JP2003339374A - Nucleic acid separation column, device and nucleic acid extraction method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for automatically and quickly extracting a nucleic acid for a template of PCR method, etc., by using a solid-phase extraction column while preventing the contamination of the nucleic acid. <P>SOLUTION: The solid-phase extraction column is provided with a means to separate the mist, etc., discharged through the outlet port of the column from the outlet port of other columns. The contamination is prevented and a nucleic acid having high purity is extracted by the method. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nucleic acid extraction column, an apparatus and a nucleic acid extraction method for extracting nucleic acid (DNA or RNA) from a biological sample or the like, and in particular to the purpose of amplifying nucleic acid by PCR etc. and detecting it with high sensitivity. On the other hand, the present invention relates to a column, a device, and a method for extracting a nucleic acid having sufficient purity.

[0002]

2. Description of the Related Art As an apparatus using a solid phase extraction column, W
There is a known example shown in O98 / 53912. In this known example, since a plurality of samples are processed at the same time, an integrated column in which a large number of columns are arranged two-dimensionally is used. Figure 1
Shown at 6. This integrated column is formed as a carrier body 226 carrying a plurality of columns (compartments 228). A carrier 234 for adsorbing nucleic acid is provided inside each compartment 228, and a discharge hole 232 is provided at the lower end of the compartment 228. A vacuum chamber is formed by hermetically installing this integrated column in the upper opening of the vacuum chamber. By depressurizing the inside of the vacuum chamber, the operation of filtering the solution in the column through the adsorption carrier is repeated, and the solid phase extraction by the so-called suction method is performed. When extracting a nucleic acid, a series of steps including adsorption of the nucleic acid to the carrier, washing of the carrier, desorption and recovery of the nucleic acid from the carrier are performed. In order to increase the recovery rate, the shape of each column is thick up to the part filled with the adsorption carrier, and the discharge hole part of the solution below is a cylindrical shape, and the recovery liquid is discharged to the central part of the recovery container. . The discharge hole 23 of the column of this known example
The tip of 2 is exposed.

QIAGEN Product Guide 2001 p.3
A second known example shown in 13 is shown in FIG. Also in this second known example, a carrier 234 for adsorbing nucleic acid is provided inside each column (compartment 228), and a discharge hole 232 is provided at the lower end thereof.
Is provided. An integrated column is formed as a carrier body 226 having a plurality of compartments 228 mounted therein. In this second known example, the partition wall 205 is provided around the root of the discharge hole, but the tip of the discharge hole 232 is at the partition wall 205.
Exposed to the outside.

[0004]

When nucleic acids are respectively extracted from a plurality of different biological samples using a plurality of columns according to the above-mentioned conventional technique, a solution containing the nucleic acids derived from the first sample discharged from the first column. Becomes droplets and scatters, and the droplets adhere to the vicinity of the discharge hole of the second column and finally become the second droplet.
The phenomenon of contamination between samples (cross contamination), that is, it is mixed in the recovered liquid of the column of 1 or directly into the recovered liquid of the second column, was newly found. The amount of droplets is very small, but when the nucleic acid obtained by the conventional technique is used in a method having a large amplification rate such as a PCR reaction, a small amount of cross contamination is greatly amplified. As a result, the nucleic acid derived from the first biological sample is as if it were the second nucleic acid.
The result is erroneous, as if it were present in the biological sample, and is a major error factor. In particular, in the case of use in diagnosing infectious diseases caused by viruses, for example, when the subject who provided the first biological sample is positive, even when the subject who provided the second biological sample is originally negative, cross-contamination as described above When this occurs, it causes a false positive result as the diagnosis result of the second subject, which is a big problem.

The present invention has been made in view of the above problems, and in an apparatus for automatically and rapidly extracting nucleic acids from a plurality of samples using a plurality of solid-phase extraction columns, cross contamination between the samples is performed. It is an object of the present invention to provide a device for preventing nucleic acid and for extracting a nucleic acid of high purity sufficient for highly accurate clinical diagnosis.

[0006]

According to the present invention, a separating member for separating mist is provided between a plurality of solid phase extraction columns, and mist or the like that may be generated from the discharge holes of the first column is discharged to the second column. Isolation from the holes prevents cross contamination. A partition wall, a filter, an extension cylinder, a holding chamber and the like are effective as the separating member. The partition wall and the filter are preferably formed integrally with the column, the extension cylinder is preferably provided detachably from the column, and the holding chamber is preferably provided separately from the column.

The partition wall separates the discharge holes of a plurality of columns from each other by accommodating the discharge holes of the columns and shielding them from the outside. The filter is in contact with the collection container to doubly separate the space inside the collection container from the space outside, so that the ejection holes of each column arranged inside each collection container are quadrupled. The extension cylinder is detachably provided on the outer surface of the discharge hole of the column without a gap, and shields the discharge hole to isolate the discharge holes of the plurality of columns from each other. The holding chamber has a column housing space for housing the lower part of the column independently for each column, thereby separating the ejection holes of each column from each other.

It should be noted that neither the above-mentioned first nor second known example has a partition wall covering the ejection holes of the column.

Since any of the isolation members is used to isolate the ejection holes of the column from each other, droplets or mist ejected from the ejection holes of the first column may adhere to the ejection holes of the second column. It is also possible to prevent the contamination of the recovered liquid of the second column from being mixed, and as a result, it is possible to prevent a decrease in reliability such as a false positive diagnostic result of the second sample. According to the preferred embodiment of the present invention, as compared with the conventional example which does not use the separating member, the installation of the partition wall causes the mixing of mist by about 3 digits or more, and the installation of the filter provides about 5 digits.
By suppressing the digits, and by installing the extension cylinder and the holding chamber, it was possible to substantially eliminate mixing of mist.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS [Embodiment 1] The configuration of a first embodiment of the present invention will be described below with reference to FIGS. 1 to 4.

FIG. 1 is a block diagram of a solid phase extraction column 1 according to the first embodiment of the present invention. 2 is a carrier, 3 is an injection hole, 4
Is a discharge hole, 5 is a partition wall, 6 is an upper hollow cylinder, 100 is a lower hollow cylinder, and 101 is a hollow cone. The basic skeleton of the column 1 is formed by joining three hollow bodies of an upper hollow cylinder 6, a hollow cone 101, and a lower hollow cylinder 100, and a hollow cylindrical partition wall 5 is joined on the slope of the hollow cone 101. The upper hollow cylinder 6 has an injection hole 3 at the upper end and holds the carrier 2 below the inside. The lower hollow cylinder 100 has a discharge hole 4 at the lower end.

