JPS6118015Y2 - - Google Patents

Description

[Detailed description of the invention] This invention relates to a DNA synthesis apparatus, and particularly provides a DNA synthesis apparatus capable of synthesizing different types of DNA or RNA in parallel.

DNA synthesis methods have been improved and developed into the so-called diester method, triester method, and phosphite method, and the solid support method, which utilizes these methods and uses a solid support, is often used because it has various advantages. has reached. Various apparatuses for synthesizing DNA using these methods have also been proposed.

On the other hand, as a method for isolating genes that produce a specific protein made of a mixture of various genes, the base sequence of the gene is inferred from the amino acid sequence of the protein, and DNA with a complementary base sequence to that base sequence is extracted. There is a method of synthesizing it, marking it, adding it to a mixture, binding the synthesized DNA to the target gene, and then using the mark as a guide to pick up the target gene.

However, in many cases, multiple genetic codes exist for a single amino acid in a protein, resulting in many possibilities for the base sequence of a gene. In this case, all or part of the possible DNA
It is common to synthesize and mix them together.

However, with conventional devices of this type, it was not possible to synthesize multiple types of DNA at the same time, so multiple devices of this type were required, or one
Different DNAs must be synthesized one after another on the stand.
It was extremely inconvenient.

On the other hand, in most conventional automatic synthesis devices, the amount of solid support used is on a scale of 100 mg or more, and there are issues such as the required amount of DNA to be synthesized and the high cost of reagents such as raw material nucleotides with protective groups. Therefore, there was a desire for an automatic synthesis device on a smaller scale.

As a result of various studies, the creator of this idea
Succeeded in improving the DNA synthesis equipment.

Thus, according to this invention, in an apparatus in which a reagent solution, etc. supply means is connected to a reactor, and a plurality of reagent solutions, etc. are supplied to the reactor in a predetermined procedure to synthesize DNA, etc., at least one communication First and second outer layer blocks each having a plurality of communication holes for one communication hole of the middle layer block are provided on both sides of the middle layer block having holes, and the first and second outer layer blocks are provided with respect to the middle layer block. Double flow in which an arbitrary communication hole of the first outer layer block and an arbitrary communication hole of the second outer layer block are switched and connected via the communication hole of the middle layer block by relative sliding movement of the outer layer blocks. It is equipped with a path switching valve mechanism and a plurality of reactors, wherein a plurality of supply flow paths for reagent solutions, etc. are respectively connected to each communication hole of the first outer layer block, and each of the plurality of reactors is connected to the second outer layer block. A DNA synthesis device is provided, which is connected to each communication hole of the outer layer block, thereby making it possible to synthesize many types of DNA.

The main features of the device of this invention are (i) it is equipped with a dual flow path switching valve mechanism with a specific configuration, (ii) it is equipped with a plurality of independent reactors, and (iii) Any one reagent solution etc. can be supplied to any one reactor via the dual flow path switching valve mechanism.

This invention will be explained in detail below based on the embodiment shown in the drawings. However, this invention is not limited to this.

1 shown in FIG. 1 is an embodiment of this invention, and is an automatic DNA synthesis device using the phosphotriester method.

Reactors 2A, 2B, and 2C have an inner diameter of 8 mm and a height of 10 mm.
This is a container in which mortar-shaped flanges 4A, 4B, 4C are provided above the cylindrical main body 3A, 3B, 3C.
A stopper 5 in which a number of nozzles for supplying reagent solutions, etc. is inserted into the dovetail flanges 4A, 4B, 4C.
A, 5B, and 5C are installed. Therefore, the head openings of the main bodies 3A, 3B, and 3C become reagent solution supply ports 6A, 6B, and 6C. Main body 3A, 3B, 3C
Filters 7A, 7B, and 7C, such as glass filters, are fitted in the lower part of the interior, and drain ports 8A, 8B, and 8C are provided at the bottom. The filters 7A, 7B, and 7C are capable of mounting supports A, B, and C such as polystyrene and silica beads (not allowing them to pass therethrough), and allow reagent solutions, solvents, and gases to pass therethrough. Filter 7A, 7B, 7C
The upper space of is the reaction part 9A, 9B, 9C, and about
It is a space with a volume of 450μ. 10A, 10B,
10C is an exhaust valve, and 11A, 11B, and 11C are drain valves.

The nitrogen gas supply flow path switching means 12 is composed of three valves 14, 15, and 16, and includes three valves 14, 15, and 16.
Supply nitrogen gas to B and 2C. The nitrogen gas is dried with a desiccant 66 such as calcium chloride.

