JPS58148896A - Automated microsynthesizer for dna or the like - Google Patents

Abstract

PURPOSE:The titled synthesizer that is provided with a supply port for feeding reagent solutions at the top and an effluent port at the bottom and has a microvolume of the reaction space formed with a filter on which a solid support for synthesis of DNA etc. can be placed, further having a means for feeding reagents in amounts corresponding to the amount of the support. CONSTITUTION:The reactor 2 consists of the cylindrical main body 3 and a cone-shaped flange 5 and the port for feeding reagent solutions 6 is set to the top opening of the main body 3 and the effluent port 9 is set to the bottom. Further, a filter 7 is fitted to the lower part in the main body 3 so that it can hold the support for DNA synthesis 9 and pass the reagent solutions, solvents and gases to form a reaction space 10 above it. A support to which only the terminals of DNA molecules are bonded 9 is placed in the reactor 2 and its amount signal is input to the control circuit 34 to control plunger-driving mechanisms 26-30, switching cocks 16-20 and air-releasing valve 51 to feed needed amounts of nucleotide reagents 11-14 and condensation agent 15. Further, valves 45-47 are controlled to feed required minimum amounts of deprotecting reagent 39, masking reagent 40 and condensing agent 41 for masking.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus for automatically synthesizing DNA, etc., and particularly provides a device suitable for automatically synthesizing a small amount of RNA.

The so-called diester method is a method for synthesizing DNA.

The modified JIL method has been developed into the triester method, the phosphite method, and the solid support method, which utilizes these methods and uses a solid support, has come to be widely used because it has various advantages. On the other hand, the reactor (reaction column) used in the solid support method is a type in which a solid support is placed and the reactor itself is shaken, in terms of the mixing/contact surface with the reagent solution.
Two types are known: one in which a reagent solution is circulated through the reactor, and the other in which a large amount of reagent solution is temporarily passed through the reactor. However, both types have various drawbacks. On the other hand, in conventional automatic synthesis equipment, the amount of solid support is almost always on a scale of 10011 MAvh or more (#), which is the required amount of DNA to be synthesized.

From the viewpoint of the fact that the reagents, such as the raw material nucleotides with a preservative group, are trivalent, an automatic synthesis device on a much smaller scale is desired.

As a result of various studies, the inventor of this invention succeeded in installing several omniscient automatic synthesis devices.

Thus, according to the present invention, the reactor has a reagent solution supply port at the upper part, a solid support for synthesizing KDN, etc. can be placed in the lower part of the reactor, and a filter that allows the reagent solution to pass through. , has a drainage port at the bottom, and has a volume of 80 μm or more on the filter.
A micro-volume device such as a DN membrane, which is composed of a container having a reaction space of 800 μm and is equipped with a reagent solution supply means that can supply a reagent solution in an amount corresponding to the amount of the support placed in the reactor. An automatic synthesizer is provided.

The main features of the device of this invention are (1) eliminating the need for special operations for mixing and contacting such as shaking the reactor; (M) using 1OIII as a solid support;
~50M1 scale, condensation 10 as 9DN
It is possible to provide a reaction system capable of synthesis on a scale of 0.3 to 2 s rno1.

(The amount of reagent solution to be used is 5 to 7 times the volume of the solid support.
The objective is to provide a suitable supply means that only requires about twice as much.

Hereinafter, this invention will be explained in detail based on embodiments shown in the drawings. Note that this invention is not limited to this.

(1) shown in FIG. 1 is a DN mold automatic weighing system using the phosphotriester method to which the present invention is applied.

The reactor (2) has a round body with an inner diameter of 8 cm and a diameter of 10 cm.
3) It is a container with a mortar-shaped 7/di (4) provided above it. A stopper (i) into which a number of nozzles for supplying reagent solutions and the like are inserted is attached to the dome-shaped 7 flange (1). Therefore, the head opening of the main body (3) becomes a reagent solution supply port (6). A filter (7) such as a J-Las filter is fitted inside the main body (1), and a drain port (8) is provided at the bottom. The filter (1) is one on which a support (9) such as polystyrene or silica beads can be placed (not permeable), and is permeable to the sample am solution, #I medium, and gas. The space above the filter (7) is the reaction section t16Ktkk), which has a volume of about 450 μIO.

There are 8 types of reagent solutions in total. (B) to (B) are various nutreotide reagents, each having adenine, cytosine, guani/thymine as a base.

(A) is a condensing agent, which is a pyridine solution of 2-4-6-)9methylbenzenesulfonyl-3-nitrotriazolide (8N'f'). (2) is a protective group-eliminating agent, which is a solution prepared by dissolving zinc bromide in a mixed solvent of isopropanol and methylene chloride. The ring is a mass medium reagent, which is a mixture of acetic anhydride and pyridine. - is a condensing agent for masking,
It is a mixture of dimethylaminopyridine and pyridine.

