JPS6311360B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to a device for automatically synthesizing trace amounts of DNA, etc.
In particular, the present invention provides an apparatus suitable for automatic micro-synthesis of DNA or RNA.

DNA synthesis methods have been improved and developed into the so-called phosphodiester method, phosphotriester method, and phosphite method, and solid-phase synthesis methods using solid supports that utilize these methods are widely used due to their various advantages. It has reached the point where it will be done. On the other hand, the reactor (reaction column) used in solid-phase synthesis is a type in which the reactor containing the solid support is shaken, and the reagent solution is circulated through the reactor, from the viewpoint of mixing and contacting with the reagent solution. Three types are known: a contact type (JP-A-56-138199 and JP-A-57-9797), and a type in which a large amount of reagent solution is temporarily passed through the same reactor. However, both types have various drawbacks from the viewpoints of reaction time, reaction efficiency, economic efficiency, etc. On the other hand, in most conventional automatic synthesis devices, the amount of solid support used is 100 mg or more, and the amount of DNA to be synthesized is
From the viewpoints of the required amount of nucleotides and the high cost of reagents such as protecting group nucleotides as raw materials, an automatic synthesis device on a smaller scale has been desired.

As a result of various studies, the inventor of this invention succeeded in improving a known automatic synthesis device.

1. The reactor (a) has a drain port at the bottom, (b) has a filter on the lower part of the interior on which a solid support for synthesizing DNA, etc. can be placed and can pass the reagent solution, (c) ) A container having a reaction space with a volume of 80μ to 800μ above the filter, and (d) a mortar-shaped flange and a stopper for the flange above the reaction space, DNA comprising a plug having a plurality of nozzles for supplying reagent solutions, etc. inserted therein, and a reagent solution supply means capable of supplying a reagent solution in an amount corresponding to the amount of the support placed in the reactor. An automated microsynthesis device is provided.

The main features of the device of the present invention are (i) eliminating the need for special operations such as shaking the reactor for mixing, contacting, or circulation; (ii) solid support of 10 mg ~ On a scale of about 50mg, and
It is possible to provide a reaction system that can synthesize DNA on a scale of about 0.3 to 2 μmol by about 10 condensations, and (iii) the reagent solution used is about 5 to 7 times the volume of the solid support, and The goal is to provide suitable means of supply.

Hereinafter, this invention will be explained in detail based on embodiments shown in the drawings. Note that this invention is not limited to this.

Reference numeral 1 shown in FIG. 1 is a small amount automatic DNA synthesis device using the phosphotriester method to which the present invention is applied.

The reactor 2 is a container having a cylindrical main body 3 with an inner diameter of 8 mm and a height of 10 mm, and a mortar-shaped flange 4 provided above. The dome-shaped flange 3 is fitted with a stopper 5 into which a number of nozzles for supplying reagent solutions and the like are inserted. Therefore, the head opening of the main body 3 becomes the reagent solution supply port 6. A filter 7 such as a glass filter is fitted inside the main body 3, and a drain port 8 is provided at the bottom. Filter 7
The support member 9, such as polystyrene or silica beads, can be placed thereon (not permeable), and is permeable to reagent solutions, solvents, and gases. The space above the filter 7 becomes the reaction section 10, which has a diameter of about 450μ.
is a space with a volume of .

There are 8 types of reagent solutions in total. Numerals 11 to 14 are various nucleotide reagents, each having adenine, cytosine, guanine, and thymine as bases. 15 is a condensing agent, mesitylenesulfonyl-3
-A pyridine solution of nitrotriazolide (MSNT). 39 is a protecting group removing agent, which is a solution of zinc bromide dissolved in a mixed solvent of isopropanol and methylene chloride. 40 is a masking reagent, which is a mixed solution of acetic anhydride and pyridine. 41 is a masking reagent, which is a pyridine solution of dimethylaminopyridine.

The syringe pumps 21 to 25 are connected to the reagent solutions 11 to 15 through switching tips 16 to 20, respectively.
can be sucked in and fed to reactor 2.

The plungers of the syringe pumps 21-25 are driven by plunger drive mechanisms 26-30, respectively.