In this embodiment, the discharge hole 4 is arranged inside the partition wall 5. That is, the lower end of the partition wall 5 is located below the lower end of the discharge hole 4. That is, the length of the lower hollow cylinder 100 is formed longer than the length of the partition wall 5. More specifically, the distance in the up-down (center axis) direction from the lower end of the discharge hole 4 to the lower end of the partition wall 5 is L (the downward direction is positive), and the distance from the center of the discharge hole 4 to the partition wall 5 ( When the distance in the (radius) direction is M, L = + 16 mm and M = 7 mm. Therefore, the solid angle ω formed by the opening surface at the lower end of the partition wall at the discharge hole 4 is 2π (1-cos θ) = 0.53 [sr]. From the viewpoint of preventing mist generation, it is preferable to suppress ω, in other words, to make L large and small M, but the lower limit of M is determined by the diameter of the recovery container, and the L dependence of ω is a sigmoid type and L>
In the case of 15, the effect of reducing ω by increasing L tends to be saturated, and it is necessary to consider the handleability of the column.

In this embodiment, the material of the carrier 2 for adsorbing nucleic acid is a glass fiber filter paper GF manufactured by Whatman (R).
Although the / F type is used, various types of quartz fiber filter paper, glass wool or quartz wool, particles or mesh having silica on the surface, non-woven fabric, etc. may be used.

FIG. 2 is a block diagram of an automatic extraction device 20 according to the first embodiment of the present invention. 7 is a column holder, 8 is a waste liquid tank, 9 is a recovery tank, 10 is an intake hole, 11 is a cover, 12 is a waste liquid hole, 13 is a waste liquid tube, 14 is a trap, 15,
15 'is an exhaust tube, 16 and 16' are filters, 1
Reference numerals 7 and 17 'are electromagnetic valves, 18 is a vacuum pump, 19 is a recovery container, 25 is a dust collecting mechanism, 26 is an intake mechanism, and 27 is an exhaust mechanism. The column holder 7 includes an upper part of the waste liquid tank 8 and a recovery tank 9
It can be installed on any of the upper parts of the
It is shown installed on top of. A collection container 19 is held in the collection tank 9. The above-mentioned main part of the apparatus is covered with the cover 11 airtightly when the cover is closed. Waste liquid hole 1 of the waste liquid tank 8
2 is connected to a trap 14 via a waste liquid tube 13. The trap 14 and the intake port 10 of the recovery tank 9 are provided with exhaust tubes 15 and 15 ′, filters 16 and 1, respectively.
6'and solenoid valves 17, 17 'are connected to the vacuum pump 18. When the cover is closed, the air inside the apparatus is kept clean by the dust collecting mechanism 25 and the intake mechanism 26, and the exhaust mechanism 2
By 7, the internal atmospheric pressure is maintained at atmospheric pressure. The automatic extractor 20 includes the above-mentioned components, and although not shown, a control mechanism for them and a column holder moving mechanism,
Equipped with reagents and automatic dispensing mechanism.

FIG. 3 is a schematic sectional view of the waste liquid chamber 21 and the column 1 of the automatic extraction apparatus according to the first embodiment of the present invention. The waste liquid chamber 21 includes a column holder 7 and a waste liquid tank 8. Although four columns 1 are illustrated in the cross section of FIG. 3, the number of columns can be further increased or decreased. The column holder 7 is a hollow body whose bottom surface is open, and has an opening on the top surface that holds the column 1 in an airtight manner. The waste liquid tank 8 is a hollow body whose upper surface is open,
It has a waste liquid hole 12. The joint between the column 1, the column holder 7, and the waste liquid tank 8 can be detached, and the joint is kept airtight by an o-ring or the like (not shown). FIG. 4 shows the recovery chamber 22 of the automatic extraction device according to the first embodiment of the present invention.
3 is a schematic cross-sectional view of a column 1 and a recovery container 19. FIG. The recovery chamber 22 includes a column holder 7 and a recovery tank 9. The recovery tank 9 is a hollow body having an open upper surface, and has an intake port 10. The joint portion between the column 1, the column holder 7, and the recovery tank 9 can be detached and kept airtight by an o-ring or the like (not shown). Further, in FIG. 4, the upper end of the recovery container 19 is provided above the discharge hole 4.

Next, the operation of the first embodiment of the present invention will be described with reference to FIG.
To FIG. 5 will be described. FIG. 5 is a flow chart showing a nucleic acid extraction process from a whole blood sample according to the first embodiment of the present invention. 31 is a decomposition process, 32 is an additive liquid mixing process, 33
Is an adsorption step, 34 is a cleaning step, and 35 is a recovery step,
Each process is performed in this order. The contents of each step are schematically shown on the right side of the figure.

40 is a sample (whole blood), 41 is an enzyme solution, 4
2 is a decomposition solution, 43 is an addition solution, 44 is a cleaning liquid, 45 is an eluate, 46 is a decomposition liquid, 47 is an adsorption solution, 48 is an adsorption waste liquid, 49 is a cleaning waste liquid, and 50 is a recovery liquid. Enzyme solution 4
1. As the decomposition solution 42, the addition solution 43, the washing solution 44, and the eluate 45, unless otherwise specified, the reagents having the compositions described in JP-A 8-501321 were used.

Each step of the first embodiment of the present invention will be described below. In the suction method of the present invention, it is possible to simultaneously extract the nucleic acids contained in each of a plurality of samples in parallel by using a column whose upper limit is the number of column openings on the upper surface of the column holder. For one column
A case where nucleic acid is extracted from one sample using one sample will be described below.

The decomposition step 31 includes the sample 40 and the enzyme solution 4
1. This is a step of mixing and stirring the decomposing solution 42 and heating to decompose the cell membrane, nuclear membrane, and nuclear protein, and release the nucleic acid contained in the nucleus of white blood cells into the decomposing solution 46. Sample 40
1 mL of whole blood, 0.1 mL of enzyme solution 41, and 1.2 mL of decomposition solution 42 were used, and heating was performed at 70 ° C. for 10 minutes.

