JPS6054399A - Polynucleotide synthesizer - Google Patents

Abstract

PURPOSE:The paths connecting the reactor to reagent and solvent bottles are switched by means of a valve which is operated by a finger grip, and a plurality of such systems are provided to simplify valve operation and reduce malfunctioning. CONSTITUTION:N2 gas is sent from bomb 20 through distributor 21 to reagent bottles 23, 24 and solvent bottles 25, 26. The pressure of the N2 gas send the reagents and solvents from individual bottles through switching valves 27, 28 and T-cock 29 to the reactors. The switching valve 27 is provided with reagent and solvent switching ports 37a, 37c, 37e connecting to bottles 24-26, N2 gas switching ports 37b, 37d, 37f, 37h connecting to the distributor 21, a switching port 37g connecting to the vacuum pump 33 and common port 37q connecting through T-cock 29 to the reactor 30. In the same manner, the switching valve 28 is provided with switching ports, 37i, 37m, 37k, 37o, 37j, 37l, 37n, 37p and common port 37q'.

Description

[Detailed description of the invention] The present invention relates to a polynucleotide synthesizer.

Polynucleotides, e.g. DNA (deoxyliponuclear M
) is known to use a support to which nucleotides are chemically bonded, and sequentially condense nucleotides by a phosphotriester method, a phosphodiester method, a phosphite method, etc. In this synthesis method, steps such as washing-deprotection→washing→condensation reaction-washing are repeated, and although there are not many types of steps, there are many repeated operations and it is complicated.

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, the device body is equipped with a reactor, reagent bottles and solvent bottles filled with reagents, solvents, etc. necessary for polynucleotide synthesis reaction, and a flow path connecting each reagent bottle and solvent bottle to the reactor. A cock is installed in each of the reactors, and the pressure of nitrogen gas is used to open and close each cock to sequentially feed reagents, solvents, etc. from the reagent bottle and solvent bottle into the reactor, and the process of cleaning, protection, cleaning, etc. is repeated. The composition is known.

However, the above device has a large number of cocks, and there is a risk of erroneous operation. If such an erroneous operation is made, the user must start over from the beginning, and all the work done up to that point may be wasted.

Further, in the case of automation, it is possible to replace the cock with a solenoid valve, but there are problems such as high cost and complicated control due to the use of a large number of solenoid valves.

In order to solve the above problem, the present applicant proposed a device as shown in FIG. 1 (Japanese Patent Application No. 126249/1982). In the figure, 1 is the nitrogen cylinder, 2 is the distributor 13
a to 31 are nitrogen gas flow paths, 4.5, 6, and 8 are reagent bottles, 7 and 9 are solvent bottles, 10 is a six-way switching valve, llb to l1
g is a flow path for reagents and solvents, 12 is a three-way cock, 13 is a three-way cock, 148 to 14 g is a flow path for reagents, solvent, and nitrogen gas, 15 is a six-way cock, 16 is a reactor, and 17 is a waste liquid tank.

According to the above device, for example, when the common port 10h is set to the port 1 (l) using the operating knob (not shown) provided on the six-way switching valve 10, the reagent bottle 4 is moved to the reactor 16.16.
connected to. Then, N is sent from the nitrogen cylinder 1 to the reagent bottle 4 through the distributor 2 and the flow path 3b.
Due to the pressure of the two gases, the reagent (inactivating agent) flows from the reagent bottle 4 through the channel l1l), the port 10b, the common boat lOh, the three-way cock 12, the three-way cock 13, and the channels l4a, ]4t). are fed into the reactions W16 and 16, respectively. At this time, the hexagonal cock 154 is set at the solid line position, that is, FEED.

In addition, there are 10 common boats with the operation knob! l to port 1
When the temperature reaches 0C, the reagent bottle 5 is placed in the reactor I6.16.
Similarly, the reagent (condensing agent/solvent solution) is reacted from the reagent bottle 5 by the pressure of N2 gas.
sent to. In addition, the common boat ] Oh the ball l-
10 (If set to I, the reagent (inactivation aid/solvent lI solution) will flow from reagent bottle 6, and if set to port] Oe, solvent will flow from solvent bottle 7, and if set to port 10,
When 10g is added to bottle 1, the reagent (deprotecting agent/solvent Y) is added from reagent bottle 8, and when 10g is added to bottle 1, solvent 11 is added from solvent bottle 9.
are fed into reactors 16 and 16, respectively.

