JPS5997056A - Reagent supported porous substrate and instrument for preparing the same - Google Patents

Abstract

PURPOSE:To easily obtain a carrier, especially, for a nucleotide reagent or condensing agent for the synthesis of nucleic acid, by using a specific preparing intrument obtained by freeze drying a porous substrate impregnated with a predetermined amount of a solid reagent solution in predetermined concn. to make the dried impregnated substrate preservable in a hermetically closed container. CONSTITUTION:A columnar body 35 comprising a porous filter substrate made of polyethylene, polypropylene, ceramics, glass or cotton is put in a hermetically closable tool 30 used in preparing a reagent supported porous substrate comprising a container 34 consisting of an upper lid 31, a container main body 32 and a lower lid 33 and the main body 32 thereof is immersed in a solution of a nucleotide reagent or condensing agent for DNA or RNA synthesis while a syringe S is used to impregnate the porous substrate inserted into the main body with the solution L. In the next step, the lower lid 33 is applied to the main body 32 to freeze the solution L in a cryogen and, after freeze-drying, the upper lid 31 is applied to the main body 32 to make the reagent stably preservable. In use, the upper and the lower lids are detached to add a solvent and the definite amount of the reagent can be supplied to a reactor.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent-supporting porous substrate supporting a fixed amount of a solid reagent, and an apparatus for producing the same.

Some reagent solutions are unstable. For example, among the reagent solutions used in DNA synthesis, the condensing agent solution used in the phosphotriester method, the nucleotide reagent solution used in the phosphomonotriazolide method, and the nucleotide reagent solution used in the bosphite method are unstable and cannot be prepared within a few hours. It must be used. That is, these reagents cannot be stored in the form of a solution, and must be prepared by dissolving the solid reagent in a solvent each time they are used.

However, the preparation requires three steps: (1) quantitative determination of the solid reagent, (i) quantitative determination of the solvent, and (iii) dissolution, which is time-consuming.

This invention improves li) quantitative determination of solid reagents and (iii) dissolution of the above three operations, and also improves the storage of solid reagents.

That is, in one aspect, the present invention is capable of dissolving a solid reagent in a solvent at a predetermined TEA degree, impregnating a porous base material with a predetermined amount of the solution, and removing the solvent by freeze-drying. A reagent pretreatment method for supporting a fixed amount of a reagent is provided, and a reagent-supporting porous substrate obtained thereby is provided.

The porous base material is preferably one that maintains overall integrity and has little variation in porosity. A specific example is a polyethylene filter.

Examples include polypropylene filters, ceramic filters, glass filters, and cotton.

As the solid reagent, a drug that remains as a solid upon lyophilization and is stable can be used. Specific examples include 2-4-6-drimethylbenzenesulfonyl-3-nitrotriazolide (MSIJT), which is a condensing agent;
Examples include nucleotide derivatives used in the phosphomonotriazolide method and nucleotide derivatives used in the bosphite method.

As the above-mentioned solvent, a solvent that can dissolve the solid reagent and that sublimates during freeze-drying can be used.

Specific examples include pyridine and dioxane.

In another aspect, the present invention provides a device for manufacturing the above-mentioned reagent-supporting porous substrate. That is, an upper lid having a truncated conical convex portion on the bottom surface, a recessed portion on the upper surface into which the convex portion of the upper lid is airtightly fitted, and a convex portion similar to the convex portion of the upper lid on the bottom surface. A container body further includes a through hole bored from the center of the recess on the top surface to the center of the convex part on the bottom surface, and a recess in which the bottom convex part of the container body is airtightly fitted. A device for producing a reagent-supporting porous substrate is provided, which includes a container having a lower lid, and a porous substrate held in a through hole of the container.

By immersing the convex portion on the lower surface of the container body of the above-mentioned reagent-supporting porous substrate manufacturing device in a solution in which a solid reagent is dissolved at a predetermined concentration, and connecting a syringe to the concave portion on the upper surface of the container body, the solution is sucked up with the syringe. , the porous substrate is impregnated with the solution. Then, by freeze-drying the entire container body, a porous base material supporting a fixed amount of solid reagent can be obtained. If this container body is sealed with an upper lid and a lower lid, the reagent-carrying porous substrate can be suitably stored.

