JPS6019796A - Apparatus for synthesis of polynucleotide - Google Patents

Abstract

PURPOSE:To provide the titled apparatus capable of easily checking the residual amount of the reagent and operating the apparatus, by attaching a reactor to the front panel of the main body of the apparatus, putting reagent bottles, solvent bottles, etc. detachably in the exterior holder of the case, and transferring the reagent and the solvent successively to the reactor by a change-over valve. CONSTITUTION:The reactor 2 is attached to the front panel 1 of the main body A of the apparatus, and the holder 1b is attached to the exterior part of the case B of the main body A at a directly discernible position. The reagent bottles 4-6 and the solvent bottles 7-9 containing the reagents, solvents, etc. necessary in the polynucleotide synthesis reaction are attached detachably to the holder 1b. The change-over valve 10 to switch the flow of the reagents and solvents by one operation knob 11 of the front panel 1 is inserted in the flow paths 22b-22h connecting the reagent bottles 4-6 and the solvent bottles 7-9 to the reactor 2. The reagents and the solvents are supplied from the reagent bottles 4-6 and the solvent bottles 7-9 successively to the reactor 2 by the liquid supplying means using the pressure of the N2 gas in the nitrogen bomb 20. A polynucleotide is synthesized by repeating the reaction and cleaning procedures.

Description

[Detailed description of the invention] TECHNICAL FIELD The present invention relates to a polynuc[/otide synthesizer.

Polynucleo 11-', for example [)NA (deoxyribonucleic acid), is synthesized by using -ribo1~, which is chemically bonded with nucleosides, using the phosphoric acid l-lyester method, the phosphoric diester method, or the phosphite method. A method is known in which nucleotides are sequentially condensed by a method such as a method. In this synthesis method, steps such as C1, washing, deprotection, washing, condensation reaction, washing, etc. are repeated, and although there are not many types of steps, it is complicated because it requires one repetition.

In recent years, various DNs have been developed for the purpose of eliminating the troublesomeness of synthesis operations.
A synthesizer has been proposed.

For example, there are many known DNA synthesis apparatuses in which reagents, solvents, etc. necessary for synthesis reactions are fed into the reactor by manually operating a cock, and cleaning, protection, washing, condensation reactions, washing, etc. There was a risk of operating the cock incorrectly. If you make such a mistake, you will have to start over from the beginning, and all the work you have done up to that point will be wasted.

In addition, in the above device, the reagent bottles, solvent bottles, etc. are stored in the case, so in order to check the remaining amount, it is necessary to open the side panel of the case, which is cumbersome, and it is difficult to check the remaining amount. There was a risk that the user would perform the operation without doing anything. Also, it was troublesome to replace reagent bottles and solvent bottles.

The present invention has been made in view of the above circumstances, and its purpose is to provide a polynucleotide synthesizer that is free from the risk of manual operation and that allows for easy confirmation of the amount remaining in reagent bottles, etc. It is.

That is, the present invention has a reactor equipped on the front panel of the apparatus main body, and a reagent bottle or solvent bottle filled with reagents, solvents, etc. necessary for polynucleotide synthesis reaction in a directly visible part on the case side of the apparatus main body. A storage section is provided that can be detachably attached, and a flow path connecting the reagent bottle, the solvent bottle, and the reactor is provided with a switch for switching the flow path with a single operating knob disposed on the front panel. The reactor is characterized in that a valve is provided so that a reagent and a solvent are sequentially fed into the reactor from the reagent bottle and the solvent bottle by a liquid supply means.

Therefore, according to the present invention, it is easy to ascertain the amount remaining in the reagent bottle and the solvent bottle and to replenish the liquid, and the operability is good and there is no risk of erroneous operation.

An embodiment of the present invention will be described below with reference to the drawings.

FIG. 1 is an overall perspective view showing an example of the synthesis apparatus of the present invention, and FIG. 2 is a flowchart 1 to 1 of the synthesis apparatus.

In the figure, reference numeral A is the main body of the device, B is the case, and 1 is the front panel.

On one end side of the front panel 1 (left side in Figure 1), there is a G-shaped section 1a.
A reactor 2 supported by a support arm 3 of a pie break (not shown) installed in the case B is disposed within the recess 1a.

