JPH0242969A - Apparatus for treating biological specimen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a biological sample processing device, and particularly to an automated sample processing device capable of simultaneously and continuously processing a plurality of samples.

(Prior Art) When isolating and purifying a desired substance from a biological sample, various methods are employed depending on the type of the substance. For example, in the field of biotechnology, for the separation and purification of DNA from bacterial bodies and cells.

Boiling method, alkaline method, etc. are used.

According to these methods, it is possible to separate and purify DNA on a relatively large scale, and it is possible to obtain DNA in an amount of 100 μg or more. But these methods.

Both are manual processes that require complex processes and are difficult to automate. Because it is done manually, there are variations in the recovery rate and purity of ON, and there are also large disparities between individuals due to differences in technology. Furthermore, the DNA obtained by this method has
RNA, various proteins, polysaccharides, etc. are mixed in, so if you leave it as it is, it will be difficult to analyze the DNA sequence.

Various experiments such as purification of fragments from plasmid DNA cannot be performed correctly.

As a device that can automatically process biological samples and isolate and purify target substances with high purity.

Generally high performance liquid chromatography (HPLC).

It is widely used because of its high performance and excellent reproducibility. However, 2 For example, DNA from bacterial cells
When supplying a bacterial cell lysate as a sample to this IIPLc device for the purpose of separation, it is necessary to pre-treat the sample to prevent damage to the separation column due to large amounts of impurities in the sample (cell debris, etc.). be. Furthermore, operations such as column regeneration are complicated, and completely automatic separation and purification operations cannot be performed.

On the other hand, with advances in separation and analysis technology, methods are being developed to perform analysis with relatively high accuracy using simple column operations using mini-columns without using expensive equipment such as HPLC.What is a mini-column? .

It is a small open column made of plastic filled with a filler, and is commercially available as a disposable column. However, analysis using this mini column
All of this is done manually using a batch method. Therefore, when a plurality of samples are analyzed, a certain amount of each sample, washing liquid, eluent, etc. must be measured and then supplied in an orderly manner, which is complicated. It is desired to develop an apparatus that can automatically process biological samples using mini-columns and easily isolate and purify target components with high purity.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional problems.

The purpose is to provide a biological sample processing device that can simultaneously and continuously process multiple samples and isolate and purify a large amount of target components in the samples with simple operations. It is in. Another object of the present invention is to provide a biological sample processing device that can continuously process a relatively large amount of bacterial cells and easily isolate and purify DNA in the bacterial cells with high purity. It is about providing.

(Means for Solving the Problems) The biological sample processing device of the present invention is a column at least partially filled with an adsorbent.

a column connected to a column shaking means; a pretreatment sample tank for accommodating a pretreatment sample to be supplied to the column, the pretreatment sample tank being equipped with a filter for filtering the pretreatment sample; connected.

A medium storage container containing a column processing medium for performing column processing; Supply means A for supplying the pretreatment sample from the pretreatment sample tank to the column; Supply means A for supplying the pretreatment sample from the medium storage container to the column supply means B for supplying the medium; and control means for controlling the column shaking means and the supply means A and B, whereby the above object is achieved.

The biological sample processing device of the present invention is a column at least partially filled with an adsorbent.

A column connected to column shaking means; a tank for accommodating a pretreated sample to be supplied to the cough column or a sample to be pretreated, the pretreated sample or the sample pretreated in the tank; a pretreatment sample tank equipped with a filter for filtering the sample and connected to a pretreatment sample tank shaking means; a sample processing tank connected to the column and/or the pretreatment sample tank for pretreating the sample; A sample/medium storage container that individually accommodates a medium, a column processing medium for column processing, and/or a sample; a sample/medium storage container that individually stores a medium, a column processing medium for column processing, and/or a sample; transferring the sample processing medium and/or sample from the sample/media storage container to the pretreatment sample tank; Supply means C for supplying; supply means A for supplying the pretreated sample or the sample pretreated in the tank from the pretreated sample tank to the column; supply means B for supplying a column treatment medium; and control means for controlling the column shaking means, the pretreatment sample tank shaking means, and the supply means A, B and C; The above objectives are achieved.

For example, the biological sample processing apparatus of the present invention has nine examples.

As shown in FIG. 1, column 1. Pretreatment sample tank 2゜Multiple medium storage containers 31. Supply means A4. Supply means B5. and control means 7 for controlling supply means A and B. has.