When in use, by extruding the reagent-supporting porous base material from the container body and flowing a predetermined amount of solvent through the porous base material, the supported solid reagent is eluted and a reagent solution follows.

Such a reagent-supporting porous substrate can be handled as a single object, which is more convenient than handling the solid reagent itself. Particularly when the solid reagent is in the form of powder or in a small amount, it has the advantage of preventing dissipation. Furthermore, the solid reagent is distributed over a wide area by being supported on the porous base material, and the solvent flows randomly within the porous base material, resulting in significantly improved solubility. There are also advantages.

The embodiment shown in the figures will be described below. However, this invention is not limited thereby.

1 shown in FIG. 1 is an example of the device for producing a reagent-supporting porous substrate of the present invention, and the upper lid (311, container body c
Sealable container (to) consisting of lz and lower lid (2)
and a polyethylene filter (35).

The upper lid (31) has a truncated cone-shaped convex part (36j), and it is preferable that the taper of this convex part (36) matches the taper of the tip of the suction syringe (S), for example, 1/l.
It has an O taper.

The container body C32) has a recess into which the protrusion (36) is airtightly fitted and a protrusion (support) similar to the protrusion □□□, and a through hole (3 (T)) is bored in the center. It is preferable that the size of the through hole (39) is such that the filter (2) can be inserted therein, but that it will not fall out naturally.

The lower lid ■ has a recess (
40) is provided.

The polyethylene filter (to) has an outer diameter of approximately 5 mm.

It is a cylindrical body with a length of about 7 mm, a pore diameter of 10 μm, and a porosity of 40%.

Next, with reference to FIG. 2, the production of a reagent-supporting porous substrate using the above-mentioned production apparatus (1) will be explained.

For example, 2-4-6~trimethylbenzenesulfonyl-
Dissolve 3-nitrotriazolide (MSNTJ) in dioxane at a concentration of 1 mol/J7. ) and suck the solution (L) with the suction syringe [S] fitted in the recess (9). As a result, the porous substrate (G) is impregnated with about 55 μl of the solution.Second
Figure (a) shows this chemical impregnation step.

After the porous base material (G) is impregnated with the solution (L), a lower lid (33) is fitted into the container body 0, and the porous base material (G) is immersed in a cryogen (F) such as methanol/dry ice. If methanol/dry ice is used, a low temperature of about 170° C. can be obtained, and since the freezing point of dioxane is 12° C., the solution (L) is thereby frozen in the porous substrate (1). FIG. 2(b) shows this freezing process.

The frozen porous substrate containing the impregnated solution (L) is placed in the container body @ and placed in the vacuum container (7) with the lower lid attached. Since the dioxane is sublimated under vacuum, only the dried and solid MSNTs remain in the porous substrate □□□. The amount is approximately 55μm
It is ol. FIG. 2 (Q) shows this drying process.

As described above, approximately 55 μmol of MSN in dry state was obtained.
A reagent-supporting porous substrate (41) supporting T is obtained.

This reagent-supporting porous substrate (411) is placed in the container main body, attached with a lower lid (2), and furthermore with an upper lid (2), and stored in a sealed state.

(1) shown in FIG. 3 is an example of an automatic DNA synthesis device using a reagent-supporting porous substrate (41) (41') (41"λ...) of the present invention. DNA synthesis The method is the phosphotriester method.

The reactor (2) is a container having a cylindrical main body (3) with an inner diameter of 8 mm and a height of 1Q mm, and a mortar-shaped flange (4) provided above. A plug (5) into which a number of nozzles for supplying reagent solutions and the like are inserted is attached to the dome-shaped seven flange (4). Therefore, the head opening of the main body (3) becomes a reagent solution supply port (6). A filter (7) such as a glass filter is fitted inside the main body (3), and a drain port (8) is provided at the bottom. The filter (7) is one on which a support (9) such as polystyrene or silica beads can be placed (not permeable), and allows reagent solutions, solvents,
It allows gas to pass through. The space above the filter (7) becomes the reaction chamber, which has a volume of about 450 μl.

En～(13 is a solvent, pyridine is used as a reaction solvent, and tetrahydrofuran (THFJ is used as a drying solvent).
, a mixture of Improper Tool and methylene chloride was used as a cleaning solvent.