13, the cleaning solvent etc. are transferred to the reactors 2A, 2B, 2
This is a dual flow path switching valve mechanism for supplying any one of C. Basically two outer layer blocks 1
7 and 19 and one middle layer block 18, and the flow path is switched by slidingly moving the outer layer blocks 17 and 19, respectively, with respect to the middle layer block 18. This sliding movement is performed by separately driving motors 23 and 24 connected to the outer blocks 17 and 19.

Figures 2 and 3 show the above-mentioned double flow path switching valve mechanism 1.
3 is shown in more detail. The first outer layer block 17 is a disc body having a rotating shaft 25 connected to a motor 23, and has six communicating holes 20a to 20 arranged on the same circumference around the rotating shaft 25.
supply channels a to f for cleaning solvents and the like are connected to these communicating holes 20a to 20f. The second outer layer block 19 is a disc body having a rotating shaft 26 connected to a motor 24, and has three communicating holes 22α to 2 arranged on the same circumference centered on the rotating shaft 26.
2γ, and the reactors 2A to 2C are connected to these communicating holes 22α to 22γ. Middle block 18
is a cylindrical body having a partition wall 18a at the center of the internal space, and has one communication hole 21 in the partition wall 18a. The outer layer blocks 17 and 19 are slidably brought into close contact with both sides of the partition wall 18a, respectively, and are held by retaining rings 27 and 28. Outer layer block 17
By aligning a desired one of the communication holes 20a to 20f of the outer layer block 19 with a desired one of the communication holes 22α to 22γ of the outer layer block 19 to the communication hole 21 of the middle layer block 18, a desired cleaning solvent, etc. It can be fed to the reactor.

In detail, the cleaning solvents include pyridine 35 as a cleaning solvent, tetrahydrofuran [THF] 36 as a cleaning and drying solvent, a mixed solution of isopropanol and methylene chloride as a cleaning solvent 37, and isopropanol as a protecting group removing agent solution. These are a solution 38 in which zinc bromide is dissolved in a mixed solution of methylene chloride, a mixed solution 39 of acetic anhydride and pyridine as a masking reagent solution, and a mixed solution 40 of dimethylaminopyridine and pyridine as a masking condensing agent. These liquids are fed by nitrogen gas pressure.

The dual flow path switching valve mechanism 13' for supplying nucleotide reagent solution, etc. is the dual flow path switching valve mechanism 13'.
It has a similar structure. Therefore, corresponding elements are given the same number and a dash symbol. The outer layer block 1 of this double flow path switching valve mechanism 13'
The communication holes 20a' to 20f' of the outer layer block 19' are connected to supply paths a' to f' for nucleotide reagent solutions, and the communication holes 22α' to 22γ' of the outer layer block 19' are connected to the respective reactors 2A to 2C. are connected. Therefore, we used pyridine 35 as a washing solvent, 41, 42, 43, 44 nucleotide reagent solutions with adenine, cytosine, guanine, and thymine as bases, or 2-4-6-trimethylbenzenesulfonyl as a condensing agent solution. A pyridine solution 45 of -3-nitrotriazolide (MSNT) was added to the reactor 2A,
Can be supplied to 2B or 2C. These reagent solutions 41 to 45 are supplied in predetermined amounts by syringe pumps 51 to 55, respectively.

The plungers of the syringe pumps 51 to 55 include, for example, a pulse motor 61, a screw shaft 62, and a nut 6.
Plunger drive mechanism 56-6 consisting of 3
Driven by 0.

65 is a control circuit such as a microcomputer, which controls the valves 10A, 10B, 10C, 11A,
11B, 11C, 14, 15, 16, 33, 3
4, motors 23, 24, 23', 24', switching cocks 46-50, and plunger drive mechanism 56-
60. It also interacts with the operator via the console 46.

During DNA synthesis, first each reactor 2A, 2
Remove plugs 5A, 5B, 5C of B and 2C,
Support A to which only the terminal part of the DNA molecule is bound,
Insert B and C. This amount is, for example, support A,
If B and C are polystyrene powders, 10mg to 50mg
is suitable. After returning the plugs 5A, 5B, and 5C to their original positions, check the amount of supports A, B, and C that were put into each reactor 2A, 2B, and 2C, and each reactor 2A, 2B, and 2C.
The target DNA to be synthesized, including its base sequence, is input to the control circuit 65 via the console 64, and then a start command is input.

As a result, the control circuit 65 operates valves, switching switches, etc., and synthesizes the target DNA in parallel in each of the reactors 2A, 2B, and 2C.

