JPH0465839B2 - - Google Patents

Description

[Detailed Description of the Invention] (a) Industrial Application Field The present invention relates to a DNA synthesis device for synthesizing DNA and RNA.

(B) Prior art The synthesis of DNA, etc. includes a step that requires avoiding the presence of water or moisture, and for this reason, for example, Japanese Patent Application Laid-Open No. 58-148897 and Japanese Patent Application Laid-open No. 58-148898
It has been proposed that a DNA synthesis device be equipped with a drying process, as disclosed in the issue.

However, the conventionally proposed drying process
This takes into consideration the period during which DNA synthesis is in operation from the start of synthesis to its completion, and does not take into consideration the period during which DNA synthesis is stopped, from the completion of DNA synthesis to the start of the next DNA synthesis.

However, in DNA synthesis equipment, even after DNA synthesis is complete, liquid remains in the nucleotide reagent container, etc., and this liquid absorbs and retains moisture, which may cause problems when starting the next DNA synthesis. be.

(c) Purpose of the Invention The present invention provides a DNA synthesis apparatus having a function of performing a drying process while taking into account the suspension of DNA synthesis.

(d) Structure of the Invention The apparatus for synthesizing DNA, etc. of the present invention automatically supplies each nucleotide reagent solution from a large number of nucleotide reagent containers to a reactor according to a predetermined procedure.
In an apparatus for synthesizing nucleotide reagents, a means for supplying a solvent for washing and drying the container to any one of a large number of nucleotide reagent containers, and a means for supplying a drying gas for discharging the solvent and providing a drying function. and a switching means for making the two supply means correspond to other nucleotide reagent containers in sequence, further comprising an operation control means capable of controlling the operation of the two supply means and the switching means at a desired time interval. The apparatus is characterized in that each nucleotide reagent container after the completion of the synthesis reaction can be automatically washed and dried with a solvent and then with a drying gas by the operation of the attached operation control means.

(e) Example 1 shown in FIG. 1 is an example of the present invention, which is an automatic DNA synthesis apparatus using the phosphotriester method.

2 to 6 are reagents or solvents stored in tanks, respectively. 2 is a masking reagent consisting of a mixture of acetic anhydride and pyridine, 3 is a masking condensing agent consisting of a mixture of dimethylaminopyridine and pyridine, and 4 is a masking reagent consisting of a mixture of dimethylaminopyridine and pyridine. A reaction solvent consisting of pyridine,
5 is a protecting group removal reagent consisting of a mixed solution of trichloroacetic acid and methylene chloride, and 6 is a washing/drying solvent consisting of methylene chloride.

A solvent valve 7 is a six-way rotary valve, and is connected to any one of the ports 7a to 7f, 7g.

8 is a liquid sensor that detects the presence or absence of liquid in the flow path.

9 to 13 are three-way solenoid valves, respectively.

14 is a reient valve consisting of a 10-way rotary valve with two flow paths, and its ports 14a and 14
A, 14b and 14B..., 14j and 14J, one of the port pairs 14k and 14K
The two are connected to each other. Note that in FIG. 1, ports 14e to 14J are omitted for convenience of drawing.

16 to 19 are nucleotide reagent preparation containers. There are also nucleotide reagent adjustment containers 20 to 25 corresponding to ports 14e to 14J (not shown) of the reagent valve 14, but they are not shown in FIG. 1 for the sake of simplification.

26 is a reactor. 27 and 28 are 2 each
It is a solenoid valve.

Reference numeral 29 is a control circuit for controlling the above-mentioned valves, etc., and 30 is an operation console for the control circuit 29.

When synthesizing DNA, as shown in Figure 2,
First, two types of powder reagents A and B are contained in the nucleotide reagent preparation container 16, supported by filters 16a and 16b. Powder reagent A in the lower row is a crystalline condensing agent [2-4-6-trimethylbenzenesulfonyl-3-nitrotriazolide (MSNT)],
Powder reagent B in the upper row is a nucleotide reagent in a crystalline state.

Condensing agents and nucleotide reagents are similarly stored in other nucleotide reagent preparation containers 17 to 25.

Next, a support to which only the terminal portion of the DNA molecule is bound is placed in the reactor 26, and a start command is given via the operator console 30.