The addition solution mixing step 32 is a step of adding the addition solution 43 to the decomposition solution 46 to solubilize lipids and the like, reduce the solubility of nucleic acids, and enhance the efficiency of solid phase adsorption and washing of nucleic acids. is there. 1 mL of the additive solution 43 was added to the cooled decomposition solution 46, and the mixture was stirred to adsorb the adsorption solution 47.
Got

In the adsorption step 33, the adsorption solution 47 is applied to the column 1.
In this step, the nucleic acid in the adsorption solution 47 is adsorbed on the surface of the carrier 2 by passing through the carrier. As shown in FIG. 3, the column holder 7 in which the new column 1 is installed is placed on the waste liquid tank 8 to form the waste liquid chamber 21. The adsorption solution 47 was injected into the column 1 through the injection hole 3 and suction filtered. The suction operation will be described with reference to FIG. Operate the vacuum pump 18,
The electromagnetic valve 17 was opened, and the pressure inside the trap 14 was reduced via the filter 16 and the exhaust tube 15. Waste liquid tube 1
The inside of the waste liquid chamber 21 is also depressurized via 3. Therefore, due to the pressure difference between the injection hole 3 of the column 1 and the atmospheric pressure, the adsorption solution 47 inside the column 1 moved to the discharge hole 4 through the carrier 2. In this process, the nucleic acid in the adsorption solution 47 is adsorbed by the carrier 2, and the components other than the nucleic acid are absorbed in the adsorption waste liquid 4.
8 leaked from the discharge hole 4. The adsorption waste liquid 48 drops into the waste liquid tank 8 and then passes through the waste liquid tube 13 to the trap 1
Move to 4 and store. After the adsorption solution 47 passed through the column and the adsorption waste liquid 48 dropped into the waste liquid tank 8, the electromagnetic valve 17 was closed and the flow of gas to the vacuum pump 18 was shut off. The atmosphere flowing from the injection hole 3 of the column 1 returned the atmospheric pressure to the section from the waste liquid chamber to the solenoid valve 17. Even if the installation environment of the device is contaminated with nucleic acid-containing mist or the like, the contamination mist outside the device is not mixed into the inside by the action of the cover 11 and the dust collecting mechanism 25, so that mixing of exogenous mist through the atmosphere is prevented. .

In the washing step 34, a unit washing operation of injecting the washing liquid 44 into the column 1 and then aspirating is carried out a plurality of times to wash away coexisting components other than nucleic acid from the carrier, and purify nucleic acid on the carrier surface. Is the process of leaving. The cleaning liquid 44 has the composition described in Tokuhyo 8-501321 (cleaning liquid A).
And Tween (R) as a surfactant while complying with it
Two types were used, one containing 20 wt% of 5 wt% (cleaning liquid B).
First, the unit washing operation using 3 mL of the washing liquid B was performed twice, and then the unit washing operation using 3 mL of the washing liquid A was performed twice. The cleaning waste liquid 49 containing coexisting components other than nucleic acid is the discharge hole 4
After leaking from the tank and dropping into the waste liquid tank 8, the waste liquid tube 13
To the trap 14 via. The recovery step 35 is a step of injecting the eluate 45 into the column 1, incubating after incubating, and aspirating the eluate 45 to recover it in the recovery container 19 as the recovery solution 50 containing the purified nucleic acid. Using the column holder moving mechanism provided in the automatic extraction device, the column holder 7
Was moved above the recovery tank 9 to form the recovery chamber 22 shown in FIG. A recovery container 19 is installed in the recovery tank 9, and the upper end of the recovery container 19 is fitted into the space between the lower hollow cylinder 100 of the column 1 and the partition wall 5. In other words, the upper end of the collection container 19 is arranged inside the partition wall 5 of the column 1, and the discharge hole 4 of the column is arranged inside the collection container 19. Next, the eluate 45 is injected from the injection hole 3 into the column 1
Incubated for 5 minutes, the nucleic acid adsorbed on the carrier was dissolved and then aspirated. The suction operation in the recovery step 35 is the same as the suction operation in the adsorption step 33 described above, but the vacuum chamber used is the recovery chamber 22, and the exhaust system is the intake hole 10 ′, the exhaust tube 15 ′, the filter 16 The difference is that it is a ", solenoid valve 17". The eluate containing the nucleic acid is collected from the ejection hole of the column in the collection container
It dropped to 9 and was recovered as a recovery liquid 50. 1 per time
Using the mL of the eluent 45, the above unit operation was repeated twice to obtain 2 mL of the recovered solution 50 in the recovery container 19. Although the case where the nucleic acid is extracted from one sample using one column has been described above as an example, it is also possible to simultaneously extract the nucleic acids contained in each of the samples from a plurality of samples in parallel at the same time. . For example, in order to evaluate the reproducibility, 1 mL of the same whole blood is used as the sample 40, and when the number of simultaneous treatments is 24, the nucleic acid recovery amount is about 25 μg on average, or 260
The ratio of absorbance at nm and 280 nm was about 1.9. Therefore, highly pure nucleic acid was obtained with a high recovery rate. In general, individual samples are, for example, different whole blood samples collected from different subjects, and extraction operations are carried out independently and in parallel using different columns to purify the corresponding nucleic acids and separate collection containers. Get to. When nucleic acids are amplified and used for applications such as diagnosis as described in the section of the problem, it is extremely important that there is no cross-contamination between a plurality of samples even though the amount is extremely small.

As will be described later in the section of "Effects of the invention", in this embodiment, due to the novel construction in which the discharge holes 4 are arranged and shielded inside the partition walls 5 of the column, the cloth which has been a problem of the conventional column is crossed. Avoided contamination. Even if a solution such as the adsorption waste liquid 48, the cleaning waste liquid 49, and the recovery liquid 50 discharged from the discharge hole of the first column forms droplets, mist, foam, etc. (hereinafter collectively referred to as mist), these partition walls Is held inside the column to prevent scattering and adhesion to the second column (the discharge hole of this column is also shielded inside the partition) and the collection container. Therefore, in this embodiment,
It is possible to completely eliminate the risk of contamination due to the first contamination route in which the mist derived from the first sample is directly scattered and adheres to the second column or the like. A second contamination path in which a part of the mist derived from the first sample diffuses inside the vacuum chamber, floats in the chamber, and then indirectly mixes into the second column is also conceivable. It is considered that the probability of contamination of the chamber by the mist based on the second path depends on the solid angle of the column opening surface. The solid angle formed by the column opening surface at the lower end of the partition wall of the present embodiment over the discharge hole is about 0.5 [sr]. On the other hand, the column according to the prior art has no partition, or even if it has the partition, the lower end of the column is located above the discharge hole, so that the solid angle ω formed by the column opening surface to the discharge hole is at least 2π = 6.28.
[sr], generally L = -18 mm, M = 6.5 m
The solid angle ω in this case reaches 12.2 [sr]. Therefore, the probability of contamination of the chamber by the mist by the second route in this example is as low as about 1/12 to 1/24 compared with the conventional column. Further, since the discharge hole of the second column is also shielded inside the partition wall, the mist suppression effect by the partition wall doubles, and the mist mixing probability by the second path is 140 to 530 of the conventional example. . Further, in the present embodiment, there is a preferable feature that there is a flow of liquid or gas from the discharge hole of the column toward the vacuum chamber, which is the only outlet, and the mist does not easily approach the discharge hole of the column. As a result of the above effects, the risk of mixing in the first and second routes is extremely low in this embodiment as compared with the conventional example. Contamination of the partition walls in the adsorption process is completely removed because the cleaning waste liquid rinses away the adsorption waste liquid in the cleaning process. In the recovery step, since the upper end of the recovery container 19 is arranged inside the partition wall 5 of the column 1 and the discharge hole 4 is arranged inside the recovery container 19, the mist caused by the eluate containing the nucleic acid of another column is generated. Is a collection container 1
It is extremely unlikely that it will enter the inside of 9 and be mixed into the main recovery liquid 50. Further, with the above arrangement, even if the recovery liquid discharged from this column is scattered, almost all of it collides with the corresponding inner wall of the recovery container and falls inside the recovery container, so the recovery efficiency is high and the mist is not outside the column. There is an effect that it is difficult to leak.

