DE3425763A1 - Method for the synthesis of polynucleotides - Google Patents

Abstract

A device for the synthesis of polynucleotides comprises a reactor device (2) which is mounted on the front plate (1) of an apparatus body (A); the device furthermore has a receiving arrangement (1b, 30a, 30b), which is located in an inspectable position on the outside of the housing (B) of the apparatus body (A), to receive detachable bottles which are filled with liquids such as, for example, a reagent or solvent, which are necessary for the reaction to synthesise the polynucleotides. The device furthermore comprises a selection valve device which is located in a passage between the bottles (4-9) and the reactor device (2) in order selectively to connect one of the bottles to the reactor device, and an introduction device (18) for introducing the liquids into the reactor device. <IMAGE>

Description

The invention relates to apparatus for synthesizing polynucleotides, and more particularly to apparatus for synthesizing of polynucleotides that is easy to use.

A well known method for synthesizing polynucleotides such as DNA (deoxyribonucleic acid) includes Process in which a carrier chemically linked to nucleosides is used and in which nucleotides by means of a phosphorus triester process, a phosphorus diester process, a phosphide process, inter alia, are sequentially condensed. In this synthesis method, processes such as washing - * ■ removal of protective groups ■> Washing - * ■ Condensation reaction - »■ Washing, etc. with little variation the processes repeated. In many cases, however, complicated work processes that have to be carried out repeatedly are also required.

Recently, there are various synthesizing devices of DNA has been proposed to simplify the synthetic process.

For example, such a known device for Synthesize DNA on a manually operated valve that allows the reagent and solvent used for the synthesis reaction is required to be fed to a reactor (a reaction vessel) by processes such as washing - »- Protection group removal -> - washing - »■ condensation reaction - * ■ Washing to be carried out. However, since it is necessary to operate many valves, there is a possibility of incorrect operation. When such misoperation occurs, all operations must be carried out again from the beginning, which makes all previous workflows superfluous.

There continue to be the reagent bottles and the solvent bottles are accommodated in a housing of the above-mentioned device, is to determine the remaining amount in the bottle an annoying opening of the side panel of the housing is necessary, and there is a possibility that the process could be continued without making sure of this residual amount. Farther it is difficult to replace the reagent bottle and the solvent bottle.

Accordingly, it is an object of the present invention to provide a new apparatus for synthesizing polynucleotides to specify.

Another object of the invention is to provide a device for synthesizing polynucleotides that is easy to handle can.

Finally, it is an object of the invention to provide a device for the synthesis of polynucleotides in which the possibility an incorrect operation is switched off, and it also allows a determination of the remaining amount in the reagent bottle etc. in an easy way.

This object is achieved with a device specified in the preamble of claim 1, which according to the invention is designed according to the manner specified in the characterizing part of this claim.

Further, advantageous refinements of the invention emerge from the subclaims.

An apparatus for synthesizing polynucleotides according to the present invention The invention comprises a reactor device mounted on the faceplate of the body of the apparatus and a receptacle device in a visible position on the outside

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a housing of the body to detachably receive bottles filled with liquids such as a reagent and a solvent, necessary for the polynucleotide synthesis reaction are filled; it also includes a valve selector at a passage between the bottles and the reactor device to selectively use one of these bottles to connect the reactor device; finally it has a supply device for supplying the liquids to the Reactor device on.

Accordingly, the present invention can keep the remaining amount of liquid in the reagent and solvent bottles easy to determine, and liquid can be easily introduced into the bottles. Furthermore, the operational readiness improved, and there is no possibility of incorrect operation.

In the following, the invention is illustrated with reference to the figures Embodiments described and explained in more detail.

Fig. 1 is a perspective view of an embodiment the synthesis apparatus according to the present invention;

Figure 2 is a flow diagram for the embodiment of Figure 1; Figure 3 shows details of a cross section through the top of the reactor;

Fig. 4 shows a side view of the reactor with a heating device thereon;

Fig. 5 shows a cross section corresponding to Fig. 4;

Fig. 6 shows the flow chart of a further embodiment a synthesizer with two reactors; 7 shows a perspective view of the synthesis device according to Fig. 6; and

Fig. 8 shows a perspective view of a further embodiment of the synthesis device.