For example, as shown in FIG. 1, the column 1 includes an adsorbent JilO filled in one space of the column,
and a reservoir part 11 consisting of a space formed in the other part of the column. A pretreatment sample and a column processing medium can be held in the reservoir portion 11 . Components in the sample are adsorbed in the adsorbent layer 10 . between this adsorbent layer 10 and the reservoir portion 11, and the adsorbent J
Filters 13 and 12 are installed between iiiO and the column end. At the bottom of the column 1 (meaning the end on the adsorbent layer side), an eluent outlet 14° protruding downward, a collection tube 15 connected to the outlet 14, and a valve 16 are arranged. ing. Column 1
A column stopper 17 capable of airtightly sealing the inside of the column 1 is attached to the upper end of the column 1, and a tube 18 and a valve 19 are also attached. The tube 18 and the valve 19 allow the medium remaining in the column 1 to be removed from the system when the medium is replaced, and the inside of the column 1 to be an open system. Tubes 41 and 51 are connected to the column 1 for supplying a pretreatment sample, a column processing medium, and the like.

Column 1 is held on a column holder 100.

The column holding stand 100 has a pair of holders 101 and 101 that hold the column 1 in a vertical state, and a fixing plate 102 with each holder 101 attached to one side thereof. This column holding stand 100 is connected to shaking means 8. The shaking means 8 is 1, for example, the fixing plate 10 of this column holding stand 100.
The rotary shaft 84 has a rotary shaft 84 with a rotary shaft 84 attached to the center thereof, and a support body 85 that rotatably supports both ends of the rotary shaft 84. In this column holder 100 and shaking means 8, each holder 101 is rotatably supported by a support 85 via a fixed plate 102 and a rotation shaft 84. therefore,
In this shaking means 8, the column 1 can be shaken (rotated) by rotating the three rotation shafts 84. The column I can rotate up to 1809 degrees around the rotation axis 84. By rotating the column l, it is possible to stir the pretreatment sample injected into a part of the reservoir, and by rotating it more than 90 degrees, it is possible to stir the column processing medium remaining in a part of the reservoir. can be quickly removed from the system.

The pretreatment sample tank 2 is a tank for accommodating a pretreatment sample, and includes a pretreatment container 20 and a filter 21.

The pretreatment sample tank 2 includes a tube 41 as shown in FIG.
(included in the supply means A4) to the column 1. A pretreatment container cap 22 that can airtightly seal the inside of the container is attached to the upper end of the pretreatment container 20, and a tube 23 and a valve 24 for making the inside of the container an open system are attached. The filter 21 is a cell shell or the like in a pretreated sample.

It has the function of filtering substances that cause clogging of the adsorbent in the column during column operation, and its shape and installation location are not particularly limited. here.

The filter 21 has a cylindrical shape with a bottom, and the pretreatment container 20
It is attached to the tip of a tube 41 that extends through the pretreatment container cap 22. In addition, for example, a spherical filter may be used. The pore size of the filter is 0
．． The range is 0.05-1iar, preferably 0.1-0.5am. If it is less than 0.05+n+a, clogging is likely to occur, and if it exceeds 1 mm, solids cannot be effectively removed. The material of the filter is not particularly limited, but metal, plastic, or sintered materials such as ceramics are suitable. In the later-described sample processing operation, if the filter 21 is not present, the solid components contained in the C2 pretreated sample will be dissolved and left in the molten solution. There is a risk of it getting mixed in. In the present invention,
Since a filter is provided in the pretreatment sample tank 2 outside the column, such contamination is avoided.

Each of the plurality of medium storage containers 31 accommodating the column processing medium is a tube 51 (included in the supply means B5).
is connected to the reservoir portion 11 of the column 1 via. These multiple medium storage containers.

For example, container 311.312.313.314.315
Each contains a cleaning solution, an eluent, a regeneration solution, etc. These solutions are column processing media for column processing of samples.