In addition, when the reactor 16 is drained υ1 from the reactor 16, the hexagonal cock 15 is set to the dotted line position, that is, BLOW, and the cocks 16a and 16a are opened.

As mentioned above, the flow path is controlled by - operation knobs], 1
b to l1g can be switched, and compared to conventional devices that open and close a large number of cocks provided in each flow path, erroneous operations can be reduced and automation can be easily performed.

However, looking at the operation of the six-way switching valve 10 in one condensation process, the common boat 10h, port 1, Og-"
10 d-10b→10 g→10 e→10f→10
It is designed to switch in the order of e→]Og→IOC (
Note that when switching ports 10b to 10g, first switch to boat 10a before switching). In other words, it is necessary to repeat forward and reverse rotation of the six-way switching valve 10 (knob), and when switching from port 10b to port 10g, it passes through two ports 10C, 10d, and also port 1.
When switching from 0g to 10e, one port 10f is passed.

For this reason, there is a problem in that the control of the motor that operates the six-way switching valve 10 becomes complicated.

The present invention has been made in view of the above circumstances, and its purpose is to reduce the number of erroneous operations.Secondly, control can be simplified during automation. The objective is to provide a polynucleotide synthesis device that can perform the following steps.

That is, the present invention provides a reactor, a reagent bottle and a solvent bottle filled with reagents, solvents, etc. necessary for a polynucleotide synthesis reaction and removably taken into a directly visible part; It has a plurality of reagents connected to a solvent bottle, a solvent port, and a common boat connected to the reactor, and the flow path connecting each reagent bottle, solvent bottle, and reactor can be controlled by rotating a knob. It is characterized by comprising a plurality of fiM switching valves and a liquid supply means for feeding reagents, solvents, etc. from each reagent bottle and solvent bottle into the reactor using the pressure of an inert gas.

Therefore, according to the present invention, it is possible to further reduce the number of erroneous operations that occur in conventional devices, and the switching valve can be easily controlled during automation.

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

FIG. 2 shows a flow sheet of the entire synthesis apparatus of the present invention. In the figure, 20 is a nitrogen cylinder, 21 is a distributor 123, 24 is a reagent bottle, 25.26 is a solvent bottle, 27.28 is a switching valve, 29 is a three-way cock, 30 is a reactor, 31 is a six-way cock, and 32 is a A waste liquid tank, 33 is a vacuum pump, and 34 is a safety valve.

Bottle 23 is filled with an inactivating agent and an inactivating aid, bottle 24 is filled with a deprotecting agent (detritylation agent)/solvent ■ solution, bottle 25 is filled with solvent I, and bottle 26 is filled with solvent ■. There is.

In this embodiment, the reagent bottle 5 (condensing agent/solvent "solution") shown in FIG. .

The N2 gas in the nitrogen cylinder 20 is supplied to the distributor 2.
1, and after being diverted here, the reagent bottles 23.24
and sent to solvent bins 25 and 26.

The 1r force of this N2 gas flows from each bottle 23 to 26 to the reactor 30.3 via the switching valves 27, 28 and the three-way cock 29.
Reagents and solvents are sent to 0.

The nitrogen cylinder 20 may be installed inside the case of the device. In this way, the trouble of connecting the nitrogen cylinder 20 each time during use can be saved.

The switching jp21.28 is a characteristic part of the present invention, and the bin 2
Channels 35a-35 between 3-26 and reactor 30.30
e, switch 36. The switching valve 27 has a bin 24.
Reagent/solvent switching boat 37a connected to 25.26
, 37c, 37e, and N2 gas switching boards 1-37b, 37d, 37f connected to the distributor 21.
, 37h and the switching board 1- connected to the vacuum pump 33.
37g, and a common boat 37Q connected to the reactors 830 and 3G via a three-way cock 29.