Preferred embodiments will now be described with reference to the drawings.

Figs. 1 and 2 show an embodiment of a synthesis device according to the present invention, wherein the reference character A denotes the apparatus body or body of the device, the reference character B denotes the housing and 1 the front panel.

At one end part of the faceplate (on the left side in Fig. 1) there is provided a recess 1a, and in the recess 1a there is a reactor 2, which is contained in the housing B by a support arm 3 of a (not shown) Vibrator is held.

Furthermore, in the central part up to the other end (on the right-hand side in FIG. 1) of the front panel there is a recess 1b provided as a receiving part for reagent and solvent bottles serves. The recess 1b formed in the central part takes a reagent bottle 4 for a deactivator, a reagent bottle 5 for a solution of a condensing agent with a solvent II, and a bottle 6 for a solution from a deactivation tool in a solvent II among others, these reagent bottles have a little content. Furthermore, the recess 1b at the other end takes reagent and solvent bottles off large contents, a bottle 7 a solvent, a bottle 8 a solution of a protective group removing agent in a solvent I and a bottle 9 a Contains solvent II or similar. Since the solvent I, which prevents the condensation reaction, is widely used the solvent II, which does not hinder the condensation reaction, should be used in the condensation process will. Furthermore, mounts 1c-1h are provided in the ceiling part of the recess 1b, albeit these mounts

are not shown in detail, these brackets serve to detachably hold the openings of the bottles 4-9.

Furthermore, in the central part of the front panel 1, there is an operating button 11 for an eight-way selector valve, an actuator button 13 for a two-way selector valve 12, an operating button 15 for a four-way selector valve 14, an operating button 16 for a timer (not shown) of a vibrator, a main switch and others.

The housing B contains a portable nitrogen cylinder 18 small content and a manifold 19 in addition to the eight-way selector valve 10, the two-way valve 12, the four-way valve 14, the vibrator (not shown) and others, as above was shown. Since, as described above, the nitrogen cylinder 18 is in the housing B, is there is no need to use a large nitrogen cylinder when using it e.g. to wear near body A of the device or to carry body B of the device to one point which is the nitrogen cylinder. Furthermore it is not necessary to use the nitrogen cylinder with the manifold connect to.

The nitrogen cylinder 18 is connected to the inlet port through a pipe 21a 19a of the distributor 19 connected, and if that Valve 2 0 of the nitrogen cylinder 18 is opened, so will N "gas is supplied to the inlet port 19a. The N ^ gas is from the inlet port 19a out to each outlet port 19b-19i of the distributor 19 distributed.

The openings 19b-19g are connected to the holders 1c-1h via pipes 21b-21g, and N ₂ ~ gas is supplied to the bottles 4-9 mounted in the holders 1c-1h. The opening 19h is via a pipe 21h with the

The inlet port 10a of the eight-way selector valve 10 is connected, and the port 19i is connected to an opening via a pipe 21i 14a of the four-way valve 14 connected.

The other inlet ports 10b-10g of the eight-way selector valve 10 are connected to the holders 1c-1h through pipes 22b-22g, and when N ~ gas is supplied to the bottles 4-9, liquids in the bottles 4-9 become , such as a reagent and a solvent, due to the pressure of the N ₂ gas to the openings 10b - 10g conveyed out.

A common outlet port 10h of the eight-way selector valve

10 is connected to the two-way valve 12 via line 221, and an outlet of the two-way valve 12 is connected to the upper inlet 2a of the reactor 2 via a pipe 22a.

If, for example, according to FIG. 2, the common opening 1Oh due to the actuation of the operating button

11 is brought into connection with the opening 10b, so becomes the deactivator located in the bottle 4 is fed to the reactor 2 due to the pressure of the N ^ gas. Although at In this embodiment, an eight-way selector valve 10 is used corresponding to the number of bottles 4-9, is in a case where the reagent is a polynucleotide raw material contains, it is necessary to use a selection valve with a larger number of selection positions.