That is, in the initial state, all the valves are "closed", the communication holes of the dual flow path switching valve mechanisms 13 and 13' are not aligned, and no reagent solution is supplied, but when the start command is issued, Control circuit 65
First, the communication hole 20 of the dual flow path switching valve mechanism 13 is
c and the communication hole 22α are aligned with the communication hole 21, and the valve 10A is opened. As a result, the cleaning solvent 37
is supplied to reactor 2A. After a predetermined period of time, the double flow path switching valve mechanism 13 and the valve 10A are returned to their initial states, and after a while, the valves 11A and 14 are opened.
As a result, the cleaning solvent 37 is drained from the reactor 2A. After a predetermined time, valves 11A and 14 are closed. This operation is repeated several times to perform the cleaning process shown at 70 in FIG. Thereafter, steps 71 to 75 in FIG. 2 are performed in the same manner in the reactor 2A.

Next, in the dual flow path switching valve mechanism 13, the communication hole 20c and the communication hole 22β are aligned with the communication hole 21, and the valve 10B is opened. Similarly, reactor 2B
In this step, the processes shown in FIGS. 70 to 75 are performed.

In this way, in the reactor 2B, 70 to 70 in FIG.
At the same time as performing the process in step 75, the control circuit 65 operates the double flow path switching valve mechanism 13' to switch the reactor 2A.
Supply the nucleotide reagent solution to the nucleotide reagent solution. For example, if a nucleotide having adenine as a base is needed next as a DNA base sequence, the communicating hole 20b' and the communicating hole 22α' are aligned with the communicating hole 21', and the switching cock 46 and the plunger drive mechanism 56 are operated. A predetermined amount of the nucleotide reagent solution 41 is supplied to the reactor 2A. After supplying the nucleotide reagent solution, the communication hole 20f' and the communication hole 22α' are aligned with the communication hole 21', and a predetermined amount of the condensing agent solution 45 is supplied to the reactor 2A using the syringe pump 60.

The amounts of the nucleotide reagent solution and condensing agent solution supplied are controlled such that their total amount is the minimum amount sufficient to swell the support. Specifically, for example, when the support is polystyrene powder, the amount of the nucleotide reagent solution and the condensing agent solution is about 3 ml and about 3 ml per 1 g of the support, respectively. This causes the support to swell approximately 5 to 7 times. Needless to say, it is necessary to adjust the concentration so that the minimum amount of the reagent solution contains a sufficient amount of the reagent.

When the processes shown in FIG. 4 70 to 75 are completed in the reactor 2B, the double flow path switching valve mechanism 13 for supplying cleaning solvent etc. next selects the reactor 2C and similarly performs the processes shown in FIGS. 4 70 to 75. . At the same time, the nucleotide reagent solution etc. supply channel switching means 13' selects the reactor 2B and performs the process 76 in FIG. 4.

The same applies to the processes 77 to 80 in FIG.
DNA synthesis is performed in parallel in C.

Now, in the automatic DNA synthesis apparatus 1 of the above embodiment, by using the double flow path switching valve mechanism 13, 13', a plurality of reactors 2A to 2 can be easily configured.
It is now possible to supply reagent solutions etc. to C.
As a result, DNA can be synthesized in parallel in a plurality of reactors 2A to 2C. In other words, since the channels are switched by a single dual-channel switching valve mechanism, the dead volume of reagent solution, etc. remaining in the switching valve mechanism is smaller than when switching channels using multiple switching valves. At the same time, the flow path configuration of this device can be made compact. Another advantageous effect is that the number of valve switching drive devices required for flow path switching can be reduced.

First, in the apparatus 1, the reactors 2A, 2B, 2
The reaction parts 9A, 9B, 9C of C are miniaturized, and supports A,
Place B and C, supply reagent solution etc. from above,
Reactors 2A, 2B, 2C so as to drain from the bottom.
It consists of Therefore, the drain valves 11A, 11B,
If a reagent solution, etc. is supplied from above while 11C is closed, the reagent solution, etc. will be contained in the supports A, B, and C and will swell them, as well as filters 7A, 7B,
It stays in the reaction parts 9A, 9B, and 9C above 7C and does not fall downward. Therefore, all the supplied reagent solutions participate in the reaction, and nothing accumulates in the dead space. As a result, DNA can be synthesized in small amounts, the minimum amount of supply is sufficient, and mixing and contacting operations such as shaking the reactor to promote the reaction are no longer necessary. In addition, this apparatus 1 cleans and dries the insides of the reactors 2A, 2B, and 2C with THF36 and blows them with dry gas before the reaction of connecting new nucleotides, completely cleaning the insides of the reactors 2A, 2B, and 2C in a short time. Designed to dry. As a result, water that inhibits the condensation reaction can be completely removed, so the reaction efficiency does not decrease.
No extra reagents required.