As a result, the control circuit 29 connects the ports 7f and 7g with the solvent valve 7, and the three-way solenoid valve 9,
12 and 13 in the flow path position shown by the solid line in Figure 1, and 1
0 and 11 are in the flow path position indicated by the broken line, and the two-way solenoid valve 2
Since 7 and 28 are closed, the nitrogen gas pressure is used to clean and
A drying solvent 6 is supplied to the reactor 26 and stored therein. After a predetermined period of time, the three-way solenoid valve 9 is moved to the flow path position indicated by the broken line to stop the solvent 6, and the two-way solenoid valve 27 is opened to remove the solvent 6 that has entered the flow path from the three-way solenoid valve 9 to the reactor 26. are all fed into the reactor 26, and the two-way solenoid valve 27 is closed. After leaving it as it is for a while, the three-way solenoid valve 13 is set to the flow path position indicated by the broken line, the one-way solenoid valve 28 is opened, and the solvent 6 in the reactor 26 is discharged under nitrogen gas pressure. As described above, the support is preliminarily washed.

While draining the solvent 6 from the reactor 26 in the pre-wash, the solvent valve 7 connects ports 7a and 7.
g is communicated, the three-way solenoid valve 9 is placed in the flow path position indicated by the solid line, and the 3-way solenoid valve 12 is placed in the flow path position indicated by the broken line.
The solvent 6 remaining in the flow path from the solvent valve 7 to the three-way solenoid valve 9 will be discharged.

After preliminary cleaning, the masking reagent 2 is supplied to the reactor 26 by operating the same valves, etc.
Also, a masking condensing agent 3 is supplied. This blocks the reactive groups at the ends of the support and the DNA molecules bound thereto with protecting groups. In other words, it is masked.

After masking, a cleaning/drying solvent 6 is supplied to the reactor 26 to perform cleaning.

Next, when the protecting group removal reagent 5 is supplied to the reactor 26, the protecting group blocking the reactive group at a predetermined site on the end portion of the DNA molecule bonded to the support is removed, making it possible to react. becomes.

After removal of the protecting group, a washing/drying solvent 6 is supplied to the reactor 26 for washing, and then nitrogen gas is passed through the flow path and the reactor 26 for a predetermined period of time. As described above, the reactor 26 and the like are cleaned and dried.

After washing and drying, ports 7d and 7g are communicated with solvent valve 7, three-way solenoid valves 9 to 13 are placed in the flow path position shown by the solid line, and ports 14a and 14k, 14A and 14K are communicated with regent valve 14, respectively. The reaction solvent 4 flows through the nucleotide reagent preparation container 16, and at this time, the nucleotide reagent A and the condensing agent B are dissolved and supplied to the reactor 26 as a reagent solution. After a predetermined period of time, the three-way solenoid valve 9 is moved to the flow path position indicated by the broken line to stop the reaction solvent 4, and the two-way solenoid valve 27 is opened to enter the flow path from the three-way solenoid valve 9 to the reactor 26. All of the reaction solvent 4 is sent to the reactor 26, and the two-way solenoid valve 2
Close 7.

In the reactor 26, the condensation reaction progresses over time, and a new nucleotide is linked to a predetermined site at the end of the DNA molecule bound to the support.

After a predetermined condensation reaction time, the three-way solenoid valve 9 is placed in the flow path position indicated by the solid line, and the two-way solenoid valve 28 is opened to allow the reaction solvent 4 to flow through and clean the flow path and the reactor 26.

Thereafter, the process returns to the masking process and repeats the following series of processes. However, nucleotide reagent preparation containers 17 to 25 are used in sequence.

As a result, the nucleotide reagent adjustment container 16
DNA having the same base sequence as that of the nucleotide reagent placed in ~25 is synthesized.

Now, when the final condensation reaction is completed and the subsequent washing with the reaction solvent 4 is completed, the control circuit 29 connects the ports 7f and 7g with the solvent valve 7,
The three-way solenoid valves 9 to 13 are placed in the flow path position shown by the solid line, and the regent valve 14 is connected to ports 14a, 14k, and
4A and 14K are connected to each other, and the two-way solenoid valve 28 is opened. As a result, the washing/drying solvent 6 flows through the nucleotide reagent preparation container 16 and is washed. After cleaning for a while, connect ports 14b and 14k, 14B and 14K with regent valve 14.
When these nucleotide reagent preparation containers 17 are communicated with each other, the next nucleotide reagent preparation container 17 is washed. Thereafter, all the nucleotide reagent preparation containers are sequentially washed by switching the reagent valve 14 while the washing/drying solvent 6 is kept flowing in the same manner.