The switching valve 28 also includes switching balls l-37i, 37m, 37, and 370 for reagents and solvents connected to the bottles 26, 23.25, and N-370 connected to the distributor 21.
2 gas switching bow 1-373, 3? 1 y 37
n * 37 p and three-way cock 2 in reactor 30.30
A common boat 37q' connected via 9 is provided. Note that the part indicated by a black circle in the figure is the liquid supply stop part.

These switching ports 1-37a to 37p are arranged in the order of liquid supply/drainage operation, and the switching ports 1-37a to 37 of one of the switching valves 27 are
? The liquid supply and drainage operation in one condensation process can be completed by switching the switch 1-37i to 37p of the other switching valve 28. In addition, when switching to the switching boat 37g, the vacuum pump 3
3 to perform azeotropic hydrophilization.

3a and 3b show an example of the structure of the switching valve 27. FIG.

A common boat 37q is formed at the center of one end surface of the valve body 3B, and switching boats 37a, 37c, and 376537g are formed on the circumference around this common boat 37q, and switching boats 3? b, 37d, 37f, and 37h are formed.

A common bow) 37q and a switching port 1-37 extend from one end surface to the circumferential surface of the rotor portion 39 rotatably attached to the valve body 38.
A communication groove 39a is formed to communicate with a to 37h.

A spring 40 is provided between the valve body 3B and the rotor portion 39.
A thrust ball bearing 41 and a click mechanism 42 are provided.

The spring 40 urges the rotor portion 39 toward the valve body 38 to prevent a gap from forming therebetween, thereby ensuring sealing between the switching boats 378 to 37g.

The thrust ball bearing 41L and the spring 40 prevent one rotation of the rotor portion 39 from becoming heavy.

The click mechanism 42 includes a ball plunger 43 that is provided on the valve body 38 and slides on the other end surface when the rotor section 39 rotates, and a switching ball 1-378 to 37g and a liquid supply at a position where the ball plunger 43 on the other end surface slides. It is composed of four parts 44 provided with "C" corresponding to the stop part, and the rotor part 3
9 is positioned at each of the switching boats 37a to 37g and the liquid supply stop portion.

An operating knob 45 is attached to the end r of the rotor portion 39 protruding from the valve body 38, and by rotating the rotor portion 39 with the operating knob 45, the common boat is connected to the common boat via the communication groove 3.98. Switching boards I.3.7a to 37g communicating with 37q are switched.

When automating the apparatus, a stepping motor or a servo motor is connected to the rotor section 39, and the motor is sequentially controlled according to a program installed in a control device. At this time, since the reagent and solvent boats L 37a to 37g are arranged in the order of liquid supply operation, there is no need to pass one or two boats as in the device shown in Fig. 1, and the motor control is controlled. It becomes easy. Note that a drum-type sequencer may be used to drive the motor.

The structure of the switching valve 27 is not limited to the above-mentioned one, and the point is that the switching valves 1-37a to 37g may be arranged in the order of liquid supply/drainage operation.

Note that the switching valve 28 is configured in the same manner as the switching valve 27, so a description of its structure will be omitted.

The six-way cock 31 serves as the six-way cock 15 shown in Fig. 1 and the cocks 16a, 16a of the reactors 16, 1'6, and connects the top of the reactor 30, 30 with outside air during liquid supply.
At the same time, close the bottom (see the solid line in Figure 2), cut off the communication between the m part of the reactor 3 (1;
(Dotted line box 11?A in the same figure).

As with the switching valves 27 and 28, this six-way cock 31 can also be automated by connecting a stepping motor or the like and driving the motor according to a program built into a control device.

FIG. 4 shows the external appearance of the synthesis apparatus. case 46
The reactors 30, 3 are placed in the recess 47 provided on the front surface of the
0 is attached via an arm 48a of a shaker 48 (see FIG. 2). Further, on the front of the case 46, there are operating knobs 45, 49, 50, 51 for the switching valves 27, 28, three-way cock 29, six-way cock 31, a pressure gauge 52, and an operating knob 53 for a timer (not shown). , and a power switch 54.