The reagent solution Qυ~ can be sucked into the reagent solution Qυ~ through the switching cocks, respectively, and supplied to the reactor (2).

The plungers of the syringe pumps (c) to (7) are driven by granger drive machines W (e) to (7), respectively.

Granger drive machine m (E) consists of a pulse motor 6◇, a screw shaft (2) rotated by it, and the screw shaft (2).
A pulse motor (b) is pulse-controlled by a control circuit such as a microcomputer. The other plunger drive mechanisms - - (2) have similar structures.

The solvents are a volatile drying solvent, tetrahydrofuryl (THF), a cleaning solvent, viridi, and a mixture of isopropanol and methine chloride, which are also cleaning solvents. These ILI medium and the reagent solution can be supplied to reaction a(2) through valves, respectively, under nitrogen gas pressure.

The valve reacts with nitrogen gas! (Nitrogen gas is supplied directly to the reactor (2) while blowing inside the reactor (2). The nitrogen gas is dried with a desiccant such as calcium chloride.

The control circuit includes the gradation/jar control mechanism (2) as described above.
In addition to controlling ~ (to), the switching cock (2) ~ Zeng, valve ~
~-2 Controls the drain valve ring and exhaust valve f4o operation. It also interacts with the operator via the console.

When synthesizing DN5, first remove the stopper (6) and place a support (9) into which only the end portion of the DNA molecule is bound into the reactor. ) is polystyrene powder, 10111-50q is suitable. After returning the stopper (5) to its original position, input the amount of support (9) etc. to the control circuit @ via the operating wheel (to). input, then input the start command.

The control circuit then operates the valves w, f4. - is operated to supply a mixed solvent of isopropanol and methylene chloride to the reactor (2) K, and the support (9) is washed. Tube 9, open the valve 2 to supply the solvent, close the valve 1, wait a while, open the valve 1 to drain the liquid, valve II.
tClose. Do this several times.

After this washing, operate the valve-1- to release the protective group release agent OS.
t to the reactor (2), and after a predetermined period of time, the valve-2
Activate f4 to drain the liquid.

The reactive group at the end of the nine DNA molecules bound to the support (9) is blocked by adding a dimethoxytrityl group (DM'I'r) as a protecting group in advance.
However, the above operation causes DMTr at a predetermined portion to be detached.

Then the control circuit again switches the valves, M, f4. - is activated to supply a mixed solvent of isopropanol and methylene chloride to the reactor (2) and wash the support (11).

Furthermore, actuate valves -, w, to, and u to add pyridine (2
) to clean the inside of the reactor (z).

Just activated the f valve -, m, (II, @ and 'THF@
The inside of the reactor (2) is washed with a water bottle, and then valve-1 is activated to blow nitrogen gas into the inside of the reactor (2) for several minutes. As a result, the inside of the reactor (2) is completely dried.

The control circuit uses four nucleotide reagents (b) to convert different bases based on the base sequence of the DNA to be synthesized.
Select one nucleotide reagent from ~(b), actuate the corresponding switching tip and plunger drive mechanism, and actuate the front exhaust wheel to supply the nucleotide reagent solution to reaction e).

For example, a nucleotide with adenine as a base is D
Next, if necessary as a NA base sequence, select the nucleotide reagent solution (b), operate the switching cock (to) and the plunger drive mechanism, operate the exhaust front wheel, and react the shaft with the cylinder/dibon pump. ! (supply to ri.

At the same time, switch switch #J and plunger drive
Turn on the reactor and supply the condensing agent (2) to the reactor using the syringe pump.

The feed rate is controlled such that the content of the nucleotide reagent solution and condensing agent is the minimum amount sufficient to swell the support (9). Specifically, for example, when the support (9) is a polystyrene powder, the support 116 has a nucleotide reagent of 3s/, and a mixture of about 2-. Needless to say, it is necessary to adjust the minimum amount of the reagent solution so that it contains a sufficient amount of the reagent.

By the above operation, the friend DN is connected to the support (9).
A nucleotide having a desired base is linked to a predetermined site on the terminal portion of the A molecule. Note that the pentahydroxyl group of the nucleotide is previously blocked by D M Tr, which is a protective group.

When the support (9) is polystyrene powder, the terminal part of a DNA molecule of about 0.1 m vnoll is bound to each support II, and a new nucleotide is added to each KU as described above. Concatenated. However, the number 9! The phlegm may remain unreacted. This rounding control device axis masks unreacted pentahydroxyl groups as follows. That is, after a certain period of time, the inside of the reactor (2) is washed with pyridine (2) by operating valves (1m, 1, ti). Next, the valve-
2@ is activated to supply the bean paste reagent to the reactor (2). At the same time, the valves are operated to supply the condensing agent (c) for mass media to the reaction (11).

Pressure fitting and control path 1 which performs masking by the above operation are as follows:
Valve-1-1-1 Activate 1 ring to pump pyridine (in the reactor body).
2) Wash.