Supply means A4 is provided to supply the pretreated sample in the pretreated sample tank 2 to the column 1. This supply means A4 is composed of two, for example, a tube 41 and a pump 42. One end of the tube 41 is inserted through the column plug 17 of the column 1 and connected to the reservoir part 11, and the other end is inserted through the pretreatment sample tank 22 and opens into the pretreatment container 20. The supply means B5 is provided to supply the medium in the medium container 31 to the column 1. This supply means B5 includes one tube, for example, tubes 51 and 511-515. Pump 53.
and a valve 52. One end of the tube 51 is inserted through the column stopper 17 of the column 1 and connected to the reservoir part 11.
The other end is connected to the tube 51 via the valve 52.
1 to 515. Tubes 511-515
are connected to containers 311 to 315, respectively. The pump 53 is provided at any location on the tube 5. Inside the container, the tubes 511 to 5 are connected by the power of the pump.
15 and 51 and is fed from vessel 31 to column 1. The valve 52 is a switching valve for communicating a predetermined container among the plurality of containers 31 with the column 1 . The pump 53 does not need to be a high-grade pump such as that used in IIPLC, and may be a normal multiple tube pump. The valve 52 has a function of selectively communicating any one of the plurality of containers 31 connected to the column 1 via tubes 511 to 515 and 51 to the column depending on the purpose of sample processing. 2 normal for this valve.

A switching valve is used.

The control means 7 consists of a computer 9, for example, and the pump 42
．． 53. Valve 16.19.24.52. It is also connected to a column shaker 8 and a fraction collector 70, which will be described later. This control means 7 is provided to control pump flow rate adjustment, valve switching, shaking of column 1, fraction collector, etc. This control means 7 allows selective communication between the container and the column by the pump.
done automatically. For example, the control means made of a computer can control multiple or multiple control units based on information input in advance. Even when a plurality of samples are analyzed using a plurality of columns using a fraction collector, a desired collection container 700 (described later) can be selected by the control means 7.

FIG. 2 is similar to the device of FIG. 1, but differs in the connection state of supply means A and B. In the device shown in Figure 2,
One end of the tube 51 is not directly connected to the column 1, but is connected to the tube 41 (included in the supply means A) by a pump 42.
are connected via a valve 54 on the upstream side (pretreatment sample tank side). A portion of the tube 41 downstream of the valve 54 is shared by supply means A and B. By switching the valve 54, the pretreatment sample from the pretreatment sample tank 2 or the medium from the medium storage container 31 is transferred to the pump 4.
2 to column 1.

Another embodiment B of the apparatus of the present invention is shown in FIG.

In this apparatus, an untreated sample is processed in a pretreatment sample tank 2 and is automatically supplied to a column 1.

It is possible to perform column processing. The pretreatment sample tank 2 of this device is held on a pretreatment sample tank holding stand 200. The pretreatment sample tank holding stand 200 includes a pair of holders 201 and 201 that hold the pretreatment sample tank in a vertical state, and a fixing plate 202 with each holder attached to one side thereof. This pretreatment sample tank holding stand 200 is connected to the shaking means 9. The shaking means 9 is 9, for example, this pretreatment sample tank holding stand 2.
The rotating shaft 91 has a fixed plate 202 of No. 00 attached to the center thereof, and supports 92 rotatably support both ends of the rotating shaft 91. In the pretreatment sample tank holder 200 and the shaking means 9, each holder 201 is rotatably supported by a support 92 via a fixed plate 202 and a rotating shaft 91.

Therefore, in this shaking means 9, the pretreatment sample tank 2 can be shaken (rotated) by rotating the rotating shaft 91. This pretreatment sample tank 2 is supplied with a sample and a sample processing medium from a sample/medium container described later, and as the pretreatment sample tank rotates, the supplied sample and medium are stirred, and the sample is Pretreatment is performed.

Furthermore, the apparatus shown in FIG. 3 is provided with a sample processing medium for pre-processing the sample and/or a plurality of sample/medium containers 32 for individually accommodating the samples. For example, these plurality of containers 1.

321, 322.323.324.325 have samples respectively.

It contains a sample processing medium and, if necessary, a cleaning liquid. These samples and sample processing media.

The sample is supplied to the pretreatment sample tank 2 by the supply means C6.

The supply means C6 is one, for example, tubes 61 and 611.
615. It is composed of a pump 63 and a valve 62.

One end of the tube 61 is connected to the pretreatment container frame 2 of the pretreatment sample tank 2.
2 is inserted into the pretreatment container 20, and the other end is opened into the pretreatment container 20.

It is connected to tubes 611 to 615 via valve 62. Each of the tubes 611 to 615 is connected to the container 32.
1 to 325 are connected. The sample and sample processing medium in the container 32 are passed through the tubes 611 to 615 and 61 by the power of the pump 63 to the pretreatment sample tank 2.
supplied to The valve 62 is a switching valve for communicating a predetermined container among the plurality of containers 32 with the pretreatment sample tank 2 . As for the pump 63.