Further, the side surface of the case 4G is provided with recesses 55 and 56, and the upper four mounting parts 55 are equipped with bins 24, 25...26, and the lower four mounting parts 56 are equipped with bins 24, 25... Equipped with a bottle 23, the remaining amount of reagents and solvents can be directly viewed from the outside, and it is easy to replenish the liquid and has good operability.
There is no risk of incorrect operation. Although not shown in detail, mounting portions 23a to 26a for airtightly mounting the openings of the bottles 23 to 26 are arranged on the ceiling portions of the first recesses 55 and 56.

Next, the operation of synthesizing DNA using the above synthesizer will be explained.

First, the reactor 30.30 is filled with Zabo 1- (silica gel, etc.) to which nucleosides are bonded, and the Zabo 1- (silica gel, etc.) is wetted with solvent I.

After completing the preparatory operations in this manner, detritylation is performed. In this detritylation step (1), the common boat 37q of the switching valve 27 is switched to the switching boat 37a, and the deprotecting agent/solvent solution is sent from the reagent bottle 24 to the reactor 30.30. When the predetermined amount is reached, switch to boat 37b and press N.
The deprotecting agent/solvent solution remaining in the flow path 36 is sent to the reactor 30.30 by the 2 gas. Next, it is switched to the liquid supply stop section 3 2 . After that, the shaker 48 is operated, and after a predetermined period of time, the hexagonal cock 31 is set to T3 L OW (second
(dotted line condition in the figure). Then, switch to boat 37 b,
The tax protection agent/
? Drain the 8th agent f6 liquid into the waste liquid tank 32°32.

υ[When the liquid is finished, switch to BO 1-37c, set the hexagonal cock 31 to FEED (solid line state in Fig. 3),
Washing is carried out with solvent l. In this cleaning step (IT), the switching valve 27 is switched in the order of boat 37c → 37d → liquid supply stop part to send solvent I from solvent bottle 25 to reactor 3 (1, 30) without leaving it in flow path 36. After operating the shaker 48 for a predetermined time, the hexagonal cock 31 is turned on.
W, then switch the switching valve 27 to the boat 37d,
Add the solvent l to FJF, and after the completion, turn the hexagonal cock 31 to F.
Make it EED.

The detritylation step (■) and the washing step (n) may be repeated multiple times if necessary.

After this, cleaning is performed with solvent H. In this cleaning step 4 (ITI), the switching valve 27 is
→ Switch to 37'f-liquid supply stop part, and
from the reactor 30.30 without leaving any solvent in the flow path 36.
send to. After operating the shaker 48 for a predetermined time,
The six-way cock 31 is set to BLOW, and then the switching valve 27 is switched to 1-37f to drain the solvent, and after completion of this, the six-way cock 31 is set to FEED.

This washing step (■) may also be repeated multiple times if necessary.

After doing this, raw materials are injected. In this raw material injection step (rV), the tops of the reactors 30' and '30 are opened, and a raw material solution in which a monomer salt represented by the following structural formula is dissolved in a solvent (2) is injected.

Note 1 DMTr ・(R1)dB-0-P-0"'N"H(
R2)3O-φC1 Here, R1 is a protecting group (such as a benzoyl group), R2 is a haalkyl group, and B- is a nucleic acid such as adenine (A), guanine (G), cytonine (C), or thymine (T). It is a base.

Note that a dimer salt may be used instead of a monomer salt.

Next, water in the raw material is azeotropically dehydrated under reduced pressure. In this dehydration step (V), the six-way cock 31 is fully closed (located between the solid line and the dotted line shown in FIG. 2). Then, the switching valve 27 is switched to the boat 37g, and the vacuum pump 33 is operated. As a result, the pressure inside the reactor 30.30 is reduced, and the water dissolved in the solvent (1) from the raw material is azeotropically dehydrated.

When dehydration is completed, switch to boat 37h and fill it with N2 gas. After this, the switch is switched to stop the liquid supply, and the six-way cock 31 is set to FEED.

After this, condensation occurs. In this condensation step (VT), the tops of the reactors 30, 30 are opened, the condensing agent Zfg agent (2) is introduced, the shaker 48 is operated, and the heater (1!
The reactor 30.30 is heated by. After the condensation reaction is complete,
Shaker 48 stops the operation of the heater. Then, the hexagonal cock 31 is set to BT, 05W, the switching valve 27 is switched to the boat 37h, and the liquid is drained. After draining is completed, turn the six-way cock 31 to FEED.