The effect peculiar to the present embodiment is that, with a simple structure, it is possible to separate the splashes and mists from other samples and prevent cross contamination. Therefore, there is an effect that it is possible to provide a nucleic acid extraction device that is easy to handle, quick, simple, and highly accurate, and can improve throughput, reduce cost, and achieve high reliability. [Embodiment 2] FIG. 6 is a schematic view of the configuration of a solid phase extraction column 1'according to a second embodiment of the present invention. The configuration of the column 1'is basically the same as that of the column 1 in the first embodiment, except that a filter 23 is provided between the lower hollow cylinder 100 having the discharge hole 4 and the partition wall 5. .

FIG. 7 is a sectional view showing the mutual relationship between the column 1'and the recovery container 19 in the recovery chamber according to the second embodiment of the present invention. The filter 23 of the column 1 ′ is pressed against the collection container 19, in other words, the space inside the collection chamber and the space inside the collection container are doubly separated by the filter 23.

The filter 23 is the same as the carrier 2 Whatman®.
A GF / F type glass fiber filter paper manufactured by the same company was punched into a donut type for use. According to the company's catalog, this GF / F type has a particle retention capacity of 0.7 μm, that is, the transmittance of particles larger than 0.7 μm is 2%.
It is the following. That is, this filter allows gas to pass therethrough, but has a function of trapping 98% or more of particles of the μm order among particles such as droplets and mist. Furthermore, since this filter paper has the ability to adsorb nucleic acids, even if the captured particles contain nucleic acids, they have the function of adsorbing and removing the nucleic acids.

In the recovery step, the space inside the recovery container and the space inside the recovery chamber are doubly separated by the filter 23. Therefore, it is possible to prevent double leakage of the mist from the inside of the collection container to the collection chamber. Similarly, it is possible to prevent double mixing of mist from the recovery chamber into the recovery container. That is, the space inside the two recovery containers is quadruple isolated by the filter. Therefore, the transmittance of 0.7μm particles becomes 0.02 ⁴ That 8ppm below, substantial problems are suppressed to a level that does not cause even if the PCR amplification. That is, even when the nucleic acids contained in a plurality of samples are extracted using a plurality of independent columns, a very small amount of cross-contamination can be prevented and highly purified nucleic acid can be purified.

Although not shown, in this embodiment, a structure similar to that of FIG. 7 was applied not only to the recovery process but also to the adsorption process and the cleaning process. At this time, a recovery container 19 ′ similar to the recovery container 19 was installed inside the waste liquid chamber 21 and used. Similar to FIG. 7, the filter 23 has a collection container 19 '.
The pair of the recovery container 19 'and the column 1'was installed in the waste liquid chamber 21 while being supported so as to be pressed against the column, and the adsorption step and the washing step were performed. The operation in this case is basically the same as that in the recovery process described above, except that in the adsorption process and the cleaning process, the adsorption waste liquid and the cleaning waste liquid move to the recovery container 19 ′ through the discharge hole 4. At this time, cross-contamination between the columns is prevented by the action of the filter 23 as in the above. In addition, the waste liquid is in the collection container 1
Since it collects in 9 ′, it is possible to easily remove the contamination. As a modified example, it is also possible to connect the lower part of each recovery container 19 ′ to a common waste liquid reservoir, provide a backflow prevention mechanism in the connection flow path, and integrate the waste liquid container.

The effect of this embodiment is that, with a simple structure, by collecting and adsorbing and removing the droplets and mist derived from other samples,
It is also possible to prevent the risk of extremely small amounts of cross contamination. Therefore, there is an effect that it is possible to provide a nucleic acid extraction device that is easy to handle, quick, simple, and highly accurate, and can improve throughput, reduce cost, and achieve high reliability. [Third Embodiment] A third embodiment of the present invention will be described below. FIG. 8 is a schematic view of the configuration of a column 1 ″ according to the third embodiment of the present invention. The configuration of the column 1 ″ is basically the same as that of the column 1 ′ in the second embodiment, but the outer surface of the discharge hole is The difference is that the extension tube 24 is detachably provided without a gap. Extension tube 24 is basically a hollow cylinder, column 1 "
The outer surface of the discharge hole at the tip of the lower hollow cylinder 100 and the inner surface of the hollow portion at the upper part of the extension cylinder 24 are fitted together without a gap. Therefore, the outer surface of the discharge hole of the column is shielded from the outside by the extension cylinder and isolated. By the combination of the column 1 ″ and the extension cylinder 24, the discharge hole of the column is substantially extended to the tip of the extension cylinder, and there is no opening in the middle. The lower end of the extension cylinder 24 is below the lower end of the partition wall 5. The extension tube 24 is preferably provided integrally with the column 1 ".

FIG. 9 is a schematic sectional view of the waste liquid chamber 21 in the modification of this embodiment. In this modification, an extension cylinder
There is a claw 67 near the lower end of 24, and it is a stopper inside the waste liquid tank 8.
It differs from the third embodiment in that it has 68. The claw 67 has elasticity, the lower end of the claw is fixed near the lower end of the extension tube 24,
In the natural state, the upper end is open as shown in FIG.
Due to the elasticity of the claw 67, the upper end can be compressed close to the surface of the extension tube 24. The stopper 68 has an opening through which the extension tube 24 with the tab 67 compressed can freely pass, but the extension tube 24 with the tab 67 open can only pass downward. In other words, the stopper 68 forms a latch mechanism in combination with the pawl 67. The stopper 68 is removable from the waste liquid tank 8.