Similar to the pump 53 described above, for example, a multiple tube pump or the like is used.

In the apparatus shown in FIG. 3, the control means 7 is as follows.

Furthermore, it is connected to the above-mentioned shaking means 9 and supply means C6, and controls the flow rate of the pump and switches the valve.

Controls the shaking of the pretreatment sample tank 2, etc.

The apparatus of FIG. 4 is substantially similar to the apparatus of FIG. 3, but differs in the connection form of the supply means A, B, C and the container for containing the medium. In the apparatus shown in FIG.

One end of tube 51 is not directly connected to column l.

Pump 42 of tube 41 (included in supply means A)
is connected to the upstream side of the valve 54 via a valve 54.

In this device, the portion of the tube 41 downstream of the valve 54 is shared by supply means A and B. By switching the valve 54, the pretreated sample from the pretreated sample tank 2 or the medium from the medium container 31 is supplied to the column 1 via the pump 42.

In the apparatus shown in FIG. 5, a plurality of sample/medium storage containers 3 are provided in place of the medium storage containers 31 and sample/medium storage containers 32 shown in FIGS. 3 and 4.

This plurality of containers 32, for example, 33L 332.333.3
34 and 335 individually accommodate a sample, a sample processing medium (for example, a bacteriolytic agent), a washing liquid, an eluent, a regenerating liquid, and the like. These containers 3 are connected to the pretreatment sample tank 2 by supply means C. The supply means C is here a tube 601. 602.603゜604,6
05 and 610. Valve 600. and pump 63. The pump 63 is provided anywhere on the tube 610, and the tube 51 is connected between the pump 63 and the valve 600 via a valve 55.

By switching valves 600 and 55 accordingly.

For example, it is possible to supply the sample and sample processing medium in the containers 331 to 335 to the pretreatment sample tank 2; and to supply the column processing medium in the container into the column 1.

In the apparatus shown in FIG. 6, one end of the tube 51 shown in FIG.
2 via a valve 56. Valve 6
By manipulating 00 and 55.

Supplying the sample in container 3 and the sample processing medium to pretreatment sample tank 2 ; By operating valves 55 and 56, the pretreated sample is transferred from pretreatment sample tank 2 to column 1.
and valves 600° 55 and 56;
By switching the column processing medium in the container 3, it is possible to supply the column treatment medium to the column 1.

The apparatus of the present invention can also be configured to include a plurality of columns 1 and pretreatment sample tanks 2 and to be able to process a plurality of samples at the same time. FIG. 7 shows an example of an apparatus having a plurality of columns 1 and pretreatment sample vessels 2 and in which a fraction collector is arranged. In the apparatus of FIG. is connected to a plurality of pretreatment sample tanks 2 through a plurality of tubes 41.
is connected from the pretreatment sample tank 2 to a plurality of columns 1 via a pump 42. Tubes 610 and 41 are each connected by a plurality of tubes 51. Fraction collectors 70 are downstream of the plurality of columns 1, and the columns 1 and the fraction collectors 70 are connected to each other in a portion 7.
Ten collection containers 700 are arranged to correspond to each other. FIG. 8 shows a partial cross-sectional plan view of the plurality of pretreatment sample vessels 2 in FIG. 7.

(Hereinafter, blank spaces) The apparatus and method of the present invention are used for analyzing samples derived from various living organisms, isolating target components from the samples, and the like. Since the apparatus of the present invention has a pretreatment sample tank equipped with a filter, sample analysis, isolation, etc. can be performed effectively.

Here, the sample pretreatment step is a step of lysing cells such as microbial cells.

Refers to the protein dissolution process, extraction process, etc.

The apparatus of the present invention is suitably used for isolating DNA from a sample containing DNA, particularly from a sample containing cells such as bacterial cells.

In particular, plasmid DNA (10' Dalton order) from E. coli cells and even higher molecular weight DNA^ (108
Dalton order). The device can also be used for cells other than E. coli cells, such as eukaryotic cells.

In the above apparatus, the adsorbent and column processing medium (e.g., washing liquid, eluent) to be packed into the column used
The type of the device varies depending on the purpose of use of the device, and is selected as appropriate depending on the purpose.

The adsorbent for column 1 is 9, for example, ion exchange gel,
There are reversed phase gels and normal phase gels. Ion exchange gel has
DEAE Sepharose, QAIE Sepharose,
There are 0M Sepharose, SP Sepharose, etc.