As a modified example, the reaction time shown in FIG.
Since it takes about 10 minutes, operations such as supplying reagent solutions to other reactors are performed during this time.Similarly, the reaction time shown in Fig. 4, 78, takes about 10 minutes, so operations to other reactors are performed during this time. An example of this is something that allows you to do this.

Further, in the dual flow path switching valve 13, the outer layer block 17 is fixed, and the middle layer block 18 and the outer layer block 19 are rotatably slidable.

Another example of the dual flow path switching valve mechanism is one having a configuration like 29 shown in FIG. A double flow path switching valve mechanism such as 29 allows simultaneous switching of a plurality of flow paths.

In addition, reactor 2 can be funnel-shaped or barrel-shaped, and the internal volume of the reaction section can be 80μ to 800μ.
Here are some things that have changed between.

In addition, Kel-F・g styrene was used as a solid support.
Examples include those using silica gel, polyacrylic morpholide, etc. These supports have a particle size of 30
A thickness of approximately 300 μm is preferable.

Other examples include those in which this invention is applied to a DNA synthesis apparatus using the phosphomonotriazolide method, the phosphite method, or the diester method.

As understood from the above explanation, this idea
According to the DNA etc. synthesis device, it is possible to easily supply reagent solutions etc. to multiple reactors using a relatively simple dual flow path switching valve mechanism, and DNA etc. can be synthesized in parallel in multiple reactors. You will be able to perform synthesis. Therefore, the ability to synthesize DNA, etc. will be greatly improved, and in particular, it will become possible to synthesize different types of DNA, etc. in parallel. As mentioned above, this is very useful when separating a specific gene from a mixture of many types of genes.

Furthermore, when synthesizing long-chain DNA (for example, chain length 200), continuous synthesis is not normally done from the viewpoint of yield and time;
Multiple pieces of DNA are synthesized and linked together, but with this device, multiple pieces of DNA can be linked together.
It is also very useful in this case because DNA can be synthesized in parallel.

[Brief explanation of the drawing]

Figure 1 is an explanatory diagram of the configuration of an automatic DNA synthesizer, which is an embodiment of the DNA synthesis apparatus of this invention.
The figure is a perspective view of a specific example of a dual flow path switching valve mechanism.
FIG. 3A is a vertical sectional view of the double flow path switching valve mechanism shown in FIG. 2, and FIGS. The outer layer block is viewed from the left side in FIG. 2, FIG. 4 is a flowchart of the operation of the device shown in FIG. 1, and FIG. 5 is equivalent to FIG. 3 of another specific example of the dual flow path switching valve mechanism. It is a diagram. 1...DNA automatic synthesizer, 2A, 2B, 2C
...Reactor, 12...Nitrogen gas supply channel switching means, 13...Double channel switching valve mechanism for supplying cleaning solvent, etc., 13'...Dual channel switching valve mechanism for supplying nucleotide reagent solution, etc., 17, 17 '...1st
Outer layer block, 18, 18'...middle layer block,
19, 19'...Second outer layer block, 20a-2
0f, 20a' to 20f'...Communication hole, 21, 21'
...Communication hole, 22α~22γ, 22α'~22
γ′...Communication hole.

Claims (1)

[Scope of claim for utility model registration] A means for supplying reagent solutions, etc. is connected to the reactor, and a plurality of reagent solutions, etc. are supplied to the reactor according to a predetermined procedure.
In an apparatus for synthesizing DNA, etc., first and second outer layer blocks each having a plurality of communicating holes for each communicating hole of the middle layer block are provided on both sides of a middle layer block having at least one communicating hole. By rotating and slidingly moving the first and second outer layer blocks relative to the middle layer block, an arbitrary communication hole of the first outer layer block and an arbitrary communication hole of the second outer layer block are formed. and a cylindrical return flow path switching valve mechanism which is switched and connected through the communication hole of the middle layer block, and a plurality of reactors, each of which has a plurality of supply flow paths for a plurality of reagent solutions, etc. A plurality of reactors are connected to each communication hole of the outer layer block, and a plurality of reactors are respectively connected to each communication hole of the second outer layer block, and various types of reactors are controlled in parallel or simultaneously by controlling a dual flow path switching valve mechanism.
Characterized by making it possible to synthesize DNA, etc.
DNA synthesis equipment.