 100 μm thickness of the membrane.

The nucleic acid-adsorbing porous membrane through which the solution can pass inside has a water permeability of 1 to 5000 mL per minute per 1 cm ^{2 of} membrane when water is passed at 25 ° C. at a pressure of 1 kg / cm ^2. Preferably there is. More preferably, the amount of water permeation when water is passed at 25 ° C. at a pressure of 1 kg / cm ² is 5 to 1000 mL per minute per 1 cm ^{2 of} membrane.

  The nucleic acid-adsorbing porous membrane through which the solution can pass is preferably 0.1 μg or more of nucleic acid adsorbed per 1 mg of the porous membrane. More preferably, the amount of nucleic acid adsorbed per 1 mg of the porous membrane is 0.9 μg or more.

  The nucleic acid-adsorbing porous membrane through which the solution can pass is not dissolved within 1 hour but immersed within 48 hours when a square porous membrane with a side of 5 mm is immersed in 5 mL of trifluoroacetic acid. Cellulose derivatives are preferred. Further, it is more preferable to use a cellulose derivative that dissolves within 1 hour when a square porous membrane having a side of 5 mm is immersed in 5 mL of trifluoroacetic acid but does not dissolve within 24 hours when immersed in 5 mL of dichloromethane.

  When passing a sample solution containing nucleic acid through a nucleic acid-adsorptive porous membrane, passing the sample solution from one surface to the other surface allows the solution to contact the porous membrane uniformly. It is preferable. When a sample solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane, it is preferable that the sample solution is passed from the side having the larger pore diameter of the nucleic acid-adsorbing porous membrane to the side having a smaller pore size.

The flow rate when the sample solution containing nucleic acid is passed through the nucleic acid-adsorbing porous membrane is ² to 1500 μL / sec per cm ^{2 of} the membrane in order to obtain an appropriate contact time of the liquid with the porous membrane. Things are preferable. If the contact time of the liquid with the porous membrane is too short, a sufficient separation and purification effect cannot be obtained, and if it is too long, it is not preferable from the viewpoint of operability. Furthermore, the flow rate is preferably 5 to 700 μL / sec per cm ^{2 of} the membrane area.

  Further, the number of nucleic acid-adsorbing porous membranes through which the solution to be used can pass may be one, but a plurality of nucleic acid-adsorbing porous membranes can also be used. The plurality of nucleic acid-adsorbing porous membranes may be the same or different.

The plurality of nucleic acid-adsorptive porous membranes may be a combination of an inorganic material nucleic acid-adsorptive porous membrane and an organic material nucleic acid-adsorptive porous membrane. For example, a combination of a glass filter and a porous membrane of regenerated cellulose can be mentioned. The plurality of nucleic acid-adsorptive porous membranes may be a combination of an inorganic material nucleic acid-adsorptive porous membrane and an organic material non-nucleic acid-adsorptive porous membrane, for example, a glass filter and nylon or A combination with a porous membrane of polysulfone can be mentioned.
The nucleic acid-adsorptive porous membrane described above can be in a form other than the membrane shape according to the shape of the cartridge. For example, a chip shape or a block shape can be used.

Next, the properties of the cleaning liquid W and the recovery liquid R will be described.
As the cleaning liquid W, for example, a liquid having the following composition can be used, and the viscosity is preferably 0.2 to 20 mPa · s, and the surface tension is preferably 20 to 45 mN / m.
(Cleaning solution)
100 mM NaCl
10 mM Tris-HCl
65% ethanol

As the recovery solution, a nucleic acid solubilizing reagent solution having the following composition can be used, and the viscosity is preferably 0.5 to 50 mPa · s, and the surface tension is preferably 40 to 80 mN / m.
(Nucleic acid solubilizing reagent solution)
Guanidine hydrochloride (Life Technology) 382g
Tris (Life Technology) 12.1g
TritonX-100 (ICN) 10g
1000ml distilled water

It is a schematic diagram explaining the dripping prevention means by the automatic nucleic acid extraction apparatus of this invention. 1 is a perspective view showing an embodiment of a nucleic acid extraction apparatus with a cover removed. FIG. It is a schematic block block diagram of a nucleic acid extraction apparatus. It is a perspective view of the rack in a mounting mechanism. It is a perspective view which shows the use condition of a rack. It is process drawing of extraction operation | movement. It is a perspective view of a nucleic acid separation and purification cartridge.

Explanation of symbols

1 Nucleic acid extraction equipment (nucleic acid separation and purification equipment)
2 Device main body 3 Mounting mechanism 4 Pressurized air supply mechanism 5 Dispensing mechanism 6 Rack 11 Nucleic acid separation and purification cartridge 11b Nucleic acid-adsorptive porous membrane 11c Discharge part (the other opening)
11e Upper opening (one opening)
12 Waste container 13 Collection container 40 Pressure head 51w, 51r Dispensing nozzle 52w, 52r Supply pump (pressure generating mechanism)
53w, 53r Pump motor (pressure generation mechanism)
55w, 55r switching valve 56w, 56r bottle 70 control unit S sample liquid W cleaning liquid R recovered liquid

Claims (8)

A nucleic acid separation / purification cartridge containing a nucleic acid-adsorbing solid phase in a container having at least two openings, and a pressure generation mechanism for sending a liquid to the nucleic acid separation / purification cartridge, and a liquid pressurized by the pressure generation mechanism An automatic nucleic acid separation and purification apparatus for separating and purifying nucleic acid from a sample containing nucleic acid injected into the nucleic acid separation and purification cartridge from a dispensing nozzle,
The dispensing nozzle is connected to the pressure generating mechanism via a switching valve, and the pressure generating mechanism is connected to the liquid tank via the switching valve;
A liquid that sucks a small fixed amount of liquid from the dispensing nozzle by the same pressure generating mechanism as that used for discharging after discharging a certain amount of liquid from the dispensing nozzle by the pressure generating mechanism. An automatic nucleic acid separation and purification apparatus comprising a drooling prevention means.   2. The automatic nucleic acid separation and purification apparatus according to claim 1, wherein the minute fixed amount sucked from the dispensing nozzle is 0.1 to 50 [mu] l.   3. The automatic nucleic acid separation according to claim 1 or 2, wherein the nucleic acid-adsorbing solid phase is a porous body made of an organic polymer on which nucleic acid is adsorbed by a weak interaction not involving ionic bonds. Purification equipment.   4. The automatic nucleic acid separation and purification apparatus according to claim 3, wherein the porous material comprising the organic polymer comprises an organic polymer having a hydroxyl group.   The automatic nucleic acid separation and purification apparatus according to any one of claims 1 to 4, wherein the nucleic acid-adsorbing solid phase is made of an organic material obtained by saponifying a mixture of acetylcelluloses having different acetyl values.   6. The automatic nucleic acid separation and purification apparatus according to claim 5, wherein the saponification rate of the mixture of acetylcelluloses having different acetyl values is 5% or more.   The automatic nucleic acid separation and purification apparatus according to claim 5 or 6, wherein the organic material obtained by saponifying the mixture of acetylcelluloses having different acetyl values is a saponified product of a mixture of triacetylcellulose and diacetylcellulose.   The automatic nucleic acid separation and purification apparatus according to any one of claims 1 to 7, wherein the nucleic acid adsorbing solid phase is a front and back asymmetric porous membrane.

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