Examples of the reversed phase gel include Cl11 silica gel and Fukonyl silica gel. Examples of normal phase gels include silica gel. Other adsorbents that can be used include:
Hydroxyapatite starch.

Contains cellulose and activated carbon. For example, when selectively adsorbing only double-stranded DNA (dsDNA) of a sample containing DNA, hydroxyapatite is preferably used.

Pretreatment solutions used for sample pretreatment (bacterial lysis, protein lysis, extraction, etc.) (used when pre-processing samples or as a sample treatment medium) include detergents, salts, lytic enzymes, alkaline agents, etc. .

Solutions containing chelating agents, chaotropic agents, urea, etc. may be used. The chelating agent or lytic enzyme may be one, e.g.
In the method of isolating DNA from cells.

Used to weaken cell walls during cell lysis.

Chelating agents include EDTA, 8-hydroxyquinoline, and the like. Harizozyme as a lytic enzyme.

Examples of proteinases include proteinases. Detergents are used to lyse cell walls weakened by lytic enzymes or chelating agents. For example, sodium dodecylbenzene sulfonate (SOS).

These include Triton X-100, Non1det, and lauroylsarcosine. For example, the concentration of SOS used is in the range of 0.1 to 2%, preferably in the range of 0.5 to 1%. Urea is usually used at a concentration of 2-8M. 8M urea corresponds to approximately the saturation concentration. When the sample to be treated is E. coli, the preferred combination of pretreatment liquids is: 0.
2M NaOH, 1% SO3, 50mM) Lis HC
I (pl+ 8.0), 10+wM EDTA, 1
mg/ml lysozyme. If the sample is eukaryotic cells, IMNaCl, 1% SOS, 50mM
) Lis-1 (C1 (pH 8,0), 10n+M
II! l0TA, 1 mg/m! It's proteinase.

9 as an eluent used as a column processing medium.
For example, a solvent can be used that has a strong interaction with at least one, preferably both, of the components (anions and cations) constituting the adsorbent.

For example, when isolating and purifying DNA using hydroxyapatite (the main component of which is calcium phosphate), a solvent that has a strong interaction with at least one, preferably both, of the phosphate component and the calcium component of the calcium phosphate, Especially.

Salt solutions capable of forming sparingly soluble salts may be used.

Among such salt solutions, those with a small dissociation constant and high affinity with nucleic acids such as DNA are preferred. Furthermore, a salt is selected that is soluble in water or alcoholic solvents and does not precipitate out by ethanol precipitation. For example, when hydroxyapatite is used as an adsorbent, carbonic acid; and organic acids such as formic acid, acetic acid, and propionic acid are suitable as the anion component of the salt used. Suitable cationic components include ammonia; alkylamines such as diethylamine and triethylamine; alkanolamines such as ethanolamine and propatoolamine; and aromatic amines such as pyridine and aniline. A triethylamine carbonate buffer is particularly suitable as an eluent containing a salt made of such components. Since triethylamine carbonate buffer is volatile,
By distilling off the solvent of the eluate containing DNA or freeze-drying it, it becomes possible to collect only the DNA in the mouth N with high purity.

The concentration of the eluent varies depending on the type of eluent used; for example, when triethylamine carbonate buffer is used, it is in the range of 10mM or more, preferably 50mM to 2M, and more preferably 100mM to LM.

In this way, even at low concentrations below 50mM, DNA
It is possible to elute

The cleaning liquid used for cleaning the adsorbent layer generally contains a chelating agent and a chaotropic agent.

These chelating agents and chaotropic agents include:

Any of the reagents used in the pretreatment solution may be used.

Processing of biological samples using the device of the present invention.

This will be explained using the apparatus shown in FIG. 1 and the apparatus shown in FIG. First, pretreatment of the sample, such as bacteriolysis and extraction, is performed outside the system, and the pretreated sample is placed in the pretreatment sample tank 2 of the apparatus shown in FIG.

The pretreated sample is supplied to the column 1 via supply means A4. The sample is then sent to the adsorbent layer 10, where desired components in the pretreated sample are effectively adsorbed onto the adsorbent. Next, if necessary, a cleaning liquid is supplied from the container 311 to the column 1 via the supply means B5 to perform cleaning. Further, the valve 52 is switched to supply the eluent from the container 312 to the column I. As a result, the target component adsorbed on the adsorbent layer IO is eluted. Should I introduce this to a suitable measuring device and measure it?