The operation of the adsorption and cleaning process of this modification is basically the same as that of the first embodiment, but the following points are different.
Column 1 "with extension tube 24 before the adsorption process starts
Is installed inside the waste liquid chamber 21. Ie column 1 ”
By pushing in from above, the tab of the extension tube 24 is compressed and passes through the stopper. The column 1 "is airtightly fixed to the column holder 7 in the state shown in Fig. 9. The subsequent operation is the same as that of the second embodiment up to the middle, but the result of the suction operation mainly in the adsorption and cleaning steps is Different: That is, when the adsorption solution 47 or the cleaning liquid 44 is sucked, the adsorption waste liquid 48 or the cleaning waste liquid 49
Leaks from the lower end of the extension cylinder 24, which is a substantial discharge hole.
The lower end of the extension cylinder 24 projects below the lower end of the partition wall 5 of the column, and is blocked by the stopper 68. Therefore, the adsorption waste liquid 48 and the cleaning waste liquid 49 are near the lower end of the extension cylinder 24 and the stopper 68.
Although it partially adheres to the lower surface, it does not adhere to the column body above the stopper 68. The outer surface of the discharge hole 4 of the column is covered with a filter 23 and an extension cylinder 24. In other words,
The discharge hole of the first column is isolated from the discharge hole of the second column, and there is a risk that mist derived from the adsorption waste liquid 48 and the cleaning waste liquid 49 from the second column will adhere to the vicinity of the discharge hole of the first column. There is no Therefore, even if the nucleic acids contained in a plurality of samples are extracted using a plurality of independent columns, cross contamination can be prevented.

After completion of the washing process and before proceeding to the recovery process, the column 1 "is moved from the waste liquid chamber to the recovery chamber together with the column holder 7. At this time, the claw 67 of the extension cylinder 24 is caught by the stopper 68 and extended. The cylinder 24 is detached from the column 1 ″ and remains in the waste liquid tank. Therefore, the extension cylinder 24 contaminated with the waste liquid is not brought into the recovery chamber. Further, as described above, the waste liquid or the mist derived from the waste liquid does not adhere to the column. Therefore, the waste liquids are not brought into the recovery chamber, and the risk of self-contamination (the first waste liquid is mixed with the first recovery liquid to reduce the purity) and cross-contamination is significantly reduced.

In the above modification, the extension cylinder 24 is individually formed and provided in the state of being integrated with the column 1 ", but the extension cylinder may be provided separately from the column 1". In this case, the operability is good if the extension cylinders are connected to each other and are integrally formed with the stopper 68. Waste chamber
The integrated extension tube is installed in advance inside 21 and then the column is installed, and the extension tube and the column are connected and used.

The effect peculiar to this embodiment and the modification is that contamination prevention in the adsorption and cleaning steps can be achieved at an extremely high level and the analysis result can be highly reliable.

[Embodiment 4] A fourth embodiment of the present invention will be described below. FIG. 10 is a block diagram of an automatic extraction device according to a fourth embodiment of the present invention. The basic configuration of this embodiment is similar to that of the first embodiment, but the pressurization mechanism 2 is used instead of the suction mechanism.
8 is different. The pressurizing mechanism 28 is attached to the column holder 7
The pressure chamber 29, which is an airtight closed space, is formed together with the column holder 7 and the column 1 by installing the pressure chamber 29 in the upper part.

The operation of this embodiment is the same as that of the first embodiment, except that when the reagents are passed through the column, the liquid is fed not by vacuum suction but by pressurization.

The effect peculiar to this embodiment is that the appropriate pressure mechanism 2 is used.
By adopting 8, the injection hole 3 and the discharge hole 4 of the column
Since a pressure difference of 1 atm or more can be applied between and, the passage time of the solution through the column can be shortened and the process can be speeded up. It is also possible to use a highly viscous adsorption solution. Further, as a structure of the pressurizing mechanism 28, a pressure chamber is formed by ensuring airtightness for each column opening of the column holder, and a mechanism for applying pressure independently to each pressurizing chamber and a sensor for applying pressure. Another feature is that each column can be individually controlled by providing a mechanism for observing the pressure in the pressure chamber and controlling the pressure. In this case, the pressurizing chamber corresponding to the unused column is simply kept airtight and the preparation is easy.

As a modification of this embodiment, a pressure mechanism
Instead of 28, it is also possible to use a pressurizing / depressurizing mechanism capable of depressurizing as well as pressurizing. In this case, various solutions can be moved in two directions through the carrier in the column. A specific example of the pressurizing / depressurizing mechanism is an automatic pipettor, and a column having an adjusted inner diameter can be used instead of the pipette tip. A related method is disclosed in JP-A-11-
No. 127854. When the ejection hole at the bottom of the column is projected below the partition or the extension tube is projected below the partition, various solutions can be sucked into the column from the bottom of the column, eliminating the pipette tip for sample and reagent injection. it can. The cleaning solution can be injected from the upper part of the column by providing a branch channel for liquid injection inside the automatic pipettor, so that the inside of the column can be cleaned efficiently. The effect peculiar to this modification is that operations such as adsorption, desorption, and washing of nucleic acids can be performed a required number of times by passing the liquid in two directions, and the liquid sending conditions can be precisely controlled. [Fifth Embodiment] A fifth embodiment of the present invention will be described below. FIG. 11 is a schematic diagram of the main part of an automatic extracting apparatus according to the fifth embodiment of the present invention. The basic configuration of this automatic extraction device is similar to that of the first embodiment, but the configurations of the column, the waste liquid chamber 21 and the recovery chamber 22 are mainly different. That is, in the present embodiment, the integrated column 66 in which a plurality of columns are integrally formed and the column holder 7'which holds the column in an airtight manner are used. The integrated column has a shape compatible with, for example, a multititer plate, and the columns can be arranged two-dimensionally. An integrated holding chamber 60 and an integrated holding chamber 60 ′ were provided and used for the waste liquid chamber 21 and the recovery chamber 22, respectively. In the integrated holding chamber 60, a column storage space for storing the lower part of the integrated column is provided independently for each column,
The respective column storage spaces are separated from each other by an integrated holding chamber. The bottom of each column storage space,
A flow passage 61 connects the opening to the bottom of the integrated holding chamber 60. The bottom opening of the integrated holding chamber 60 faces the bottom opening of the waste liquid tank 8 and communicates with the waste liquid hole 12. Integrated holding chamber
In 60 ', a storage space for storing a pair of the lower part of the integrated column and the recovery container 19 is independently provided, and the bottom of each storage space and the opening provided in the bottom of the integrated holding chamber 60'. The flow path 61 'is connected between the and. The bottom opening of the integrated holding chamber 60 ′ faces the bottom opening of the recovery tank 9 and communicates with the intake hole 10. Since the upper portions of the integrated holding chambers 60 and 60 ′ are in airtight contact with the column holder 7 ′, the column storage spaces do not directly communicate with each other, but communicate with each other only through the flow passages 61 and 61 ′.