C14) is a test solution for removing protective groups, and is a solution in which zinc bromide is dissolved in a mixed solvent of inpropatol and methylene chloride. Q51 is a masking reagent, which is a mixture of acetic anhydride and pyridine. (10 is a masking condensing agent, which is a mixed solution of dimethylaminopyridine and pyridine.

The above solvent (11) ~ αJ and reagent solution α4) ~ 06)
are supplied to the reactor (2) through the valves (1η~ (Company)) by nitrogen gas pressure. The valve @ is a valve that directly supplies nitrogen gas into the reactor (2), and (to) is a drain valve, and (c) is an exhaust valve.These valves (17+-i are
Its operation is controlled by a control circuit such as a microcomputer. Note that the nitrogen gas is dried with a desiccant such as calcium chloride.

An operator may interact with the control circuitry via console 661.

The reagent-supporting porous substrate (41) contains approximately 55 μmo of MSNTs.
10,000, reagent-supported porous substrate (41 pieces)
41') ... is about 25 μm of nucleotide reagent
It carries ol. MSNT-supported porous group +, ttat
+ and nucleotide reagent-supporting porous substrate (41') or (
Pyridine (111) can be made to flow to any pair by switching the valve 1291 (29'J).The nucleotide reagent is For example, in the case of monomers, there are four types depending on the base.Each porous substrate (41 (
The bases of the nucleotide reagent of 41"..." are matched in order to the base sequence of the target DNA.

Reagent-supporting porous substrate (41+ (41') (41")
... may be set to the device (1) at any time less than mJ from the time when DNA formation is actually started. This is because both are set in a crystalline state and are therefore not unstable.

When synthesizing DNA, D is placed in the reactor (2) in advance.
A support (9) to which only the terminal portion of NA is bound is placed.

For example, when the support (9) is polystyrene powder and the supported amounts of MSNT and nucleotide reagent are about 55 μmol and about 25 μmol, respectively, the IT of the support (9) is about 5.
0 mg is appropriate.

The basic operation of this device (1) is basically the same as that of a known device of this type using the phosphotriester method, so the general explanation will only be given as the fourth factor, flow, and the characteristic synthesis Only the operation of the process will be explained in detail.

In the synthesis process, the control circuit (55) drives the motor to rotate the valve w (29'), and also opens the valve (5) and the valve to transfer pyridine (Ill) into the reagent-supporting porous hole. The pyridine av dissolves the nucleotide reagent and the condensing agent MSNT,
The reagent solution is supplied to the reactor (2).

This causes a condensation reaction in the reactor (2), and a new nucleotide is linked to the terminal portion of the DNA.

Thereafter, by switching the valve (5) (29) and continuing synthesis in the same manner, the target DNA can be synthesized.

According to the DNA microautomatic synthesizer (1) of the above embodiment, MSNT as a condensing agent is stocked in a stable crystalline state, and is turned into an unstable solution state immediately before use. Therefore, even if DNA synthesis is started at any time, stable synthesis is ensured, which is very convenient.In other words, the operator does not have to prepare a reagent solution every time DNA synthesis is started, which greatly reduces maintenance. becomes easier.

In addition, in the above-mentioned apparatus (1), ``reaction part 0 of reactor (2)
The reactor (2) is miniaturized, and the support (9) is placed on the filter (7), and the reagent solution (σ1) to 05) is supplied from above and drained from the bottom. ) to Mlf
It's 55 years old. Therefore, if the reagent Z solution is supplied from above with the drain valve (241 closed), the reagent solution will flow to the support (9
), it swells and remains in the reaction section (101) above the filter (7) and does not fall downward. Therefore, all the supplied reagent solution participates in the reaction, and what accumulates in the dead space is As a result, the minimum amount of supply (approximately 5 to 7 times the volume of the support) is sufficient, and mixing and contacting operations such as changing the reactor to promote the reaction are no longer necessary. In addition, before the reaction to link a new nucleotide, the inside of the reactor (2) is washed and dried with a drying solvent such as THIP (121) and blown with dry gas to dry the inside of the reactor (2) in a short time. It is configured to allow complete drying, and as a result, water that inhibits the condensation reaction can be completely removed, so the reaction efficiency does not decrease and no extra reagents are required.

Other examples include those in which the present invention is applied to a DNA synthesis apparatus using the phosphomonotriazolide method, the yaphosphite method, or the diester method.