Collect the elution fractions to obtain the desired component. After elution.

When the column is regenerated and reused, the valve 52 is
The regenerating liquid is supplied to the column 1 from the container 313 by switching. When switching from a washing solution to an eluent, or from an eluent to a regeneration solution, or when changing the type of washing solution or eluent, the column 1 is rotated approximately 180 degrees by the column shaking means 8, and the medium in the column is transferred to the tube. Removal through 18 allows rapid exchange of the medium.

The apparatus shown in FIG. 6 is used when pretreatment of a sample is also performed. First, put a sample such as a bacterial suspension into container 3.
31 through tubes 601 and 610 to pump 6
3 to the pretreatment sample tank 2.

Next, the valve 600 is switched and a sample processing medium (for example, a lytic agent) is supplied from the container 332 to the pretreatment sample tank 2 . The sample in the pretreatment sample tank and the medium are mixed by the shaking means 9, and the sample is pretreated. Furthermore, the valve 600 is switched as necessary to supply another sample processing medium (for example, a neutralizing agent) from the container 333 to the pretreatment sample tank 2, and mix it by shaking.

The pre-treated sample prepared in this way was filtered through the filter 2.
1 and fed via tube 41 to column 1 by pump 42. In column 1, valve 6
By switching 00.55 and 56, container 3
34, 335, etc., into the column 1 via tubes 610.51.41.

By treating the eluate eluted from column 1 with appropriate means, the desired substance is isolated and purified.

For example, when DNA is isolated from bacterial cells using the above device, a suspension containing bacterial cells is used as a sample, and a lytic enzyme (lysozyme, proteinase, etc.) and a chelating agent (EDT) are used as a pretreatment liquid. Such).

A solution containing a detergent (such as sodium dodecyl sulfate) is used. By supplying such a pretreatment liquid, the bacterial cells are lysed in the pretreatment sample tank 2. In other words, the cell walls of bacterial cells are weakened by chelating agents and lytic enzymes, and lysed by detergents. The obtained lysate is supplied to the adsorbent layer of column I and comes into contact with the adsorbent of the adsorbent layer. Hydroxyapatite or the like is preferably used as the adsorbent.

For example, by passing a 0.15 to 0.25 M phosphate buffer as a solvent, ds DNA in the lysate is selectively adsorbed. RNA, proteins, polysaccharides, etc. in the lysate are discharged outside the column 1 without being adsorbed. next.

An eluent is supplied from the container 3 and brought into contact with the adsorbent that has adsorbed and supported the DNA. For example, adsorbing DNA
When triethylamine carbonate buffer is brought into contact with supported hydroxyapatite, carbonate ions combine with calcium in hydroxyapatite to produce poorly soluble calcium carbonate, while triethylamine forms a salt with the phosphate moiety of hydroxyapatite, resulting in , DNA is desorbed from hydroxyapatite. For example, by passing a triethylamine carbonate buffer in an amount twice the bed volume of a hydroxyapatite column, the adsorbed DN
Almost the entire amount of A can be recovered. Purified DNA can be obtained by subjecting the eluate thus collected to a conventional ethanol precipitation treatment.

Since triethylamine carbonate in the eluate is dissolved in ethanol, it does not mix with the DNA and reduce its purity. Triethylamine carbonate can also be volatilized and removed (desalination is achieved) by distilling off the solvent under reduced pressure or freeze-drying instead of ethanol precipitation, resulting in highly pure D.
NA is obtained.

The sample processing apparatus of the present invention is used not only for the isolation and purification of NA as described above, but also for the dissolution and extraction of proteins.

1 serving■ The invention will be explained below with reference to examples.

The apparatus shown in FIG. 6 and having a plurality of columns and pretreatment sample vessels (the number of columns and pretreatment sample vessels are 10 each) was used.

Plasmid DNA was isolated from E. coli as follows.

(A) Culture of bacterial cells Escherichia coli HB 101 containing plasmid pBR322
Stocks.

The cells were cultured overnight in 200mfLB medium. Add this culture solution to 6
The cells were centrifuged at 00 rpm for 10 minutes to precipitate the bacterial cells.