□Next, wash with solvent ■. In this cleaning step (■), the switching valve 28 is switched in the order of boats 37i-=37j--pickup stop and liquid supply. The operation details are cleaning process (ITI)
is the same as

This washing step (■) may also be repeated multiple times if necessary.

Next, capping is performed. This camping process (
■) Then, connect the switching valve 28 to the boat 37! - The inactivating agent disappears from the reagent bottle 23 in the order of the liquid supply stop part.
Deactivation auxiliary agent/solvent ■ The molten liquid is fed into the reactors 3'0, 30. Next, after operating the shaker 48 for a predetermined time, the hexagonal cock 31 is set to BLOW, and the switching valve 28 is set to BLOW 1-3.
7j! Switch to drain the liquid. After draining is completed, turn the six-way cock 31 to FEED. - Then wash with solvent ■. In the rice cleaning step (IX), the switching valve 28 is switched in the order of port 3fm -> port 37n - supply 7 6 liquid stop part. The operation details include the cleaning process (
IIT).

Then wash with solvent I. This cleaning worker fs! (X), the switching valve 28 is connected to the boat 37 Q->37 r+-.
Switch in the order of the liquid supply stop section. The operation details are the same as the washing step (nT).

Note that the cleaning steps (T)0 and (X) may be repeated multiple times if necessary.

The above steps (T), (TI), (II), (IV
).

(■), (IX), and (X) are repeated multiple times if necessary, but at this time, only the six-way cock 31 may be operated. For example, when repeating step (1),
While keeping the bow set to 1-37C, switch the hexagonal cock 31 in the order of FEED-4BLOW → FEED → BLOW. This makes it possible to reduce the number of operations.

By repeating steps (1) to ('X) in this manner, the nucleotide chains are sequentially condensed.

In this embodiment, the three-way cock 12 and reactor 1 shown in FIG.
Since the six and sixteen cocks 16a and 16a are also used as the eight switching valves 27 and 28 and the six-way cock 31, the number of valve operations can be reduced.

For example, when cleaning with a solvent (■), in the apparatus shown in FIG.
■ Operate the three-way cock 12 to close-open-close,
■Set the six-way cock 15 to FEED-4BLOW; ■Close-open-close the cock 16a; ■change the six-way cock 15 from BLOW to FIEIED.

On the other hand, in this embodiment, as described above, ■ the switching valve 27
3 boats? Switch in the order of e → 37f → liquid supply stop part, ■ Set the six-way cock 31 to FEED-BLOW, ■ Switch the boat to 37f, ■ Turn the six-way cock 31 to BLOW.
-Set to FEED.

That is, in this embodiment, the number of valve operations can be reduced by three.

In the embodiment described above, the switching valves 27 and 28 were each provided with eight switching boats, but the present invention is not limited thereto. For example, the boats 37C, 37d, 31m, and 31n can be omitted. The reason for this is that the deprotecting agent used in the detritylation process, the deactivating agent and the inert aid used in the camping process are mixed into Solvent I and Solvent II, respectively.
;To understand. That is, in the above embodiment, after the detritylation step (1), a washing step (II ), but immediately after this cleaning process (1), there is a cleaning process (II + > in which cleaning is performed with solvent ■), so cleaning process (11)
Even if this step is omitted, the deprotecting agent can be dissolved and removed in the washing step (TIT). In addition, after the camping step (■), a cleaning step (IX) is provided in which cleaning is performed with a solvent (■) in order to remove the inactivation aid remaining in the reactor 20.20. Solvent ■ immediately after (IX)
Since the cleaning step (X) is provided, the deactivating agent and the deactivating aid can be dissolved and removed in the cleaning step (X) even if the cleaning step (IK) is omitted.

In addition, the boats 30g and 30h can be omitted when there is no need to depressurize (boiling water).In this case, the boats 37o and 37p of the switching valve 28 can be omitted.
7, the boats 37o-"37p→37a→37b may be incorporated in this order.