When applied to the phosphomonotriazolide method, the above method @(
To explain based on 1), reagent-supporting porous substrate (4
For the compound (41''J...), a nucleotide derivative of formula (i) in a crystalline state is used.

[Basθ (base) +1 adenine, guanine, cytosine or thymine] A reagent-supporting porous substrate (not used. In addition, the valve (2) is not connected to pyridine a1), and the phosphorylation reagent solution is supplied via a metering pump. For example, connect to a tank containing O-chlorophenyl phosphoroditriazolide. When synthesizing, (
0-chlorophenylphosphoroditriazolides 9 to 10 are introduced into the nucleotide derivative 1o of the iJ formula, and the resulting reagent solution is supplied to the reactor (2).

A nucleotide reagent solution containing 0-chlorophenylphosphoroditriazolide and a nonucleotide derivative of formula (i) is unstable, but a nucleotide reagent solution of formula (i) and O-chlorophenylphosphoroditriazolide alone is unstable. Since it is stable, the desired effect can be obtained.

To similarly explain the case where the method is applied to the phosphite method, a reagent-supporting porous substrate (41'J (41'')...) supporting a nucleotide derivative of the [u] formula is used.

P-0-aH3(i)! [Ease (base) is adenine, guanine, cytosine, or thymine] The reagent-supporting porous substrate (41) is not used. Also, the valve surface is not connected to the pyridine α but connected to the TuF03t.

This allows unstable nucleotide reagent solutions to be prepared and used immediately.

As understood from the above explanation, according to the present invention,
The reagent can be handled in a dry state supported on the porous substrate, making it easier to handle even a small amount of the reagent. Furthermore, since the reagent is distributed over a wide area by being supported on the porous substrate, the solubility is the same as above. Furthermore, it is convenient because the amount of reagent can be increased to some extent by selecting the number of reagent-supporting porous substrates to be used. It is preferable if the porous substrate can be cut with a cutter or the like, since this allows the amount of reagent to be adjusted by adjusting the length of the reagent-supporting porous substrate.

[Brief explanation of drawings]

FIG. 1 is an exploded perspective view of an embodiment of the reagent-supporting porous base material manufacturing device of the present invention. However, the container body is a partially sectional view. FIGS. 2(a) to 2(a) are diagrams showing the steps of manufacturing the reagent-supporting porous substrate of the present invention. FIG. 3 is an explanatory diagram of the configuration of an example of an automatic micro-synthesizer using the reagent-supporting porous substrate of the present invention, and FIG. 4 is a flowchart of its operation. Rigid... Instrument for producing reagent-supporting porous substrate, (31)...
Upper cover, G2)...container body, mortar)...lower cover, (3ri...container, ■...porous base material, C36+ C(81...convex part, (371+40)
)...Recess, (39)...Through hole, (41)
...Reagent-supporting porous base material. -Figure 7Figure 2 (G) (b) (C) (d)↑

Claims (1)

[Scope of Claims] 1. A reagent-supporting porous substrate obtained by impregnating a porous substrate with a predetermined amount of a solid reagent solution at a predetermined concentration and freeze-drying the impregnated porous substrate. 2. The reagent-supporting porous substrate according to claim 1, wherein the solid reagent is a nucleotide reagent or a condensing agent for DNA or RNA synthesis. 3. The drug-supporting pore according to any one of claims 1 to 2, wherein the porous substrate is any one of a polyethylene filter, a polypropylene filter, a ceramic filter, a glass filter, and cotton. sexual base material. 4. An upper lid having a cone-shaped convex portion on the bottom surface, a concave portion on the upper surface into which the convex portion of the upper lid is airtightly fitted, and a convex portion similar to the convex portion of the upper lid on the bottom surface. A container body further includes a through hole bored from the center of the recess on the top surface to the center of the convex part on the bottom surface, and a recess in which the bottom convex part of the container body is airtightly fitted. It consists of a container consisting of a lower lid held in a container, and a porous base material held in a through hole of the container, and the porous base material is impregnated with a predetermined amount of a solid reagent solution at a predetermined concentration and freeze-dried. A device for producing a certain reagent-supporting porous substrate. 5. The device for producing a reagent-supporting porous substrate according to claim 4, wherein the porous substrate is any one of a polyethylene filter, a polyproblene filter, a ceramic filter, a glass filter, and cotton.