Also, from the center of the front panel 1 to the other end (right side in Figure 1)
is provided with four parts 1b which serve as storage parts for reagents and solvent bottles. The four parts 1b on the center side contain a deactivating agent, a condensing agent/
Small-capacity reagent bottles 4, 5, and 6 containing solvent 1 solution, inactivation aid/solvent 2 solution, etc. are stored. In addition, in the recess 1b on the other end side, solvent 1, deprotecting agent/solvent ■, and solvent ■
Large capacity reagent and solvent bottles 7.8.9 are stored. Incidentally, since solvent I is often used to inhibit the condensation reaction, during condensation, a solvent (I) that does not inhibit the condensation reaction is used. In addition, although not shown in detail, the openings of the bottles 4 to 9 are attached to the ceiling of the fourth part 1b so that they can be attached and removed.
Sections IC to lh that can be taken by one person are provided.

In addition, in the center of the front panel 1, there are an operation knob 11 for the six-way switching valve 10, an operation knob 13 for the one-way switch 12, an operation knob 15 for the four-way switch 14, and a timer device for the vibe 1/- evening. An operation knob 16 (not shown), a power switch 17, etc. are arranged.

1, 1 inside the case 13. The above-mentioned six-way switching valve 10, three-way cock 12, four-way cock 14, and pie break (not shown)
In addition to the equipment IIh, it is also equipped with a portable small-capacity nitrogen cylinder 18 and a distributor 19. If the nitrogen cylinder 18 is installed in the case B in this way, it will be necessary to carry the large nitrogen cylinder near the main body of the apparatus each time it is used, or to move the main body A of the apparatus to the place where the nitrogen cylinder is installed. ! l! There is no need to run. Further, there is no need to connect each nitrogen cylinder to the distributor 19.

The tube 2]a is connected to the bow) 19a on the inlet side of the distributor 19.
18 is connected to the nitrogen cylinder through the nitrogen cylinder 1B, and when the on-off valve 20 of the nitrogen cylinder 1B is opened, N2 gas is supplied to the boat 19a. This N2 gas is distributed from the boat 19a to each boat 1-19b to 19i on the outlet side of the distributor 19.

The boat t-19b-19g is connected to the above-mentioned takeaway parts 1c to 1h via tubes 2]b to 21g, and the N2
Bottles 4 to 9 that were separated from the gas section IC to 1h
send to. In addition, bows 1 to 19h are connected to boat Oa on the inlet side of the six-way switching valve 10 via tubes 21h, and boats 1 to 19 + are connected to boat 1 of the four-way cock 14.
4a via tube 2]i.

The other boats 10b to 10g on the inlet side of the six-way switching valve 10 are connected to the other boats R1 (1c to 1
h is connected, and when N2 gas is fed into bottles 4-9, reagents and solvents are fed from these bottles 4-9 into boats 1 (b) to 10 [ζ] by the pressure of the N2 gas.

Common bow 1-10 of 11 cases of hexagonal 1i1J switching valve 1()
The three-way connector 12 described in &f7 is connected to 22A by L, and the output 1 of the connector 12 is connected to the reactor 2 by the chain 1 and the connector r12a by the connector 2211. For example, as shown in FIG.
h and bo ) 10h and i1n - I! Then, the deactivating agent is fed into the reactor 2 from the bottle 4 by the pressure of N2 gas. Here, the six-way switching valve 10 is used for IJi, but this is due to the number of bottles 4 to 9. Also, a switching valve with a large number of switches is used.

Four sides: The bow 1- + 4b of 14 (111) is connected to the upper population 2a of reactor 2 via tube 22+, and also 14cm to bow 1: 1゛The bow 1- + 4b of reactor 2 is connected via tube 22j to the upper part 2a of reactor 2. It is connected to the bottom outlet 2b.
The boat 14d is J)l:IIIr:l, and the tube 22k is connected to it. In the state shown in Fig. 2 (when the operating knob 15 is set to 111., OW and 7), N2 gas is supplied to the upper part 2a of the reactor 2 from the tube 22+ through the 1h a and 14b, and the reaction starts. After passing through the inside of the vessel 2, the tube 22j3 boat I 4 c is inserted from the bottom outlet 2b, 14. d and is discharged by the tube 22k. Turn control knob 15 to F
When set to EED, the boats 14a and 14c are in communication, and the boats 1-14b and ]4d are in communication,
N2 gas flows from the bottom of the reactor 2 toward the -F section.

