JPS6121639B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus for synthesizing DNA, etc., and in particular, an apparatus for preparing an unstable reagent solution and supplying it to a reactor.
It relates to a DNA synthesis device capable of synthesizing DNA and RNA.

DNA synthesis methods have been improved and developed into the so-called phosphodiester method, phosphotriester method, and phosphite method, and solid-phase synthesis methods using solid supports that utilize these methods are widely used due to their various advantages. It has reached the point where it will be done. Various apparatuses for synthesizing DNA using these methods have also been proposed. All of these devices supply multiple reagent solutions to a reactor in a predetermined procedure.
What they have in common is that they synthesize DNA.

On the other hand, some reagent solutions used for DNA synthesis are unstable. For example, 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. .

Therefore, in conventional devices of this type, there was an inconvenience in that the operator had to prepare a reagent solution and set it in the device when starting DNA synthesis. In practice, it was sometimes prepared and set in advance, but in that case there was a risk that stable synthesis could not be achieved.

As a result of intensive research, the inventor of this invention discovered the publicly known
Succeeded in improving the DNA synthesis equipment.

Thus, according to the present invention, in an apparatus for synthesizing DNA, etc. by supplying a plurality of reagent solutions to a reactor according to a predetermined procedure, a sealable device having a removable rubber septum part and a device for individually supplying a plurality of reagents or solvents can be used. At least one reagent solution preparation container capable of preparing a reagent solution by simultaneously supplying the reagent solution to the reactor, a discharge nozzle fixed to the reactor, a needle that can be inserted into and removed from the septum, and a preparation container from the needle. Preparation using the preparation container comprises a liquid feeding means capable of inhaling a reagent solution therein and discharging it from the discharge nozzle, and a moving means for moving a needle relative to the preparation containers and inserting and removing the needles in the case where there are multiple preparation containers. There is provided an apparatus for synthesizing DNA, etc., characterized by comprising a reagent solution supply means for supplying a reagent solution to a reactor.

The main features of the device of this invention are (i) the device itself is equipped with a means for preparing a reagent solution, which allows it to be prepared within the device immediately before use; and (ii)
The prepared reagent solution is supplied to the reactor by the supply means of the apparatus itself.

Hereinafter, this invention will be explained in detail based on embodiments shown in the drawings. However, this invention is not limited thereby.

Reference numeral 1 shown in FIG. 1 is an embodiment of the present invention, which is an automatic DNA microsynthesizer using 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 10 mm, and a mortar-shaped flange 4 provided above. The dovetail 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 serves as 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. 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 diameter of about 450μ.
is a space with a volume of .

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 test solution for removing a protecting group, which is a solution in which zinc bromide is dissolved in a mixed solvent of isopropanol and methylene chloride. 15 is a masking reagent, which is a mixture of acetic anhydride and pyringe. 16 is a masking reagent, which is a pyridine solution of dimethylaminopyridine.

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 27 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 2 via the console 28.
Can have a dialogue with 7.

The preparation containers 29, 29', 29'', . . . are tubular containers 30, 30', each having an internal capacity of about 100μ to 500μ.
30″, ... and silicone rubber septum 31,3
It consists of 1′, 31″, .... Sebutam 3
1, 31', 31'', . . . are removable and into which a solvent supply needle 35 or the like can be inserted from the outside.These preparation containers 29, 2
9′, 29″, . . . are holder holes 3 on the turntable 32 whose operation is controlled by the control circuit 27.
3, 33', . . .

The preparation container 29 moved to a predetermined position by the turntable 32 has a needle up and down mechanism 37.
The needles 34 to 36 are inserted by. In that case, the needle 34 supplies and exhausts nitrogen gas, the needle 35 supplies the solvent, and the needle 34 supplies and exhausts nitrogen gas.
6 is for taking out the solution inside.

The syringe pump 40 includes the plunger drive mechanism 3
8 drives the plunger 40a.
Then, pyridine 11 is inhaled through the valve 45,
The pyridine is discharged via valve 44 into preparation vessel 29 .

The syringe pump 41 is connected to the plunger drive mechanism 3
9 drives its plunger 41a, sucks in the solution in the preparation container 29 through the valve 46,
Discharge via valve 47 to reactor 2. or,
Discharge via valve 46 to another preparation container.

