JPS6094993A - Device for synthesizing polynucleotide - Google Patents

Abstract

PURPOSE:A device wherein a reagent, etc. will not be discharged from an introduction passage even if a filter is set, by setting the filter at the side of a liquid feed part of a reactor to send the reagent, solvent, etc., equipping the liquid feed part with the introduction passage for the reagent, solvent, etc. and an exhaust passage for a generated gas. CONSTITUTION:A main body of a device is equipped with the reactor 11 and the plural storage part 3-8 charged with a reagent, solvent, etc., respectively, necessary for polynucleotide synthesis. The filter 24 capable of being deformed elastically, consisting of fluorosilicone rubber, etc. is set at the side of the liquid feed part 22 of the reactor 11 to send the reagent, solvent, etc. Further, the liquid feed part 22 is provided with the introduction passage 22c piercing the filter 24 and introducing the reagent, solvent, etc. to the reactor 11, and with the exhaust passage 22d discharging an gas, etc. passing from the filter 24 through the reactor 11 to the outside out of the reactor 11. Consequently, even if the passage 22c and the passage 22d set close to each other, there is no fear of direct flow of the solvent, etc. from the passage 22c to the passage 22d.

Description

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

Polynucleotides, e.g. DNA (deoxyliponucleotide #
) is known to use a support to which nucleosides are chemically bonded, and sequentially condense nucleotides using a phosphotriester method, a phosphodiester method, a phosphatide method, or the like. In this synthesis method, steps such as washing, deprotection, washing, condensation reaction, and washing are repeated, and although there are not many types of steps, there are many repeated operations and it is complicated.

In order to eliminate the troublesomeness of the above-mentioned synthesis operation, the applicant has
For example, Japanese Patent Application No. 58-126249 and Japanese Patent Application No. 58-16
No. 1637, etc., proposed a polynucleotide synthesizer.

According to this polynucleotide synthesis apparatus, the main body of the apparatus includes a reactor, a plurality of storage sections each filled with reagents, solvents, etc. necessary for polynucleotide synthesis reaction, and a flow path connecting each storage section and the reactor. It is equipped with a switching valve that is switched by rotating an operating knob, and a liquid supply means that feeds reagents, solvents, etc. from each storage section to the reactor using inert gas pressure.

In the above-mentioned apparatus, it is often necessary to relieve the pressure inside the reactor when feeding a solvent or the like into the reactor. For this reason, pressure relief passages are provided at appropriate locations in the reactor, but if this passage must be provided close to the reagent introduction channel due to space constraints, etc., the reagent, etc. A problem arises in that the gas is directly discharged from the pressure relief passage without entering the reactor.

In such a case, it is conceivable to connect a tube facing into the reactor to the introduction passage. In this way, the introduction port of the introduction passage is located at the tip of the tube, away from the pressure relief passage, and the above-mentioned problem can be solved. However, if a filter is also installed on the liquid supply side of the reactor in order to protect the valve installed in the flow path of the main body of the device, the tube becomes an obstruction, and the tube is connected to the introduction path and the reagent is removed from the pressure relief path. etc. cannot be prevented from being directly discharged.

The present invention has been made in view of the above circumstances, and its purpose is to prevent reagents from being discharged directly from the introduction passage through the pressure release passage even if a filter is provided on the liquid supply side of the reactor. It is an object of the present invention to provide a polynucleotide synthesis apparatus equipped with a reactor without a reactor.

゛ That is, the present invention provides a filter on the liquid supply side of the reactor into which reagents, solvents, etc. are sent, and an introduction passageway that penetrates the filter and introduces the reagents, solvents, etc. into the reactor. The reactor is characterized by being provided with a derivation passage (pressure relief passage) for deriving gas etc. from the inside of the reactor through a filter to the outside of the reactor.

Therefore, according to the present invention, reagents and the like are not discharged directly from the introduction passage through the outlet passage, and the valve provided in the passage of the main body of the apparatus can be protected.

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

First, an outline of the entire synthesis apparatus of the present invention will be explained with reference to FIG. In the figure, 1 is a nitrogen cylinder, 2 is a distribution valve, 3-
6 is a bottle, 7 and 8 are reserve tanks, 9 and 10 are switching valves, 11 is a reactor, 12 and 13 are three-way cocks, 14 and 15
16 is a four-way cock, 16 is a waste liquid tank, 17 is a line filter, 18 is a pressure needle, and 19 is a safety valve.