The operation of this embodiment is similar to that of the first embodiment, but firstly the difference is that the integrated column 66 is used. However, the individual columns that make up the integrated column are
Since it has the same configuration and function as the first embodiment, the operation is also the same except that a plurality of simultaneous parallel processings are possible.

Secondly, the paths of the waste liquid, the collected liquid, and the exhaust that have fallen in the column are different. For example, in the waste liquid chamber 21, the dropped adsorption waste liquid or cleaning waste liquid first drops into each column storage space of the integrated holding chamber 60. Then, from the bottom of each column storage space, through the flow passage 61, the bottom opening of the integrated holding chamber 60, the bottom opening of the waste liquid tank 8 and the waste liquid hole 12. The subsequent steps are the same as those in the first embodiment.

In the adsorption step and the washing step, the column storage space is independent for each column and is isolated from each other.
The discharge holes of the individual columns are also isolated from each other. Therefore, the adsorption waste liquid and the cleaning waste liquid from the individual columns fall only inside the column storage space corresponding to each column,
Do not mix in the column storage space corresponding to another column. The adsorption waste liquid and the cleaning waste liquid collected at the bottom of the column storage space finally flow out to the waste liquid hole 12 through the flow passage 61. Since the break of depressurization is not performed from the pump side but is performed by leaking from the column, backflow of liquid or gas from the waste liquid hole 12 toward the integrated column 66 is prevented. Further, since the length of the flow passage 61 is sufficiently longer than the inner diameter, the flow passage 61 itself has a function of preventing backflow. Further, a backflow prevention mechanism such as a trap or a check valve can be added in the middle of the flow passage 61. The recovery process in the recovery tank 9 is also the same. In this embodiment, the integrated holding chamber in which a plurality of holding chambers are integrated is adopted, but it goes without saying that the holding chambers corresponding to the individual columns may be independently formed.

The effect peculiar to this embodiment is, firstly, that the column is integrated so that it is easy to handle and is suitable for automation. Secondly, since the column storage space in the waste liquid or recovery chamber is independent for each column, the risk of cross contamination between columns and between samples is extremely low. Thirdly, the member that forms the column storage space with which the sample mainly contacts is the integrated holding chamber 6
0 and 60 'are independent of the waste liquid tank 8 and the recovery tank 9, and not only the column but also the integrated holding chambers 60 and 60' are disposable, so that the risk of cross contamination from the previous sample is extremely high. It has the effect of being low.

Next, the effect of the present invention will be described.

Here, a conventional example will be shown for comparison with the present invention. This conventional example is a method based on the publicly known example shown in WO9853912, while maintaining the technical characteristics thereof, and applying other conditions to the present invention for comparison with the present invention. In the present invention, there is a means for preventing mist that may occur during suction from mixing into another column or a collection container, but in the conventional example, there is no such mixing prevention means, or its action is Not enough. For example, the first aspect of the present invention
The column 1 of this embodiment has the partition wall 5, and the discharge hole 4 has the partition wall 5.
However, the conventional column does not have the partition wall 5 and the discharge hole 4 is directly exposed to the waste liquid tank.

First, the column 1 described in the first embodiment of the present invention is mounted on the automatic extraction apparatus 20 to extract nucleic acid from whole blood, and in the process, it is transferred to other columns or recovery containers by droplets or mist. The situation of contamination was investigated. Further, as a conventional example, the same operation was performed for a column in which the discharge holes 4 were exposed to the outside of the partition wall 5 and which did not have the partition wall 5, and the results were compared with the results of the column of the first embodiment of the present invention.

FIG. 12 shows a trace of a photograph of the state of the column according to the first embodiment of the present invention at the end of the adsorption step 33. Columns used for nucleic acid extraction are 3 from the left
In order to investigate the influence on the other columns, one column was used, but columns for which nucleic acid extraction was not performed were placed side by side in the column holder. FIG. 13 shows the result of performing the same experiment using the column based on the conventional example.

In the present embodiment, as shown in FIG. 12, the coloring derived from the adsorption waste liquid and the mist based on the adsorption waste liquid is provided inside the column, in the vicinity of the discharge hole 4, and inside and the tip of the partition wall 5.
There are 70. However, no coloring was observed on the outer surface other than the tip of the partition wall 5, and the adjacent column and dummy. Therefore, in this example, the adsorption waste liquid and the mist based on the adsorption waste liquid were limited to the inside of the used column, and the adhesion and the mixture to other columns were effectively prevented. On the other hand, in the conventional example, as shown in FIG. 13, coloring 70 'was observed not only in the inside of the column 71 and the vicinity of the discharge hole 4, but also on the entire outer surface of the column. Further, not only the adjacent columns but also the lower hollow cylinders of the columns on the right of the two columns were colored in a wide range. Therefore, in the conventional example, it is understood that the adsorption waste liquid and the mist based on the adsorption waste may adhere not only to the used column but also to other columns to cause cross contamination. From the observation results of the above experiment, in the present invention, the degree of scattering and adhesion of mist to other columns is about 3 as compared with the conventional example.
It is thought that it was suppressed more than an order of magnitude.

In the present example, most of the contamination generated on the column during the adsorption step was removed by the washing step 34. Therefore, after the washing step, the appearance of the column returned to the state without coloring. Therefore, not only cross-contamination to other columns but also mixing of adsorption waste liquid to the recovery liquid corresponding to the column (self-contamination)
Was also effectively prevented. On the other hand, in the conventional example, although the inside of the column and the vicinity of the tip of the discharge hole were washed in the washing step, the contamination on the outer surface of the column and other columns was not completely removed. In the example of FIG. 13, the mixed liquid of the adsorption waste liquid and the cleaning waste liquid remained in the connection portion between the third lower column hollow cylinder and the hollow cone used from the left. Further, about 1 μL of the adsorption waste liquid adhering to the lower hollow cylinder of the column on the right next to the second column remained as it was.

In the recovery step as well, as in the adsorption step, droplets and mist due to the recovered solution were generated from the tip of the column. If this mist adheres to the upper side of the outer side of the discharge hole of the column and merges with the remaining adsorption waste liquid, there is a risk of self-contamination in which the mist flows along the adsorption waste liquid along the column surface and merges with the collected liquid. Further, similarly, there is a risk that contaminants adhering to the upper outside of the ejection holes of the other columns (in this example, the column on the right of the two columns in the present example) may mix with the recovery liquid corresponding to the column and cause cross contamination.