After cleaning the nucleotide reagent adjustment containers 16 to 25, the three-way solenoid valves 10 and 11 are placed in the flow path position indicated by the broken line, and the flow path between the two solenoid valves 10 and 11 is cleaned.

Next, the solvent valve 7 connects ports 7a and 7.
The solvent channel is dried by communicating with g and flowing nitrogen gas. Furthermore, after that, 3-way solenoid valve 1
0 and 11 are the flow path positions indicated by the solid line, and by switching the reagent valve 14, nitrogen gas is passed through all the nucleotide reagent adjustment containers 1 in sequence.
Dry 6-25.

After the above washing and drying is completed, the apparatus 1 is stopped, but since there is no liquid left in the nucleotide reagent preparation containers 16 to 25 due to the final washing and drying, the apparatus 1 is stopped for a long time. The liquid will not absorb or retain moisture even if the liquid is in the water, and the next operation of the apparatus 1 can be carried out suitably.

Next, another embodiment will be described. The device configuration is basically the same as the device 1 shown in FIG. 1, except that the operation of the control circuit 29 is different. When the last condensation reaction is completed and the subsequent washing with the reaction solvent 4 is also completed, the control circuit 29 issues a signal to the operator and enters a standby state. Therefore, when the operator replaces all of the masking reagent 2, masking condensing agent 3, reaction solvent 4, and protecting group removal reagent 5 with washing/drying solvents and gives instructions again from the console 30, the control circuit 29 , ports 7b and 7g are communicated with the solvent valve 7, and the cleaning/drying solvent is passed through the channel from there. When the liquid sensor 8 confirms that the cleaning/drying solvent is used up, nitrogen gas is supplied for a while. Drying is carried out by continuous flow, and this is repeated in the same manner for each port 7c to 7e of the solvent valve 7, and for the last port 7f, cleaning and drying of the nucleotide reagent adjustment containers 16 to 25 are also carried out in the same manner as in the device 1. Wash and dry.

In the apparatus of this embodiment, since no liquid remains in the flow paths of the entire apparatus, it is possible to almost completely eliminate the adverse effects of moisture absorption during the period when the apparatus is stopped.

Other specific examples of cleaning and drying solvents include volatile solvents such as dioxane, tetrahydrofuran (THF), and acetone.

Other specific examples of drying gas include argon gas.

(F) Effects of the Invention According to the DNA synthesis apparatus of the present invention, at least the nucleotide reagent container is completely dried when the apparatus is stopped, and no liquid remains. This prevents any adverse effects during the operation of the system, and enables the synthesis of DNA, etc., even after a long period of equipment shutdown.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the configuration of one embodiment of the apparatus for synthesizing DNA, etc. of the present invention, and FIG. 2 is a sectional view of one of the nucleotide reagent preparation containers of the apparatus shown in FIG. 1. DESCRIPTION OF SYMBOLS 1...Automatic synthesizer for DNA etc., 6...Washing/drying solvent, 7...Solvent valve, 14...Regient valve, 16-19...nucleotide reagent adjustment container, 26...Reactor.

Claims (1)

[Scope of Claims] 1. In an apparatus for synthesizing DNA, etc., in which each nucleotide reagent solution is automatically supplied from a large number of nucleotide reagent containers to a reactor according to a predetermined procedure, any one of the large number of nucleotide reagent containers is provided. A means for supplying a solvent for washing and drying the container and a means for supplying a drying gas for discharging the solvent and providing a drying function are attached to the container, and the two supply means are sequentially connected to other nucleotide reagent containers. A switching means corresponding to the above is provided, and an operation control means capable of controlling the operation of the two supply means and the switching means at a desired time is provided, and the synthesis reaction is automatically terminated by the operation of the operation control means. A feature of the present invention is that each subsequent nucleotide reagent container can be washed and dried using a solvent and then a drying gas.
DNA synthesis equipment.