The other opening 14b of the four-way valve 14 "is via a Pipe 22i is connected to the upper inlet 2a of the reactor 2, and an opening 14c is connected to the bottom-side via a pipe 22j Outlet 2b of reactor 2 connected. An opening 14d is a drain port, and it is connected to a pipe 22k. Under the conditions illustrated in Figure 2 (i.e. if the operating button 15 is set to "BLOW") N ~ gas is supplied via the openings 14a and 14b and via the pipe 22i

is supplied to the upper inlet 2a of the reactor 2, and after passing through the inside of the reactor 2, it flows therefrom Bottom outlet 2b via pipe 22j and openings 14c and 14d, and it is drained through the pipe 22k. When the control button is set to "FEED", the openings 14a are brought into communication with the opening 14d so that the N ~ gas can flow from the floor to the ceiling of the reactor 2.

In Fig. 3, the upper part of the reactor 2 is shown in detail. According to FIG. 3 is at the upper inlet 2a of the reactor 2, a substantially cross-shaped inlet pipe 23 is provided for starting material. If the source material, such as a nucleotide, only one cap needs to be removed without the need to put one in the top Inlet 2a to remove fitted plug 25. A branch 23a of the injection pipe 23 for starting material is is connected to the pipe 221, and a branch 23b of the pipe 23 is connected to the pipe 22i.

According to FIGS. 4 and 5, the reactor 2 is provided with a clamp-like heating device 26. The heater 26 includes a heater 26a, which consists of a flexible, plate-like heating material attached to its inner Surface for receiving electric current is attached via a lead wire 26b, so that the reactor 2 is heated and the reaction is accelerated. Although one can consider the heater to heat the reactor 2 2b around the reactor, while vibrating the reactor, it is possible that the heater 26 is separated from the reactor 2 due to the vibration. It is necessary to wind the heater 26 around the reactor 2 and securely attach the heater 26 to the reactor 2 to prevent the heater from separating from the reactor.

However, the heater 26 is not necessary if the condensate

cation reaction is carried out while it is then necessary is when washing and deprotection are carried out will. If the heater 26 is attached to the reactor 2, there is the disadvantage that the supply of liquid, such as solvents, cannot see.

Thus, in the present invention, a heater is detachably attached to the reactor, the heater as the attachment means has a part for clamping to the reactor. Accordingly, the heater can be heated while it is set in vibration. The heater can easily be removed from the reactor. You can also change the hydration conditions make visible by removing the heater from the reactor when performing the washing or the Removal of protecting groups separates.

The following is the workflow for synthesizing DNA using the synthesizer described above described.

The operating button 15 of the four-way valve 14 is on "FEED" is set and valve 2c of reactor 2 is closed. Then the one associated with the nucleotide Carrier (e.g. silica gel) is filled into reactor 2, and reagent bottles 4, 5, 6 and 8 as well as the solvent bottles 7 and 9 are mounted in brackets 1c - 1h (I).

Then, the common port 10h is brought into communication with the port 10g by operating the operation button 11, and the two-way valve 12 is opened. As a result, the solvent II in the bottle 9 is supplied to _{the reactor 2 under the pressure of the N 2 gas.} Although the two-way valve 12 operates together with the selection process of the eight-way selector valve 10

is taken, a subsequent explanation of the operation of the two-way valve 12 is omitted for simplicity of explanation. When the washing of the solvent 2 is completed, the operating button 15 of the four-way valve 14 is switched to "BLOW" and the valve 2c of the reactor 2 is opened to drain the liquid. The valve 2c is then closed and the operating button 15 is set to "FEED". The operating button 11 is operated to bring the common outlet 10h into communication with the outlet 10d, and to further bring the common outlet 10h into communication with the outlet 10b. As a result of this, a solution in the bottle 6 of a deactivating aid and a solvent II and the deactivator in the bottle 4 are successively supplied to the reactor 2 due to the pressure of the N ₂ gas. After a standstill lasting a few minutes, the actuating button 15 is switched to "BLOW" and the tap 2c of the reactor 2 is opened in order to drain the liquid (II).