FIG. 5 shows a case where the shaker 48 is not used and N2 gas can be bubbled.

N from the distributor 21 to the switching valves 27 and 28
An orifice (flow Itl1 regulator) 58 for flowing the amount of N2 gas necessary for bubbling is provided in the branch flow VR57 branched from the flow path 35e for sending two gases, and this branch flow path 57 is provided in place of the hexagonal cock 31. Connection is possible via a six-way cock 59 to the bottom of the reactor 30,30.

When the hexagonal cock 59 is set to FEED (solid line state in Figure 5), the branch flow path 57 connects to the reactor 30 via the hexagonal cock 59.
．． 30 and through the orifice 58
Two gases react! Bubbling is performed by blowing in from the bottom of 30.30.

Note that a bushing valve may be provided in the branch flow path 57, and bubbling 10 may be performed while the bushing valve is pressed.

Figure 6 shows a two-way three-way cock 0 instead of a six-way cock 31.
This shows the case where . When in solid line state, F F,
F, D, and becomes 13LOW when in the dotted line state.

FEEr), when about half of the liquid is fed into the reactor 30.30, the internal pressure becomes 1 kg/-, which is almost equal to the pressure of the N2 gas used to feed the liquid. Even without changing the valve, the internal pressure automatically stops the liquid supply, making it possible to further reduce the number of valve operations.

In the above embodiments, two reactors 30 are installed, but the invention is not limited to this, and one or three or more reactors 30 can be installed. When equipped, the configuration of the six-way cock 31 and the eight-way cock 59 will be different.

Further, although two switching valves are provided, the present invention is not limited to this, and in short, a plurality of switching valves is sufficient.

Synthesizer of the present invention! ff14 as mentioned above l) N
Not limited to the case of synthesizing A, RNA (ribonucleic acid)
It can also be used for the synthesis of

2. As explained above, according to the present invention, a plurality of switching valves are provided to switch the flow paths connecting each reagent bottle, solvent bottle, and reactor by one rotation of the operating knob. becomes easy to non-wealth, i! ! +i It is possible to reduce the number of works.

In addition, since the reagent bottle and solvent bottle are removably attached to a directly visible part, the remaining amount of the reagent and solvent can be directly viewed from the outside, and liquid replenishment is easy, and operability is good and there is no risk of erroneous operation.

Furthermore, when automating the process, there is no need to use a large number of solenoid valves; for example, it is only necessary to connect a stamping motor or a servo motor to a switching valve, and the control thereof is simple, making it easy to shift to automation.

Furthermore, unlike the case where all the reagent and solvent boats are arranged in one switching valve, the reagent and solvent boats are arranged separately in multiple switching valves, so the distance between the boats can be widened to ensure sealing performance. This improves the reliability of the valve.

[Brief explanation of the drawing]

FIG. 1 is a flow sheet of a conventional device, FIG. 2 is a flow sheet showing an embodiment of the present invention, FIG. A perspective view showing the external appearance of the device, Fig. 5 is a flow sheet showing an example of a pond capable of bubbling with N2 gas, and Fig. 6 is another example using a double three-way cock instead of a six-way cock. This is a flow sheet showing the following. 20... Nitrogen cylinder, 23-26...
Reagent, solvent bottle, 27.28...Switching valve, 30
...Reactor, 378-37p...Switching boat, 45,49...Operation knob, 55.5
6...Mounting recess. Patent Applicant Zeon Corporation Agent Hideo Takino Katsumi Ikeo 4

Claims (1)

[Claims] A reactor, reagent bottles and solvent bottles filled with reagents, solvents, etc. necessary for polynucleotide synthesis reaction and removably attached to a directly visible part, and a plurality of containers connected to the reagent bottles and solvent bottles. A plurality of tubes each having a reagent bottle, a solvent bottle 1-, and a common bottle I connected to the reactor, and the flow path connecting each reagent bottle, solvent bottle and the reactor can be switched by rotating an operating knob. 1. A polynucleotide synthesis apparatus comprising a switching valve and a liquid supply means for feeding reagents, solvents, etc. from each reagent bottle and solvent bottle into a reactor under the pressure of an inert gas.