FIG. 3 shows the upper part of the reactor 2 in detail.

According to the figure, a substantially cross-shaped raw material injection pipe 23 is provided at the -1 part 2a of the reactor 2, and when injecting raw materials such as nucleotides, it is only necessary to remove the cap 24, and the upper inlet 2a is There is no need to take the trouble to remove the inserted stopper 25. In addition, the above-mentioned tube 22h is connected to the branch part 23a of the raw material injection pipe 23, and the above-mentioned tube 221 is connected to the branch part 23b.

Furthermore, a clip-type heater 26 is attached to the reactor 2, as shown in FIGS. 4 and 5. This heater 26 has a heater 26a made of a flexible planar heating element adhered to its inner surface, and supplies power to the heater 26a through a lead wire 26b to heat the reactor 2 and promote the reaction.

Next, the operation of synthesizing DNA using the synthesizer described in - will be explained.

First, the operating knob 15 of the four-way valve 14 is set to FEED, and the cock 2C of the reactor 2 is closed. Next, the reactor 2 was filled with Zabot (silica gel, etc.) to which nucleosides were bound, and the reagent bottle 4.5°6.8 and the solvent bottle 7.9
1C to 1h (T).

Next, the common boat 10h and the boat 10g are led to oil by the operation knob 11, and the three-way cock 12 is opened. Then, the solvent ■ flows from the bottle 9 into the reactor 2 due to the pressure of N2 gas.
sent to. Although the three-way cock 12 is operated in conjunction with the switching operation of the six-way valve 10, the operation of the three-way cock 12 will be omitted in the following description for the sake of brevity. After cleaning with the solvent 11 is completed, turn the operating knob 15 of the four-way valve 14 to BLOW, open the cock 2c of the reactor 2 to remove liquid, and then turn the cock 2C on.
, set the operating knob 15 to FEEr), connect the common boat 10h with the boat 10d using the operating knob 11, then connect the common boat 10h with the boat], Ob, and pour the inactivation aid/inert from the bottle 6. Solvent ■ Molten liquid and deactivating agent from bottle 4 are sequentially added to reactor 2 under the pressure of N2 gas.
send to. After leaving it for a few minutes, turn the operation knob 15.
and BLOW and open cock 2C of reactor 2 to remove liquid (IT).

Then, adjust the operation knob 15 to FEED, connect the common boat 1-10h and the boat 10g again with the operation knob 11, send the solvent 2 into the reactor 2 for cleaning, and deliquify it by the same operation as above. Do (TIT).

Next, the common boat 10h is communicated with the boats 1-10e by the operation knob 11, and the solvent (1) is sent to the bag counter device 2 for cleaning and deliquification (TV). After this, common bow) 10
h is communicated with 1Of, the deprotecting agent 0/solvent I18 solution is sent into the reactor 2, and the liquid is removed after several tens of seconds (V). Make this M and repeat it multiple times.

In this way, shallow, ilF and common baud 1-10 h are 1
Connected with 0C) After 1η, feed l into the is container 2, wash and remove liquid.
Wash 1-10 g of water with solvent (■) and remove liquid (■).

After that, after purging (■) with N2 gas for several minutes, a solution of a monomer salt not shown in the structural formula below dissolved in solvent ■ is injected into the reactor 2 from the raw*I injection pipe 23, and then the common The boat 1011 is connected to the boat 1-10C and the condensing agent/solvent I+ solution is sent to the reactor 2 (rX). It is also possible to fill a reagent bottle with a solution prepared by dissolving 100% salt in the solvent (2), boil it, and send it into the reactor 2 under the pressure of N2 gas.

Here, R1 is a protecting group (such as a benzoyl group), R2 is an alkyl group, and B is adenine (A), guanine (
G), cy]-syn (C), and thymine (T).

After standing for 40-50 minutes, drain the liquid (X) and repeat again from step (TI) above. In this way, the nucleotide chains are sequentially condensed.

Note that a dimer salt, a trimer salt, etc. may be used instead of the monomer salt.

When the remaining amount of reagents and solvents in bottles 4 to 9 is almost gone,
Replace or refill bottles 4-9.