Needle up and down mechanism 37, plunger drive mechanism 3
8, 39, and the operation of the valves 42 to 47 is controlled by the control circuit 27.
controlled by.

When synthesizing DNA, a support 9 to which only the end portions of DNA are bound is placed in the reactor 2 in advance, and preparation containers 29, 29', 29'', . . .
..., scleotide reagent A and condensing agent [mesitylenesulfonyl-3-nitrotriazolide (MSNT)]
Add B alternately. All of these are powdered. For example, when the support 9 is polystyrene powder, the amount of the support 9 is suitably 10 mg to 50 mg. The appropriate amount of nucleotide reagent A is 3 to 5 equivalents relative to the nucleoside bound to the support. When the support 9 is polystyrene powder and the amount of nucleoside bonded is 0.1 mmol/g, it is appropriate to use 400 mg of monomer and 700 mg of dimer per 1 g of support, and in the same case, condensing agent B is 300 mg per 1 g of support.
mg is appropriate. There are four types of nucleotide reagent A, even in the case of monomers, depending on the base, but the order in which they are placed in preparation containers is the same as the base sequence of the target DNA. The same applies when using a dimer, a trimer, or a mixture thereof.

The above-mentioned nucleotide reagent A and condensing agent B may be set at any time before the time when DNA synthesis is actually started. This is because all of them are set in an unpowdered state, so they are not unstable.

Since the basic operation of this apparatus 1 is basically the same as that of a known apparatus of this type using the phosphotriester method, a general explanation will be omitted, and only the operation of the characteristic synthetic steps will be explained in detail.

In the synthesis process, the control circuit 27 operates the valve 45 and the plunger drive mechanism 38 to suck a small amount of pyridine 11 into the syringe pump 40, and the valve 44,
42 to transfer the pyridine 11 to the preparation container 2.
Supply to 9. The amount of pyridine 11 is preferably determined based on the amount of support 9, for example,
If polystyrene powder is not used, it is preferable to use about 6 ml per 1 g. Since the nucleotide reagent A in the preparation container 29 is dissolved in pyridine 11, the inside of the preparation container 29 becomes a nucleotide reagent solution C after a predetermined time. FIG. 1 shows this state.

Next, the control circuit 27 operates the valve 43 , the valve 46 , and the plunger drive mechanism 39 to draw the nucleotide reagent solution C into the syringe pump 41 . Next, operate the needle up and down mechanism 37 to move the needle 3
4 to 36 are pulled out from the preparation container 29, the turntable 32 is rotated to move the next preparation container 29' to a predetermined position, and the needles 34 to 36 are inserted into the preparation container 29'. Next, the nucleotide reagent solution C in the syringe pump 41 is transferred to the preparation container 2.
9' and dissolve the condensing agent B.

After a predetermined period of time, a reagent solution containing nucleotide reagent A and condensing agent B will be present in the preparation container 29', so the valves 43, 46, and 47 are operated to supply the reagent solution to the reactor 2.

This causes a condensation reaction in the reactor 2, and a new necreotide is linked to the end of the DNA.

According to the automatic DNA microsynthesizer 1 of the above embodiment, the condensing agent MSNTB 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, there is no need to prepare a reagent solution each 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 50 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 minimum supply amount (approximately 5 to 7 times the volume of the support) is 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 inside of the reactor 2 is filled with a drying solvent such as THF3.
The reactor 2 is constructed so that the inside of the reactor 2 can be completely dried in a short time by washing and drying with water 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.

In the end, the DNA micro-automatic synthesizer 1 of the above example
is a lean device in many respects.

Variations include those in which the reactor 2 is funnel-shaped, barrel-shaped, and the internal volume of the reaction section is 80μ.
Examples include those that vary between ~800μ.
Examples of solid supports include those using Kel-F.g-styrene, silica gel, polyacrylic morpholide, and the like. These supports have a particle size of 30~
A thickness of about 300 μm is preferable.

Still another modification is one in which the stopper 5 of the reactor 2 is a silicone rubber septum, and a reagent solution or the like is supplied by inserting a needle therethrough. On the other hand, the preparation containers 29, 29', 2
9'', . . . are fixed positions, a stopper is provided at each end, and a nozzle having the same function as the needles 34 to 36 is fixed to each stopper.

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 phosphodiester method.