(B) Lysis of bacterial cells and isolation of plasmid DNA The above apparatus shown in FIG. 6 was prepared. Column 1 contains 8M urea-
Filled with hydroxyapatite equilibrated with 0.20M phosphate buffer (pH 6.8) (bed volume 5).
ml; diameter 2.5 c+s x 1 cm). As the filter 21 of the pretreatment sample tank 2, a metal fiber tube element F^03 (pore diameter 0, 4 mm,
Fuji Filter (manufactured by ■) was used. Among the sample/medium containers 3, the container 331 contains 0.2N NaOH-0,1
% Tween 80 (Tween 80 is manufactured by Hanui Chemical Co., Ltd.) aqueous solution; Container 332 contains 0.75M potassium acetate aqueous solution; Container 333 contains 8M urea-0.24
M phosphate buffer (pH 6,8); container 334 contains 1
It contained 0mM Tris-HCl buffer (p116.8). Tubes 601-6 connected to each container 331-335
05, a tube (not shown) for feeding air into the column 1 is provided.

One end thereof is connected to the valve 600, and the other end is open to the outside air.

The bacterial cells obtained in section (A) were suspended in G buffer solution 5d. Lysozyme was added to this suspension to give a final concentration of 2/d and maintained at 37°C for 15 minutes. to this,
For the purpose of measuring the isolation yield of plasmid DNA, 31 (
('H-pB
R322) was added in an amount equivalent to 1 μC4. This mixture was placed in the pretreatment container 20 of the above apparatus, and the pretreatment sample tank 22 was closed. Open valve 24.

Valves 55 and 600 were operated to bring container 331 into communication with pretreatment sample tank 2 . From the container 331,
A 0.2N NaOH-0.1% Tween 80 aqueous solution 10- was supplied to the pretreatment sample tank 2 by a pump 63. The valve 24 was closed, and the fixing plate 202 was rotated 10 times by the shaking device 9 to stir the mixture in the pretreatment sample tank 2. Further, the mixture was left at room temperature for 10 minutes to completely dissolve the bacterial cells. Next, valve 24 is opened and valve 600 is opened.
was switched so that the container 332 communicated with the pretreatment sample tank 2. 0.75M from container 332 by pump 63
30 d of potassium acetate aqueous solution was supplied to the pretreatment sample tank 2. The valve 24 was closed, and the fixing plate 202 was rotated 10 times by the shaking device 9 to stir the solution in the pretreatment sample tank 2.

Next, the valve 24 was opened, the valve 55 was switched so that the container 3 and the pretreatment sample tank 2 were not in communication with each other, and the valve 56 was also switched so that the pretreatment sample tank 2 and the column 1 were in communication with each other. After opening the valve 19 and closing the valve 16, the pretreated sample in the pretreated sample tank 2 was supplied to the column 1 via the tube 41 using the pump 42. The pretreated sample was filtered by filter 21 and introduced into the reservoir section of column 1. Next, valve 55.
54 and 600, column 1 is connected to tube 41.
51.610 and a tube (not shown) to open to the outside air. Valve 19 of column 1 is closed, valve 16 is opened, air is sent into column 1 by pump 42, and the pretreated sample in a portion of the reservoir is heated at 40 d/hr.
was introduced into the adsorbent layer 10 at a rate of . (This is the charging operation). As a result, plasmid DNA in the pretreated sample is adsorbed onto the adsorbent layer 10.

Next, operate the valve 600 so that the container 333 communicates with the column 1, and 0.24M of 8M urea in the container 333.
Add phosphate buffer (p) 16.8) to column 1 and pump 42
Supplied by. The buffer was supplied in 200 ml at a flow rate of 40 Inl/hr. As a result, proteins, RNA, etc. that were not adsorbed to the adsorbent were washed and removed.

Next, valve 16 of column 1 is closed, valve 19 is opened, and valve 600 is connected to column 1 through a tube (not shown).
The column was switched to communicate with outside air, and air was introduced into the column 1 using the pump 42. The column 1 was rotated about 180° by the shaking means 8, and the buffer solution remaining in the reservoir portion 11 was removed from the system through the tube 18. After stopping the pump 42, the column 1 was returned to its original position. (The above is cleaning operation 1).

Next, operate the valve 600 to bring the container 334 and column 1 into communication, close the valve 19 and open the valve 16, and then remove the 10mM Tris-HCl buffer (pH 6,8) in the container 334. It was supplied via pump 42 to column 1. The buffer was supplied for 15 d at a flow rate of 40 d/hr. Subsequently, the column was rotated in the same manner as described above, and the buffer solution remaining in the reservoir 11 was removed from the tube 18 to the outside of the system. (The above is cleaning operation 2).