At this time, since the handle (-j part IC to 1h) is attached to the main body, operations such as replacing the bottle and refilling the liquid can be easily performed.

In the synthesis apparatus described above, the reactor 2 does not protrude from the front panel 1, so there is no possibility of accidentally touching the cock 2C during operation, and there is no risk of damage to the reactor 2. In order to promote the reaction, the reactor 2 may be vibrated with a pie break, heated with the heater 26, or heated with the heater 26 while being vibrated with a vibrator. The pie break can be operated for 7 hours using a timer device.

In FIGS. 6 and 7, the case where 31 reactors 2 are equipped with 21 IliI is shown. In this case, a three-way valve 27 is provided at the outlet of the three-way valve 12. In this way, reagents, solvents, etc. are simultaneously supplied to each reactor 2, 2, and it becomes possible to simultaneously operate 2111 rl of reactors 2, 2 in one operation. Furthermore, by injecting different types of raw materials (for example, injecting an A monomer salt into one and a G monomer salt into the other), it is also possible to perform two types of synthesis at once. Also, reactor 2 is 3 (1?lI
With the above equipment, three or more reactors 2 may be operated at the same time, or three or more types of synthesis reactions may be performed. In this way, it is possible to significantly shorten the synthesis time. In this embodiment, the bottles 4 to 9 are placed not in the front panel 1 but in four parts 30a, 3fl+ provided on one side of the case 13. Note that 31 in FIG. 7 is a pressure gauge for N2 gas.

Figure 8, number 1, shows yet another embodiment of the present invention.

According to the same embodiment, a partition plate 2 is placed on the back 1% 11 of case B.
A storage part 29 partitioned by 3 2 8 is provided, and in addition to the bottles 4 to 9, 18 are stored in the nitrogen cylinder. In this case, the gas cylinder 18 can be easily replaced with a nitrogen gas cylinder.

In the above embodiments, N2 gas is used as the liquid supply means, but the present invention is not limited to this, and a liquid supply pump may be used, or a head may be used.

The synthesizer of the present invention is not limited to the case of synthesizing DNA as described above, but can also be used for synthesizing RNA (liponucleic acid).

As explained above, according to the present invention, reagents necessary for a polynucleotide synthesis reaction are provided in a directly visible area on the outside of the case.
A reagent and solvent bottle storage area (recess 11b) in which a solvent bottle can be removably attached.

30a, 30b, and storage section 29), the remaining amount of reagents, etc. can be visually checked, and the bottles can be easily replaced.

In addition, a switching valve is installed in the flow path connecting the reagent/solvent bottle and the reactor to switch the tfE l/JG using a single operation knob located on the front panel of the case. ), it is possible to reduce the number of erroneous operations as much as possible.

Furthermore, since the front panel is equipped with a reactor, cleaning
・Removal and maintenance - = ・Can be operated while watching cleaning etc.

[Brief explanation of drawings]

Figure 1 shows an example of the synthesis apparatus of the present invention; Figure 5 is a side view of the heater with the heater turned 7 degrees, Figure 5 is a cross-sectional view of the same, Figure 6 is the reactor at 2 tl.
Flow Sea 1-1 when equipped with lil FIG. 7 is a perspective view of the same device, and FIG. 8 is a perspective view showing still another embodiment. A...Device body, 13...Case, 1
...Front panel, lb, 30a, 30b, 29
・-=Reagent and solvent bottle storage compartments (4 parts), 4 to 9...
... Reagent, solvent bottle, 2 ... Reactor, 10.
...Switching valve (six-way switching valve), 11...Operation knob, 22b to 22h...Flow path (tube), 18...Nitrogen cylinder. 5

Claims (1)

[Claims] A reactor is equipped on the +iit side panel of the device body, and a reagent bottle filled with reagents, solvents, etc. necessary for the polynucleotide synthesis device is installed in a directly visible area on the outside of the case of the device body.
A storage part is provided in which a solvent bottle can be freely sprayed, and a single operation knob disposed on the front panel is provided in a flow path connecting the reagent bottle, the solvent bottle, and the reactor. A polynucleotide synthesis apparatus characterized in that a switching valve for switching the flow path is provided, and the reagent and solvent are sequentially fed from the reagent bottle and the solvent bottle into the reactor by a liquid supply means.