JPH056148B2 - - Google Patents

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 phosphite method are unstable and must be used within several hours after preparation. It is something that must be done. 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 (a) quantification of solid reagents,
Three operations are required: () quantitative determination of the solvent, and () dissolution, which is time-consuming.

This invention improves () quantitative determination and () dissolution of solid reagents among the above three operations, and also improves 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 concentration, impregnating a porous substrate with the solution in a predetermined amount, and removing the solvent by freeze-drying, thereby dissolving the solid reagent into the porous substrate in a fixed amount. The present invention provides a reagent pretreatment method for supporting a reagent, and also provides a reagent-supporting porous substrate obtained thereby.

The porous base material is preferably one that maintains overall integrity and has little variation in porosity. Specific examples include polyethylene filters, polypropylene filters, ceramic filters, glass filters, cotton, and the like.

As the solid reagent, a drug that remains as a solid upon lyophilization and is stable can be used. As a specific example, 2-4-6- which is a condensing agent
Examples include trimethylbenzenesulfonyl-3-nitrotriazolide (MSNT), nucleotide derivatives used in the phosphomonotriazolide method, and nucleotide derivatives used in the phosphite 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 lower 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 is obtained.

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 powder form 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.

Reference numeral 30 shown in FIG.
1. A sealable container 34 consisting of a container body 32 and a lower lid 33, and a polyethylene filter 35
It is made up of.

The upper lid 31 has a truncated conical convex portion 36, and the taper of the convex portion 36 is preferably set to match the taper of the tip of the suction syringe S, for example, 1/10 taper.

The container body 32 includes a recess 37 into which the projection 36 is hermetically fitted, and a projection 3 similar to the projection 36.
8, and a through hole 39 is bored in the center.
It is preferable that the size of the through hole 39 is such that the filter 35 can be inserted therein, and that it will not fall out naturally.

The lower lid 33 is provided with a recess 40 into which the projection 38 can be fitted airtight.

The polyethylene filter 35 has an outer diameter of approximately 5 mm.
It is a cylindrical body with a length of about 7 mm, a pore diameter of about 10 μm, and a porosity of about 40%.

Next, with reference to FIG. 2, manufacturing of a reagent-supporting porous substrate using the manufacturing device 30 will be described.

For example, 2-4-6-trimethylbenzenesulfonyl-3-nitrotriazolide (MSNT) is dissolved in dioxane at a concentration of 1 mol/l. The convex part 38 of the container body 32 holding the porous base material 35 is immersed in the solution L, and the solution L is sucked by the suction syringe S fitted in the concave part 37 . This results in porous substrate 35 being impregnated with approximately 55 μl of solution. FIG. 2a shows this chemical impregnation step.

After the porous base material 35 is impregnated with the solution L, the lower lid 33 is fitted into the container body 32, and the porous base material 35 is immersed in a cold agent F such as methanol/dry ice. -70℃ using methanol/dry ice
On the other hand, since the freezing point of dioxane is 12° C., the solution L is thereby frozen in the porous substrate 35. Figure 2b shows this freezing process.

Porous substrate 35 frozen with impregnated solution L
is placed in the container body 32 and placed in the vacuum container V with the lower lid 33 still attached. Since the dioxane is sublimated under vacuum, only the dried and solid MSNTs remain in the porous substrate 35. Its amount is approximately 55 μmol. Figure 2c
shows this drying process.

As described above, about 55 μmol of dry
A reagent-supporting porous substrate 41 supporting MSNT is obtained. This reagent-carrying porous substrate 41 is placed in a container body 32, fitted with a lower lid 33, and further fitted with an upper lid 31, and stored in a sealed state.

1 shown in FIG. 3 is an example of an automatic DNA microsynthesis apparatus using reagent-supporting porous substrates 41, 41', 41'', etc. of the present invention. The DNA synthesis method is a 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 10 mm, and a mortar-shaped flange 4 provided above. The dome-like flange 4 is fitted with a stopper 5 into which a number of nozzles for supplying reagent solutions and the like are inserted. Therefore, the head opening of the main body 3 becomes the 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. Filter 7
The support member 9, such as polystyrene or silica beads, can be placed thereon (not permeable), and is permeable to reagent solutions, solvents, and gases. The space above the filter 7 becomes the reaction section 10, which has a volume of about 450 μl.

Solvents 11 to 13 are pyridine as a reaction solvent, tetrahydrofuran (THF) as a drying solvent, and a mixture of isopropanol and methylene chloride as a washing solvent.

14 is a protecting group removal reagent, which is a solution of zinc bromide dissolved in a mixed solvent of isopropanol and methylene chloride. 15 is a masking reagent, which is a mixed solution of acetic anhydride and pyridine. 16 is a condensing agent for masking, which is a mixed solution of dimethylaminopyridine and pyridine.

The above solvents 11 to 13 and reagent solutions 14 to 16
can be supplied to the reactor 2 via valves 17 to 22, respectively, by means of nitrogen gas pressure. Valve 23 is a valve that directly supplies nitrogen gas into reactor 2, 24 is a drain valve, and 25 is an exhaust valve. These valves 17~
The operation of 25 is controlled by a control circuit 55 such as a microcomputer. Note that the nitrogen gas is dried with a desiccant 26 such as calcium chloride.

The operator controls the control circuit 5 via the console 56.
Can have a dialogue with 5.

The reagent-supporting porous substrate 41 contains approximately 55 μmol of MSNT.
On the other hand, the reagent-supporting porous substrate 41',
41'' carries about 25 μmol of a nucleotide reagent. The MSNT-supported porous substrate 41 and the nucleotide reagent-supported porous substrate 41' or 41'' or... are paired, and multiple pairs of them are arranged in parallel. There is. By switching the valves 29 and 29', pyridine 11
can be distributed to any pair. For example, there are four types of nucleotide reagents in the case of monomers, depending on the base. Each porous base material 41', 4
The bases of the nucleotide reagent 1″...
It matches the sequence of DNA base sequences.