The plunger drive mechanism 26 is driven by a pulse motor 31
and the screw shaft 32 rotated thereby, and the plunger 21a moved by the rotation of the screw shaft 33.
The pulse motor 31 is pulse-controlled by a control circuit 34 such as a microcomputer. The other plunger drive mechanisms 27 to 30 have similar structures.

Solvents 36 to 38 are tetrahydrofuran (THF) as a volatile drying solvent, pyridine as a cleaning solvent, and a mixture of isopropanol and methylene chloride, also as a cleaning solvent. These solvents 36
38 and the reagent solutions 39 to 41 can be supplied to the reactor 2 via valves 42 to 47, respectively, by nitrogen gas pressure.

The valve 48 supplies nitrogen gas directly to the reactor 2 in order to blow the inside of the reactor 2 with nitrogen gas. Note that the nitrogen gas is dried with a desiccant 49 such as calcium chloride.

In addition to controlling the plunger control mechanisms 26 to 30 as described above, the control circuit 34 also controls the switching mechanism 16.
-20, controls the operation of valves 42-48, drain valve 50, and exhaust valve 51. The user also interacts with the operator via the console 35.

When synthesizing DNA, first, the stopper 5 is removed and a support 9 to which only the terminal portions of DNA molecules are bound is placed in the reactor 2. This amount is preferably 10 mg to 50 mg, for example, when the support 9 is a polystyrene powder. After returning the stopper 5, the support 9 inserted
The amount and the like are input to the control circuit 34 via the console 35, and then a start command is input.

The control circuit 34 then operates the valves 44, 48, 50,
51 to supply a mixed solvent 38 of isopropanol and methylene chloride to the reactor 2 and wash the support 9. In other words, open the valves 44 and 51 to remove the solvent 3.
8 and close the valves 44 and 51. After a while, the valves 48 and 50 are opened to drain the liquid, and the valves 48 and
Close 50. Do this several times. After this washing, the protecting group removing agent 39 is supplied to the reactor 2 by operating the valves 45 and 51, and after a predetermined period of time, the valve 4
8,50 to drain the liquid.

The reactive group at the end of the DNA molecule bound to the support 9 has been blocked by adding a dimethoxytrityl group (DMTr) as a protective group in advance, but the above operation protects the reactive group at the predetermined site.
DMTr is desorbed.

The control circuit 34 then again controls the valves 44, 48, 5.
0.51 to supply a mixed solvent 38 of isopropanol and methylene chloride to the reactor 2 and wash the support 9. Furthermore, valves 43, 48, 50, 51
The inside of the reactor 2 is washed with pyridine 37.

Then, operate valves 42, 48, 50, 51.
The inside of the reactor 2 is washed with THF36, and then the valve 48,
50 to blow nitrogen gas into the reactor 2 for several minutes. As a result, the inside of the reactor 2 is completely dried.

The control circuit 34 selects one nucleotide reagent from the four nucleotide reagents 11 to 14 having different bases based on the base sequence of the DNA to be synthesized, and operates the corresponding switching knob and plunger drive mechanism. Also, the exhaust valve 51 is operated to supply the nucleotide reagent solution to the reactor 2. For example, if a nucleotide having adenine as a base is needed next as a DNA base sequence, 11 nucleotide reagent solutions are selected, the switching pot 16 and the plunger drive mechanism 26 are operated, the exhaust valve 51 is operated, and the syringe pump 21 is activated. 11 is fed to reactor 2 by.

At the same time, the switching pot 20 and the plunger drive mechanism 30 are operated, and the condensing agent 15 is supplied to the reactor 2 by the syringe pump 25.

The amount supplied is controlled so that the total amount of nucleotide reagent solution and condensing agent is the minimum amount sufficient to swell the support 9. Specifically, for example, when the support 9 is a polystyrene powder, about 3 ml of the nucleotide reagent and about 3 ml of the condensing agent are used per 1 g of the support. 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.

By the above operation, it was connected to the support body 9.
A nucleotide with a desired base is linked to a predetermined site at the end of the DNA molecule. Note that the 5' hydroxyl group of the nucleotide has been blocked in advance with a protecting group, DMTr.