The operations up to this point complete one at-cycle in which one nucleotide is linked to the new nine, which has been previously bound to the support (9), at the end of the nine DNA molecule.

The control circuit operates valves 1-2 and 11 to repeat the synthesis cycle from the protective group removal operation to the masking operation for supplying the mixed solvent of Impropanol and methylene chloride; Synthesize the target DNA input on the console ■.

Now, according to the ODN synthesis apparatus (1)K of the above embodiment, unlike the conventional method, the reagent solutions QE) to (QE) are always supplied in the minimum amount calculated based on the amount of the support, and mixed.・
No contact operations are performed. Therefore, the amount of reagent consumed is saved, and the O device for mixing and contacting operations is also unnecessary.

This improvement was achieved by downsizing the reaction section of the reactor (2) and by placing a support (9) on the filter (7).
This is because the reagent solution (b) was supplied from above and drained from the bottom. In other words, if a reagent solution is supplied from above with the front drain wheel closed, the reagent solution will be contained in the support (9) and swell it, and will remain in the reaction section (a) above the filter (7). It will not fall downward. Therefore, all the supplied reagent solutions participate in the reaction, and no dead space is left behind. As a result, a small amount of moth is sufficient to be supplied, and mixing and contacting operations become unnecessary.

In addition, before the reaction of linking a new nucleotide, the interior of the reactor (2) is completely dried to remove moisture that inhibits the condensation reaction, thereby increasing the reaction efficiency and helping to save reagents.

As a modified example, the reactor (2) is funnel-shaped, barrel-shaped, and the volume of the reaction chamber is BO#Ij.
Examples include those varying between ~800 μl.

Other examples include those in which the present invention can be applied to synthetic scissors such as DN films by the phosphomonotriazolide method, the phosphatide method, or the diester method.

Solid support @) 0 and others t) f4 and Kel-F-
Examples include g-styrene, silica gel, and poly-9 ataryl mole-7 olide. These supports are grain aSO~300IISS
Degree o4o is preferred.

As can be understood from the above-mentioned findings, the automated micro-assembly of DI and the like of the present invention has the effect of suppressing wasteful consumption of expensive synthesis reagents and making running last less expensive. . Also, mixing and contacting operations are no longer necessary. Furthermore, only the required amount can be synthesized in minute quantities, eliminating waste.

[Brief explanation of the drawing]

1st I! 1 is a diagram illustrating the construction of a DN module automatic mold amount synthesis device 0III, which is an embodiment of the ζol plan ODM system. (1) DNA micro-automatic synthesizer, (reactor, (
3) Main body, (4) 2 parts of flan, (6) Reagent solution supply port, (7) Filter, (8) Exhaust eye, (9) ...Support, reaction section, 9s-4, (14--li...reagent solution, (g)-(4)
)--Switching cock, (C)~(C)...Syringe pump, (N)~)...Plunger drive mechanism,--Control circuit, (To)-(2)...Mini , -～-1f
4-... Valve. Ding Zoku Nyama +l t7M1:Ia'l
I i艮口° 6sugi servant 1 tsu 1f'1's entry of patent application No. 30887 of 1982 2.1 to Ming's 8th restriction 1 1) Δ'! ! i micro-automatic synthesis device: 3 Supplement 11 stone $19 Which relationship Patent applicant fl Ifli 5; H4 out of 4 cities? O11 Kyomachi Street 378 Toruno Funatocho Name (19
9) Stock company arrival easy 14! Production staff (and 1 other person) Proxy agent location Wanryoku, 1 generation 1! 1 person 〒i): 30 If 191 Osaka Mountain North Layer Nishijinman: 31111
3rd A-tar Wangjiru 1), 11 commands (''I・1)
(Voluntary) 0 Supplement 11", Increased 4 Excuse 9, Relationship with the case of the person who made supplements other than the above. Mizu Yakuhin Co., Ltd. representative opinion Yusui Low Sukego, I11'1oI4 Range of water 1. The reactor is connected to section V [supply f1 of reagent bath liquid, etc.] (
-7, a solid support for synthesis of NA etc. is placed in the internal L/], and a filter is provided that can pass through the reagents. , the filter Lh is filled with sardines according to the wrinkles of the support placed in the reactor (5).
The means for supplying the reagent fd and the reagent m oil are supplied using a shell pot [(shishiru L) NA'S micro-automatic synthesis scissors equipment.

Claims (1)

[Claims] 1. The reactor has a reagent solution supply port at the top, a filter on which a solid support for synthesizing DNA, etc. can be placed at the bottom, and which allows the reagent solution to pass through, and a drain port at the bottom. It is composed of a container having a reaction space with a volume of 80 μm to 800 μl above the filter, and a reagent solution supply device that supplies a reagent solution by moving according to the amount of the support placed in the reactor. A device for automatically synthesizing a small amount of DNA, etc., which is equipped with a means.