In addition to the above contamination by the adsorption waste liquid, the problem of contamination by the recovery liquid in the recovery process was also examined. FIG. 14 shows a photographic trace of the recovery container 19 and the recovery tank 9 after the first suction, using the column based on the conventional example. Also in this example, the third column from the left was used and the other columns were not used. The collected solution was collected in the collection container 19 "corresponding to the third column used from the left.
About 2 μL of the droplet 72 was attached to the upper end of 9 ′ ″. It is considered that the liquid droplets collected from the third column used were scattered and scattered, and adhered to the adjacent collection tube. That is, in the conventional example, the recovered liquid also becomes a mist and scatters in the recovery tank,
There is a risk that it will adhere to the surrounding columns and collection containers and cause cross contamination.

On the other hand, when the recovery step was carried out using the column according to this example, no splash of the recovered liquid or scattering of mist around was confirmed. That is, in the present invention, the recovery liquid does not become mist or the like and scatters in the recovery tank even in the recovery step, and the risk of adhering to the surrounding column or recovery container or causing cross contamination is extremely high. It was confirmed to be low.

Next, the influence of the inclusion of a trace amount of the recovered liquid or the adsorption waste liquid in the conventional example on the final result after amplification by PCR was examined. First, the present invention No. 1
The lower limit of detection by PCR was examined when the genomic DNA extracted from human blood was used as a template in the above Example. The PCR conditions used are described in JP-A-11-341999.
The marker was ALDOB and PCR was 60 cycles. PC
The result of confirming the presence or absence of the R product by agarose gel electrophoresis is shown in FIG. In Fig. 15, 3 lanes marked with M are size markers, and A to G are series 7 in which the amount of genomic DNA template was changed by 1/10 from 100 ng to 0.1 pg, respectively.
Seed PCR product, H is the result of electrophoresis of the PCR product without template, and the vertical axis shows the DNA fragment length. As is clear from the figure, a PCR product equivalent to the literature value of 122 bp was confirmed when the template amount was 100 ng to 10 pg, but no PCR product was detected at 1 pg or less. In other words, PCR amplification was confirmed when a minimum of 10 pg of template was present under this PCR condition, and the lower limit of template detection was 10 pg. PC
When the number of R cycles was 50, the detection limit of the template was 87 pg.

Since the amount of the template used in the above is usually about 100 ng, the lower limit of detection of 10 pg or 87 pg corresponds to this ordinary amount of use of 1/10000 or 1/1000.
In other words, if the template is mixed in at least 10,000 or 1,000 times, the negative control will become a false positive,
In addition, the result of an originally negative sample also shows a false positive. 10p
Human genomic DNAs of g and 87 pg correspond to about 3 and 25 copies of the molecule, respectively. In other words, this detection method is an extremely high-sensitivity detection method capable of detecting a very small amount of genomic DNA of the order of several to several tens of tens, and it is necessary to avoid a small amount of contamination.

As described above, it was observed that about 2 μL of the collected liquid of the column used in the conventional example was deposited as a droplet on the upper end of the collecting container corresponding to the adjacent column. There is a risk that these droplets will drop into the recovery container and mix with the recovery liquid. The mixed amount of about 2 μL is about one-thousandth of the collected solution (2 mL). Therefore, when PCR is performed under these conditions, false positives and undesired P
It gives erroneous results such as CR products. On the other hand, in the present example, as described above, no scattering or mixing of the recovered liquid was confirmed. From the comparison result of the scattering state of droplets, it is considered that the risk rate of false positives due to the mixing of the collected liquid in the present invention is lower by at least 3 digits or more than the conventional example.

Next, a similar study was conducted on the influence of the admixture of the adsorption waste liquid. As a result, PCR products were obtained only from the adsorption waste liquid diluted to one thousandth. That is, it means that when the adsorption waste liquid is mixed in about 1 / 1,000, the negative control becomes a false positive, and the result of the originally negative sample also shows a false positive.

As described above, in the conventional example, the adsorption waste liquid of the used column was scattered and adhered to the neighboring columns, and about 1 μL remained in the lower hollow cylinders of the two adjacent columns even after the washing step. In the recovery process, when the liquid drops fall into the recovery container due to vibration or due to the splash of the recovery liquid and mix in the recovery liquid, false positives such as false positives and unintended PCR products are passed through PCR. Bring results. That is, the conventional column has a risk of giving an erroneous result due to cross-contamination due to the adsorption waste liquid. On the other hand, in this embodiment, as described above, since the scattering and mixing of the recovery liquid was prevented, this danger does not occur.

Another embodiment according to the present invention has a mist mixing prevention effect equivalent to or more than that of the first embodiment. For example, in Example 2, in addition to the effect of preventing mist mixing in Example 1, the particle transmittance is suppressed to 8 ppm or less by the effect of the filter alone. The latter level is about an order of magnitude lower than the level of false positives (1/10000) due to the same concentration of template, so that the risk of false positives can be virtually eliminated with the filter alone. In Examples 3 and 5, there is virtually no risk of mixing mist, and Example 4 is equivalent to Example 1. Therefore,
Each embodiment of the present invention has a mist mixing prevention effect equivalent to or greater than that of the first embodiment. Of course, the present invention is not limited to the scope of the embodiments shown here, and a technical element common to each embodiment is to separate mist or the like that may be generated from the discharge hole of a column from the discharge hole of another column. The present invention can be applied to other embodied modifications, improvements, and combinations thereof, and exhibits similar effects.

[0058]

As described above, according to the present invention, by providing means for isolating mist or the like that may be generated from the discharge hole of a solid phase extraction column from the discharge hole of another column, contamination can be prevented and clinical There is an effect that it is possible to provide an apparatus for extracting nucleic acids of high purity that is sufficient for highly accurate diagnosis.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a solid phase extraction column according to a first embodiment of the present invention.

FIG. 2 is a schematic configuration diagram of an automatic extraction device based on the first embodiment of the present invention.

FIG. 3 is a waste liquid chamber according to the first embodiment of the present invention.
21 and column 1 cross-section schematic.

FIG. 4 is a recovery chamber according to the first embodiment of the present invention.
22, a schematic cross-sectional view of the column 1 and the recovery container 19.

FIG. 5 is a flowchart showing an outline of a solid phase extraction procedure according to the first embodiment of the present invention.

FIG. 6 is a schematic configuration diagram of a solid-phase extraction column 1 ′ according to the second embodiment of the present invention.

FIG. 7 is a schematic diagram showing a mutual relationship between a column 1 ′ and a recovery container 19 in the recovery chamber according to the second embodiment of the present invention.

FIG. 8 is a schematic configuration diagram of a column 1 ″ according to a third embodiment of the present invention.