Then the operating button 15 is set to "FEED", and the operating button 11 is operated again to the common to connect opening 10h to opening 10g and the To supply solvent II to the reactor for washing. The liquid is then drained off in the same manner as before (III).

The operating button 11 is operated to the common opening 10h to connect to the opening 10e and to supply the solvent I to the reactor 2 for washing, and then to the Drain the liquid (IV). Then there is the joint opening 10h with the opening 10f in connection to the solution from the deprotecting agent and the solution I to the reactor, and a few minutes later drain the liquid (V). This process will take some

Times repeated.

Thereafter, the common port 10h is again brought into communication with the port 10e to supply the solvent I to the reactor 2 for washing, whereupon the liquid is drained. (VI) Then the common opening becomes 10h
communicated with the port 10g to wash the reactor with the solvent II, and then the
Drain the liquid (VII).

The reactor 2 is _{clarified with N 2} gas for a few minutes (VIII), and then a solution of a monomeric salt represented by the structural formula below, which is dissolved in the solvent II, is fed into the reactor 2 through the injection pipe 23 for starting material injected. Afterward
the common opening 10h is brought into communication with the opening 10c in order to supply a solution of the condensing agent and the solvent II to the reactor II (IX).
The solution of the monomeric salt dissolved in the solvent II can be introduced into the reagent bottle and the
Reactor 2 can be fed by pressure of the N ~ gas.

DMTr. (Rl) dB-OPO ^e A

Here, R1 denotes a protecting group (e.g. benzoyl); R2 is an alkyl group, B a nucleic acid base such as adenine (A), guanine (G), cytosine (C) and thymine (T).

After a standstill for about 40 to 50 minutes, the
Chemical in the reactor 2 is discharged (X), and the above operation (II) and subsequent operations are repeated. In this way the nucleotide chain is sequentially condensed.

3 4 2 5 7 G.

The monomeric salt can be replaced by a dimeric salt or a tri. more salt to be replaced. When the reagent and solvent, which remain in bottles 4 to 9 are essentially used up, bottles 4 to 9 are exchanged, or liquid is tracked. In this case, because the brackets 1c - 1h on the apparatus body are mounted, the replacement of the bottles and the replenishment of liquid can be carried out easily.

Because in the synthesis device described above, the reactor 2 does not protrude from the front plate 1, there is no possibility of that the operator accidentally touches the tap 2c during the work process and damages the reactor 2. In order to accelerate the reaction, the reactor 2 can be made to vibrate with the vibrator or can also be heated by the heater 26. Furthermore, it can be heated with the heater 26 and at the same time exposed to vibrations with the vibrator. The use time of the vibrator can be set with a timer device can be set.

6 and 7 is a "further embodiment of the Invention shown in which two reactors are provided. The reference characters A, B and 1 denote the apparatus body the device or the housing or the front panel.

A recess 1 a is provided in the front plate 1, and two reactors 2 are located in the recess 1 a, wherein these reactors 2 are carried in the housing B by support arms 3 of a vibrator (not shown). at with this arrangement there is no possibility of touching the taps 2c of the reactors during operation, and it is further not possible to damage the reactors 2 with other instruments.

There are also recesses 30a and 30b provided on one side of the housing B, these recesses as receiving parts serve for reagent and solvent bottles. In the lower recess 30a, reagent bottles are small Contents 4, 5 and 6 for the deactivator or the solution of the condensing agent and the solvent II or the Solution of the deactivation aid and the solvent II added. In the upper recess 30b, reagent and solvent bottles 7, 8 and 9 are large for the solvent I, the solution of the deprotecting agent and the solvent I and the solvent II added. Accordingly, it is from the outside of the housing, the remaining amount in bottles 4 to 9 was visually determined.