The reagent-supporting porous substrates 41, 41', 41''... may be set in the apparatus 1 at any time before DNA synthesis is actually started. This is because all of them are in a crystalline state. This is because it is not unstable since it is set.

When synthesizing DNA, a support 9 to which only the terminal portions of DNA are bound is placed in the reactor 2 in advance. The amount of support 9 is, for example, when support 9 is polystyrene powder and the supported amounts of MSNT and nucleotide reagent are about 55 μmol and about 25 μmol, respectively.
Approximately 50mg 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 be given only by showing the flow in Figure 4, and the characteristic synthesis steps will be explained. Only the operation will be explained in detail.

In the synthesis process, the control circuit 55 drives the motor 54 to rotate the valves 29, 29', and also rotates the valve 29, 29'.
7 and valve 28 are opened to allow pyridine 11 to flow through the reagent-supporting porous substrates 41' and 41. Pyridine 11 is a nucleotide reagent and condensing agent.
The MSNTs are dissolved and turned into a reagent solution, which is then supplied to the reactor 2.

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

Thereafter, by switching the valves 29 and 29' and continuing synthesis in the same manner, the target DNA can be synthesized.

According to the automatic DNA microsynthesizer 1 of the above embodiment, the condensing agent MSNT is stored 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, making maintenance much easier.

In addition, in the above-mentioned apparatus 1, the reaction section 10 of the reactor 2
The support body 9 is placed on the filter 7.
The reactor 2 is configured so that the reagent solutions 11 to 15 are supplied from above and drained from the bottom. Therefore, if a reagent solution is supplied from above with the drain valve 24 closed, the reagent solution will be contained in the support 9 and swell it, and will remain in the reaction section 10 above the filter 7 and will not fall downward. . Therefore, all the supplied reagent solutions participate in the reaction,
There is no more stuff that accumulates in dead spaces. As a result, the supply amount is the minimum amount (approximately 5 to 7 times the volume of the support)
is now sufficient, and mixing and contacting operations such as shaking the reactor to promote the reaction are no longer necessary. In addition, before the reaction for linking new nucleotides, the interior of the reactor 2 is filled with a drying solvent such as THF1.
The structure is such that the inside of the reactor 2 can be completely dried in a short time by washing and drying in step 2 and blowing with dry gas.As a result, water that inhibits the condensation reaction can be completely removed, so the reaction efficiency does not decrease. , does not require extra reagents.

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

To explain the case of application to the phosphomonotriazolide method based on the above-mentioned device 1, the reagent-supporting porous substrates 41', 41''... support a nucleotide derivative of the formula () in a crystalline state. use

[Base is adenine, guanine, cytosine, or thymine] The reagent-supporting porous substrate 41 is not used. Also, valve 2
7 is not connected to pyridine 11, but is connected via a metering pump to a tank containing a phosphorylation reagent solution, such as o-chlorophenylphosphoroditriazolide. During the synthesis, o-chlorophenyl phosphoroditriazolides 9 to 10 are introduced into the nucleotide derivative 10 of the formula (), and the resulting reagent solution is supplied to the reactor 2.

A nucleotide reagent solution containing o-chlorophenyl phosphoroditriazolide and the nucleotide derivative of formula () is unstable, but the nucleotide derivative of formula () and o-chlorophenyl phosphoroditriazolide are each alone. Since it is stable, the desired effect can be obtained.

Similarly, when applied to the phosphite method, the reagent-supporting porous substrates 41', 41''...
A nucleotide derivative of the formula () is used.

[Base is adenine, guanine, cytosine, or thymine] The reagent-supporting porous substrate 41 is not used. Also, valve 2
7 is not connected to pyridine 11, but connected to THF12.

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

As can be understood from the above description, according to the present invention, it becomes possible to handle a reagent in a dry state supported on a porous base material, and it becomes easy to handle even a small amount of the reagent. In addition, because the reagent is supported on a porous substrate and distributed over a wide area,
Improves solubility. 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. In addition, if the porous base material can be cut with a cutter or the like,
This is preferable because the amount of reagent can 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. 2a to 2d 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 DNA synthesis device using a reagent-supporting porous substrate of the present invention, and FIG. 4 is a flowchart of its operation. 30... Instrument for producing reagent-supporting porous substrate, 31
...Top lid, 32...Container body, 33...Bottom lid, 34...Container, 35...Porous base material, 36,
38... Convex portion, 37, 40... Concave portion, 39... Through hole, 41... Reagent-supporting porous base material.

Claims (1)

[Claims] 1. A cylindrical porous base material in a predetermined amount at a predetermined concentration.
A reagent-supporting porous substrate impregnated with a nucleotide reagent solution or a condensing agent solution for DNA or RNA synthesis and freeze-dried. 2. The reagent-supporting porous substrate according to claim 1, wherein the cylindrical porous substrate is any one of a polyethylene filter, a polypropylene filter, a ceramic filter, and a glass filter. 3. An upper lid having a truncated 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, a porous base material held in a through hole of the container, and a suction syringe having a protrusion at its tip that fits into a recess in the container body, and the porous base material A device for producing a reagent-supporting porous substrate, wherein the material is impregnated with a predetermined amount of a nucleotide reagent solution or condensing agent solution for DNA or RNA synthesis at a predetermined concentration and freeze-dried. 4 The porous base is a polyethylene filter, a polypropylene filter, a ceramic filter,
The instrument for producing a reagent-supporting porous substrate according to claim 3, which is any one of glass filters.