When applying the phosphomonotriazolide method to the device 1, in Fig. 1, the valve 45 is not connected to the tank of pyridine 11, but is connected to a new tank, and the phosphorylation is carried out in that tank. A reagent such as O-chlorophenyl phosphoroditriazolide is placed in the preparation containers 29, 29', . . .
To be able to supply... Further, the nucleotide derivatives of formula (i) are placed in the preparation containers 29, 29', 29'', . . . in the order of matching the base sequence of the target DNA.

[Base is adenine, guanine, cytosine or thymine] Syringe pump 41, plunger drive mechanism 3
9. Valve 46 may be omitted. During preparation, 9 to 10 O-chlorophenyl phosphorodithriazolides are added to 10 of the nucleotide derivative of formula (i) and reacted for a predetermined period of time to obtain a nucleotide reagent solution. Although this nucleotide reagent solution is unstable, the nucleotide derivative of formula (i) and O-chlorophenylphosphoroditriazolide are each stable, so the desired effect can be obtained.

Similarly, when applied to the phosphite method, the valve 45 is connected to the tank of THF 12 without being connected to the tank of pyringe 11. Further, the nucleotide reagents of formula (ii) are placed in the preparation containers 29, 29', 29'', . . . in the order that matches the base sequence of the target DNA.

[Base is adenine, guanine, cytosine or thymine] Syringe pump 41, plunger drive mechanism 3
9. Valve 46 may be omitted. During preparation, THF is added to the nucleotide reagent of formula (ii) to prepare a nucleotide reagent solution.

As understood from the above explanation, the present invention
According to the DNA synthesis apparatus, an unstable reagent solution is prepared and used at the time of use. Therefore, even if the reagents are set in advance, it is effective to ensure stable synthesis, which is desirable from the viewpoint of maintenance. Furthermore, even if the reaction is stopped midway through, the remaining reagents can be recovered, and expensive reagents are not wasted.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the configuration of an automatic DNA micro-synthesizing device, which is one embodiment of the DNA etc. synthesizing device of the present invention.
FIG. 2 is a flowchart of the operation of the device. 1... DNA micro-amount automatic preparation device, 2... Reactor, 11... Reaction solvent, 27... Control circuit, 2
9, 29', 29'', 29... Container for preparation, 34
~36... Needle, 37... Needle up and down mechanism, 38, 39... Plunger drive mechanism, 40,
41... Syringe pump, A... Nucleotide reagent, B... Condensing agent, C... Nucleotide reagent solution.

Claims (1)

[Scope of Claims] 1. A device for synthesizing DNA, etc. by supplying a plurality of reagent solutions to a reactor according to a predetermined procedure, which is capable of being sealed and having a removable rubber septum portion, and in which a plurality of reagents or solvents are individually supplied. Or one reagent solution preparation container that can be supplied at the same time to prepare the reagent solution.
In addition to the above, a discharge nozzle fixedly installed in the reactor, a needle that can be inserted into and removed from the septum part,
It consists of a liquid feeding means that can suck in the reagent solution in the preparation container through the needle and discharge it from the discharge nozzle, and a moving means that moves the needle relative to the preparation containers and inserts and removes the needles when there are multiple preparation containers. 1. An apparatus for synthesizing DNA, etc., comprising a reagent solution supply means for supplying a reagent solution prepared in the preparation container to a reactor. 2. The preparation container is a sealable container that has a removable rubber septum part, and has a nozzle for supplying the liquid reagent or solvent and a nozzle for inhaling the reagent solution from inside, and that the reagent solution is Claim 1, wherein the supply means comprises a discharge nozzle fixed to the reactor, and a liquid feeding means capable of sucking in the reagent solution from the nozzle of the preparation container and discharging it from the discharge nozzle. equipment. 3. The reactor has a rubber septum at the top, a solid support for synthesizing DNA, etc. can be placed at the bottom, and a filter that allows the reagent solution to pass through, and the bottom has a drain port and can be sealed. The preparation container is a sealable container having a removable rubber septum part, and the reagent solution supply means is a nozzle fixed to a solvent or liquid reagent tank.
A needle that can be inserted into and removed from the septum portion, a liquid feeding means that can suck in a solvent or liquid reagent through the nozzle and discharge it from the needle, and a liquid feeding means that can suck in and discharge a reagent solution from the needle, and the needle can be connected to the reactor or the preparation. 2. The device according to claim 1, further comprising a moving means that moves relative to the container for inserting and removing the needle.