Brackets 1c-1b are provided on the ceiling parts of the recesses 30a and 30b, but these brackets are not shown in detail; These brackets are used to detachably fasten the openings of the bottles 4 to 9.

There are also actuation buttons in the central part of the front panel 11, 13, 15, 27 and 16 for an eight-way selector valve 10, a two-way selector valve 12, a three-way valve 27 (see. Fig. 6) and a (not shown) timer of the vibrator and a timer 17 attached.

The housing B contains a portable nitrogen cylinder 18 small content and a manifold 19 in addition to the eight-way selector valve 10, the two-way valve 12, the three-way valve 27, the four-way valve 14, the (not shown) Vibrator, etc. The nitrogen cylinder 18 is not necessary housed in the housing B, but if it is housed in the housing B, it is not in use necessary to carry the nitrogen cylinder next to the housing B, and it is further unnecessary to connect it to the manifold 19.

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The nitrogen cylinder 18 is connected to an inlet port 19a of the manifold 19 via a pipe 21a, and when the valve 20 of the nitrogen cylinder 18 is opened, N ₂ "gas is supplied to the port 19. The N ₂ gas is supplied from the port 19a to the outlet openings 19b - 19i of the manifold 19 distributed.

The openings 19b-19g are connected via tubes 21b-21g, respectively connected to the holders 1c-1h, and N ~ gas is supplied to the cylinders 4-9 mounted in the holders 1c-1h. Furthermore, the opening 19h is connected to the inlet opening via a pipe 21h 10a of the eight-way selector valve 10 connected, and the opening 19i is connected to the opening 14a via a pipe 21i of the four-way valve 14 connected.

Inlet ports 10b-10g of the eight-way selector valve 10 are connected to the brackets 1b-1h via pipes 22b-22g, and when N ~ gas is supplied to the pipes 4-9, then the reagent and solvent from the bottles 4-9 via the tubes 22b-22g to the openings 10b-10g due to the Pressure of the N "gas supplied.

The two-way valve 12 is via a pipe 22 with a common A discharge port 10h of the eight-way selector valve 10 is connected, and a discharge port of the two-way valve 12 is connected to an opening 27a of the three-way valve 27 is connected.

The other openings 27b and 27c of the three-way valve 27 are connected to the upper inlet openings 29 of the reactor 2 via pipes 22h. For example, when the operating buttons 12 and 16 are operated so that the eight-way selector valve 10 and the three-way selector valve 27 are brought into the state shown in FIG. 6 and the two-way valve 12 is opened, so will from the bottle 4 the deactivator is fed to the reactor 2 due to the pressure of the N ₂ gas.

3425753

The opening 14b of the four-way valve 14 is connected to the upper inlet openings 2a of the reactor 2 via a pipe 22i, and an opening 14c is connected to the lower outlets 2b of the reactor 2 via a pipe 22j. Furthermore, an opening 14d serving as a discharge port is connected to a pipe 22k. Under the conditions shown in FIG. 6 (when the actuating button 15 is set to "BLOW"), N ₃ gas is supplied to the upper inlet openings 2a of the reactor 2 via the openings 14a and 14b and the pipe 22i. It then passes through the interior of the reactor 2 and is conducted from the lower outlet 2b via the pipe 22j to the openings 14c and 14d and is drained off via the pipe 22k. When the control button. 15 is set to "FEED", ports 14a and 14c are brought into communication with each other, and ports 14b and 14d are also brought into communication, thereby _{moving the N 2} gas from the lower part to the upper part of the reactor 2 can flow.

The workflow for synthesizing DNA using the synthesizer described above is similar to that in the embodiment described with reference to FIGS. The reagent and the solvent can be fed to each reactor 2, which means that both reactors can be operated simultaneously with a single actuation 2 reached. If different raw materials are still injected (for example, an A monomer salt into one reactor, whereas a G-monomer salt is injected into the other reactor), it is possible in two ways of performing synthesis reactions at the same time. Furthermore, three reactors 2 can be installed at the same time to put all reactors 2 in operation or to carry out more than three types of synthesis reactions, so that one remarkable Reduction of the synthesis time achieved. In this embodiment, the bottles 4-9 are set back 30a, 30b housed, preferably on one side

of the housing B are formed in the front panel 1 instead. Numeral 31 in Fig. 7 denotes a manometer for N gas.