When support 9 is polystyrene powder, support 1
Approximately 0.1 mmol of the terminal portions of DNA molecules are attached per gram, and new nucleotides are attached to most of them as described above. However, about a few percent may remain unreacted. For this reason, the control device 34 masks unreacted 5' hydroxyl groups as follows. That is, after a certain period of time, valve 4
3, 48, 50, and 51 to clean the inside of the reactor 2 with pyridine 37. Next, the valves 46 and 51 are operated to supply the masking reagent 40 to the reactor 2. At the same time, the valve 47 is operated to supply the masking reagent 41 to the reactor 2.

After masking is performed by the above operation, the control circuit 34 operates the valves 43, 48, 50, and 51 to clean the inside of the reactor 2 with pyridine 37.

The operations up to this point complete one cycle in which one new nucleotide is linked to the terminal portion of the DNA molecule that has been previously bound to the support 9.

The control circuit 34 operates the valves 44 , 48 , 50 , and 51 to repeat the synthesis cycle from the protecting group removal operation to the masking operation for supplying the mixed solvent 38 of isopropanol and methylene chloride, and inputs the information on the operation console 35 . Synthesize the desired DNA.

Now, according to the DNA synthesizer 1 of the above embodiment,
Unlike conventionally, reagent solutions 11-15 are always supplied in the minimum amount calculated based on the amount of support,
and no mixing or contacting operations are performed. This saves reagent consumption and eliminates the need for mixing and contacting equipment.

This improvement was made in the reaction section 1 of the reactor 2.
This is because the support body 9 is placed on the filter 7, and the reagent solutions 11 to 15 are supplied from above and drained from the bottom. That is, if a reagent solution is supplied from above with the drain valve 50 closed, the reagent solution will be contained in the support 9 and swell it, and will remain in the reaction section 10 above the filter 7 and will not fall downward. . Therefore,
All the supplied reagent solutions participate in the reaction, and nothing accumulates in the dead space. As a result, the minimum supply amount is sufficient, expensive reagents can be saved, and loss of reagents due to adhesion to the reactor can be avoided, resulting in economic advantages. Also, mixing and contacting operations are no longer necessary, and mixing equipment can be saved.

In addition, since the reactor has a flange that spreads out in a cone-like manner above the reaction space, it is possible to install a large number of nozzles for supplying reagent solutions, solvents, etc. necessary for DNA synthesis.
Solutions and the like can be easily introduced into the reaction section, and loss of reagents can be avoided.

Note that it is also helpful to save reagents by completely drying the inside of the reactor 2 to remove moisture that inhibits the condensation reactor before the reaction of linking a new nucleotide to increase the reaction efficiency.

As a modified example, the volume of the reaction section 10 is set to 80μ.
Examples include those that vary between ~800μ.

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

Other examples of the solid support 9 include Kel-F.g
Examples include styrene, silica gel, and polyacrylic morpholide. These supports have a particle size of 30-300μ
It is preferable to have a diameter of about m.

As understood from the above explanation, the present invention
The automatic microsynthesizer for DNA, etc. has the effect of reducing wasteful consumption of expensive synthesis reagents and reducing running costs. Also, mixing and contacting operations are no longer necessary. Furthermore, only the required amount can be synthesized in minute quantities, eliminating waste.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the configuration of an automatic DNA micro-amount synthesis device, which is an embodiment of the automatic micro-amount synthesis device for DNA, etc. of this invention. 1...DNA micro-automatic synthesizer, 2...reactor, 3
...Main body, 4...Flange part, 6...Reagent solution etc. supply port, 7...Filter, 8...Drainage port, 9...Support, 1
0... Reaction section, 11-15, 39-41... Reagent solution, 16-20... Switching pot, 21-25... Syringe pump, 26-30... Plunger drive mechanism,
34...Control circuit, 36-38...Solvent, 42-4
8, 50, 51...Valve.

Claims (1)

[Scope of Claims] 1. The reactor (a) has a drain port at the bottom, and (b) has a filter on which a solid support for synthesizing DNA, etc. can be placed at the lower part of the interior and through which a reagent solution can pass. (c) has a reaction space above the filter with a volume of 80μ to 800μ, and (d) is comprised of a container equipped with a mortar-shaped flange and a stopper for this flange above the reaction space. A reagent solution supply means capable of inserting a number of nozzles for supplying reagent solutions, etc. into the flange stopper, and supplying the reagent solution in an amount corresponding to the amount of the support placed in the reactor. A device for automatically synthesizing trace amounts of DNA, etc., which is equipped with the following.