FIG. 9 is a schematic view of a cross section of a waste liquid chamber according to a third embodiment of the present invention.

FIG. 10 is a schematic configuration diagram of an automatic extraction device according to a fourth embodiment of the present invention.

FIG. 11 is a schematic configuration diagram of a main part of an automatic extraction device according to a fifth embodiment of the present invention.

FIG. 12 is a trace of a photograph taken at the end of the adsorption step when nucleic acid was extracted from whole blood using the column according to the first embodiment of the present invention.

FIG. 13 is a trace of a photograph taken at the end of the adsorption step when nucleic acid was extracted from whole blood using a column based on the conventional example.

FIG. 14 is a trace of a photograph of the state of the collection container and the collection tank after the first suction in the collection process when nucleic acid extraction from whole blood is performed using a column based on the conventional example.

FIG. 15 shows the results of agarose gel electrophoresis of PCR products when the amount of recovery solution was used as a template and the amount thereof was changed.

FIG. 16 is a view showing a conventional integrated column.

FIG. 17 is a view showing a conventional column.

[Explanation of symbols]

1, 1 ', 1 "... column, 2 ... carrier, 3 ... injection hole, 4
... discharge hole, 5 ... partition wall, 6 ... upper housing, 7 ... column holder, 8 ... waste liquid tank, 9 ... recovery tank, 10 ... suction hole, 11 ... cover, 12 ... waste liquid hole, 13 ... waste liquid tube, 14 ... Trap, 15, 15 '... Exhaust tube, 16, 16' ... Filter, 17, 17 '... Solenoid valve, 18 ... Vacuum pump, 1
9, 19 ', 19 ", 19'" ... recovery container, 20 ... automatic extraction device, 21 ... waste liquid chamber, 22 ... recovery chamber,
23 ... Filter, 24 ... Extension tube, 25 ... Dust collection mechanism, 26
... intake mechanism, 27 ... exhaust mechanism, 28 ... pressurization mechanism, 29 ...
Pressure chamber, 31 ... Decomposition step, 32 ... Additive solution mixing step, 33 ... Adsorption step, 34 ... Washing step, 35 ... Recovery step, 40 ... Sample, 41 ... Enzyme solution, 42 ... Decomposition solution, 4
3 ... Additive solution, 44 ... Washing liquid, 45 ... Eluent, 46 ... Decomposition liquid, 47 ... Adsorption solution, 48 ... Adsorption waste liquid, 49 ... Washing waste liquid, 50 ... Recovery liquid, 60, 60 '... Integrated holding chamber, 61 , 61 '... Flow path, 62 ... Charging mechanism, 63 ... Electrode, 64 ... Electromagnet, 65 ... Magnetic particle, 66 ... Integrated column, 67 ... Claw, 68 ... Stopper, 70, 70' ... Coloring, 71. .. Conventional column, 72 ... droplet, lower hollow cylinder… 10
0, hollow cone… 101, partition wall… 205, carrier body… 22
6, Compartment ... 228, Discharge hole ... 232, Carrier ... 23
Four.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Takeshi Sonehara             1-280, Higashi Koikekubo, Kokubunji, Tokyo             Central Research Laboratory, Hitachi, Ltd. (72) Inventor Takashi Irie             1-280, Higashi Koikekubo, Kokubunji, Tokyo             Central Research Laboratory, Hitachi, Ltd. F term (reference) 4B024 AA11 AA19 CA01 CA11 HA11                 4B029 AA23 BB20 CC01                 4B063 QA01 QQ41 QS12 QS14 QS39

Claims (15)

[Claims] 1. A tubular body, an injection hole provided in the tubular body for injecting a sample solution, an adsorption carrier provided in the tubular body for adsorbing nucleic acid, and provided in the tubular body. A nucleic acid separation column comprising: a discharge hole for discharging a liquid in a body; and a partition wall provided on the outer periphery of the discharge hole so as to cover the discharge hole. 2. The nucleic acid separation column according to claim 1, further comprising a filter provided so as to close the space between the outer circumference of the cylindrical body and the inner circumference of the partition wall. 3. The nucleic acid separation column according to claim 1, wherein the cylindrical body and the partition wall portion are integrally formed. 4. An extension cylinder is further provided between the discharge hole and the partition wall, and the extension cylinder is provided so as to project below a lower end of the partition wall. 1
The nucleic acid separation column described. 5. The nucleic acid separation column according to claim 4, wherein the extension cylinder is detachably attached to the cylinder. 6. The nucleic acid separation column according to claim 5, wherein a stopper is provided around the extension cylinder, and the stopper is detachably attached to the cylinder by the stopper. 7. A cylinder, an injection hole provided in the cylinder for injecting a sample solution, an adsorption carrier provided in the cylinder for adsorbing nucleic acid, and a cylinder provided in the cylinder. A nucleic acid separation column having a discharge hole for discharging the liquid in the body and a partition, the discharge hole being installed inside the partition, and a container for containing the liquid discharged from the discharge hole. A plurality of the nucleic acid separation columns are provided, and the storage container is provided for each of the plurality of nucleic acid separation columns. 8. The nucleic acid separating apparatus according to claim 7, wherein the upper end of the container is provided above the discharge hole. 9. A decompression device for accommodating the plurality of accommodating containers and having a chamber having a vent, and comprising a decompressor for reducing the pressure in the chamber to a negative pressure through the vent. Item 7. The nucleic acid separation device according to item 7. 10. The nucleic acid separation according to claim 7, further comprising: a pressurizing device that has a chamber for accommodating the injection holes of the plurality of cylindrical bodies and that makes the pressure in the chamber a positive pressure. apparatus. 11. The nucleic acid separating apparatus according to claim 9, wherein a check valve is provided between the chamber and the pressure reducing device. 12. The storage container is a container for collecting nucleic acid in the sample solution.
The nucleic acid separating apparatus as described. 13. The nucleic acid separation apparatus according to claim 7, wherein the storage container is a container for collecting the waste liquid remaining after the nucleic acid is adsorbed on the adsorption carrier from the sample solution. 14. A step of preparing a solution containing a nucleic acid, adsorbing the nucleic acid onto an adsorption carrier provided in a cylinder, and covering the discharge hole on the outer periphery of the discharge hole formed in the cylinder. And a step of ejecting the nucleic acid adsorbed on the adsorption carrier from the ejection hole and collecting the nucleic acid using a nucleic acid separation column having a partition wall provided in. 15. The nucleic acid extraction method according to claim 14, wherein there are a plurality of the nucleic acid separation columns, and different kinds of the nucleic acids are recovered for each of the nucleic acid separation columns.