8 shows another embodiment of the present invention Invention. In this embodiment, a receiving part 19 is provided, which is through a partial plate 28 on the Rear side of the housing B is divided to accommodate the bottles 4-9 and the nitrogen cylinder 18. With this arrangement the nitrogen cylinder 18 can easily be replaced.

Although all the exemplary embodiments described above, the use show of N ^ gas as a means of supplying liquid, It is clear that the liquid supply device is not limited to N ^ gas and that a liquid supply pump can also be used or can use a pressure gradient.

Furthermore, the synthesis device is according to the present invention not limited to the application for the synthesis of DNA, but also for the synthesis of RNA (ribonucleins aure) can be used.

Since, according to the present invention, the receiving part (recesses 11 11b, 30a and 30b, and the receiving part 29) for the reagent and solvent bottles used for the synthesis reaction of polynucleotides are necessary, provided in a viewable area outside the housing and the Bottles are detachably mounted in the receiving part, it is possible to visually determine the remaining amount of the reagent, among other things and the bottles can be easily exchanged.

Because still in the reagent and solvent bottles with the reactor connecting line, a selector valve is provided, which is arranged with a single on the front plate If the dial is operated, it is possible to use a

Reduce misoperation to a remarkable extent.

Furthermore, because the reactor is mounted on the front panel, it is possible to operate the reactor and at the same time visually detect an operation such as washing - deprotection - washing, etc.

Because, after all, a manifold with a number of openings provided on the outlet side of the two-way valve 13 and each reactor with each opening of the manifold for simultaneous If the supply of reagent and solvent is connected to each reactor, it is possible to use DNA with different base numbers or synthesize different basic arrangements in a single operation, increasing the efficiency of the synthesis operation is remarkably improved.

RS / bi

Claims (9)

(■ 1. ) Device for the synthesis of polynucleotides, characterized by (A) a reactor device (2) which is mounted on the front plate (1) of the apparatus body (A), (B) a receiving device located in a part that can be seen on the outside of the housing (B) of the apparatus body (A) (1c - 1h), to detachably accommodate bottles that contain liquids, e.g. for a synthesis reaction reagent and solvent required by polynucleotides are filled, (c) a selector valve device (10, 12, 14, 27) along a line between the bottles (4-9) and the reactor device (2) to selectively connect one of the bottles (4-9) to the reactor device (2), and through (d) a feed device (18) for feeding the liquids to the reactor device (2). 2. Device according to claim 1,
characterized in that the reactor device (2) comprises a number of reactors, each of which is connected to the valve device (10) via a manifold (27) which has an inlet (27a) connected to the valve device (10) and a number of outlet openings (27b, 27c) which are each connected to the reactors (2). 3. Apparatus according to claim 1 or 2, characterized in that a heater (26) is provided which has a clamping part for Clamping to the reactor by means of a fastening device and a heating element (26a) which is inserted into the Clamping part used and removably mounted on the reactor device (2). 4. Device according to one of claims 1 to 3, characterized marked, that the receiving device comprises a recess (1b, 30a, 30b). 5. Device according to one of claims 1 to 4, characterized in that that the selection valve device (10) is actuated by a button (11) on the front panel (1) of the apparatus body will. 6. Device according to one of claims 1 to 5, characterized in that the supply device (18) comprises a source of pressurized gas. 7. Apparatus according to claim 6, characterized in that that the compressed gas is N ~ gas. 8. Device according to one of claims 1 to 7, characterized in that that another valve device (14) in the passage provided between the distributor (19) and the reactor (2) is to change the direction of flow of the gas supplied from the supply device in the reactor. 9. Apparatus according to claim 8, characterized in that the valve device (14) by a button (15) is operated on the front panel (1) of the device body.