After column 1 was returned to its original position, valve 600 was operated to bring container 335 and column 1 into communication. After closing the valve 19 and opening the valve 16, the pump 4
2, the 0.2M) ethylamine-carbonate buffer (pH 8,0) in the container 335 was supplied to the column 1. The buffer was supplied at a flow rate of 40 kg/hr. The first 5- of the eluate flowing out from the tube 15 was discarded, and the first 15- of the eluate was collected. (The above is the elution operation).

In the above steps, the switching and opening/closing operations of each valve, the operation of the pump, and the operation of the shaking means were all controlled by a computer serving as the control means 7.

In each of the above steps, the eluate eluted from the tube 15 of the column 1 was monitored by ultraviolet absorbance at 260 nm (UV absorption spectrum) and radioactivity (3u counts). The results are shown in FIG.

As is clear from FIG. 9, after the start of the elution operation, ultraviolet absorption and radioactivity increased, indicating that plasmid DNA was being eluted. The increase in UV absorption during charging and washing operations is primarily due to RNA and proteins that were not adsorbed to the adsorbent.

(C) Collection of plasmid DNA: After adding 1.5 d of 3M sodium acetate to approximately 15 ml of the eluate obtained in section (B), ethanol precipitation was performed with twice the amount of ethanol, and plasmid DNA (pBR32
2) Approximately 100 ug was collected.

(Effects of the Invention) In the apparatus of the present invention, the pretreatment sample tank and the sample/medium container are connected to the column via the supply means, and their functions are controlled by the control means. Scientific samples can be processed effectively. A filter is provided in the pre-treated sample tank, so the pre-treated sample is filtered and column clogging is avoided.

Column life is extended and the possibility of contamination of target components with impurities is reduced. Among the apparatuses of the present invention, in particular, an apparatus in which a sample/medium storage container is connected to a pretreatment sample tank and the pretreatment sample tank is connected to a shaking means is useful for pretreatment of a sample and extraction of predetermined components from the sample. Purification and isolation can be carried out continuously in a short period of time without requiring complicated operations.

By using an apparatus in which a plurality of columns and pretreatment sample tanks are arranged in parallel, it is possible to simultaneously and continuously process large quantities of a plurality of samples. This device is.

In particular, it is suitably used for isolation and purification of DNA from bacterial cells.

4. F of ゛! ■ Figures 1 to 7 are schematic diagrams showing examples of the biological sample processing apparatus of the present invention, and Figure 8 is a section showing the connection state near the pretreatment sample tank in the apparatus of Figure 7. The cross-sectional plan view and FIG.
It is a graph showing the relationship with H count).

1... Column, 2... Pretreatment sample tank, 3.32...
- Sample/medium storage container, 4... Supply means A, 5...
Supply means B.

6... Supply means C, 7... Control means, 8... Column shaking means, 9... Shaking means, 21... Filter, 31... Medium storage container, 70... Fraction collector , 100... column holding stand, 200...
Pretreatment sample tank holding stand.

that's all

Claims (1)

[Claims] 1. A column at least partially filled with an adsorbent and connected to column shaking means; A tank for accommodating a pretreated sample to be supplied to the column; a pretreatment sample tank equipped with a filter for filtering the pretreatment sample; a medium storage container connected to the column and containing a column processing medium for performing column treatment; supply means A for supplying the pretreatment sample to the column; supply means B for supplying the processing medium from the medium container to the column; and the column shaking means, and the supply means A and B. A biological sample processing device comprising: a control means for controlling; 2. A column at least partially filled with an adsorbent and connected to column shaking means; a tank for accommodating a pretreated sample to be supplied to the column or a sample to be pretreated; a pretreatment sample tank, which is equipped with a filter for filtering the pretreatment sample or the sample pretreated in the tank, and is connected to a pretreatment sample tank shaking means; the column and/or the pretreatment sample tank; A sample/medium storage container that is connected to a processing sample tank and individually accommodates a sample processing medium for sample pretreatment, a column processing medium for column processing, and/or a sample; supply means C for supplying the sample processing medium and/or sample from the container to the pretreatment sample tank;
; Supply means A for supplying the pretreated sample or the sample pretreated in the tank from the pretreated sample tank to the column; Supplying the column processing medium from the sample/medium storage container to the column A device for processing a biological sample, comprising: a supply means B for controlling the column shaking means, the pretreatment sample tank shaking means, and a control means for controlling the supply means A, B, and C.