Bottle 3 is filled with a solvent 1, bottle 4 is filled with a trityl agent/solvent 1 solution, bottle 5 is filled with a solvent 2, and bottle 6 is filled with an inactivating agent and an inactivating aid.

The reserve tanks 7 and 8 are filled with a solvent suitable for the type of trityl agent, and are used when changing the detrityl agent.

The switching valves 9 and 10 switch the flow paths between the bottles 3 to 6, the reserve tank 7.8 and the reactor 11.11. The switching valve 9 is connected to reactors 1.1 and 11 via a three-way cock 12.
A common boat 9a is connected to the reagent and solvent switching boats 9b and 9, which are connected to the bottles 3 and 4 and the reserve tank 7.
d, 9f, and the gas switching board F connected to the distributor 2.
9 c * 9 e t 9 g are provided.

The switching valve 10 also includes a common boat 10a connected to the reactor 11.11 via a three-way cock 12, and reagent and solvent switching boats 10b and 10d connected to the reserve tank 8 and bottles 5 and 6, respectively. lOf, and gas switching boats 10c, 10e, and 10g connected to the distributor 2 are provided.

The nitrogen cylinder 1ON2 gas is sent to the distribution W2, where it is divided and sent to the bottles 3 to 6 and the reserve tank 7.8.

By switching the boats 9b to 9g and 10b to 10g of the switching valves 9 and 10, solvents, reagents, etc. are transferred from the bottles 3 to 6 and the reserve tank 7.8 to the three-way tank 12 and the flow path by the pressure of N2 gas. 20a through each reactor 11.11
sent to. Further, N2 gas is sent from the distributor 2 to the reactors tt and 1i through the three-way tank 12 and the flow path 20a. When it is desired to send to only one reactor 11, the three-way connectors 12 and 13 are switched.

When feeding solvents, reagents, etc., use four-way screws 14.
15 to the solid line state (FEED). As a result, the reactor 11 is
．． The pressure is released from the top of 11, and the distributor 2
, line filling 17, three-way cock 13, four-way cocks 14, 15, and flow path 20c from the bottom of reactor 11.11 to bubble cocks 14, 15 in the state shown by the dotted line (BL
OW). As a result, the solvent and the like are discharged from the bottom of the reactor 11.11 through the four-way cock 14.15 of the flow path 2001 to the waste liquid tank 16.15.

The aforementioned (7) switching valves 9, 1 o-no-hoto 9b to 9
g, 1ob-10g are arranged in the order of liquid supply/drainage operation, first switching boats 9b to 9g, then boat lo
By switching b-10g, the supply and discharge liquid operation in one condensation step is completed. It should be noted that the switching valves 9° and 10 can also be used even if the boats 9b to 9g, 11ob to 1og are not arranged in the order of liquid supply/drainage operations.

When synthesizing DNA, a reactor 11.11 is filled with a support (such as silica gel) to which nucleosides are bonded, and the support is swollen with solvent (2).

After this, detritylation → washing (solvent I) and washing (solvent ①) are performed. Then, raw materials and condensing agent are injected into the reactor 11°11. At this time, the reactors 11, 11 are heated using the heater 47 (see FIG. 4), and bubbling is performed with N2 gas.

After the condensation reaction is completed, the reactor 11.11 is drained.

Then wash (solvent 2) and cap.

By repeating this operation, the nucleotide chains are sequentially condensed.

Next, the reactor 11, which is a characteristic part of the present invention, will be explained with reference to FIGS. 2 and 3.

The reactor 11 consists of a main body 21, an upper plug 22, and a lower plug 23, with the upper plug 22 forming the top and the lower plug 23 forming the bottom.

The main body 21 is made of glass (transparent or translucent), has a silane treatment on its inner peripheral surface, and has cut sections 21a, 21a formed on the inner peripheral surface at both ends thereof. Note that the main body 21 may be made of stainless steel, polypropylene, polyethylene, fluorine resin, or the like.

The upper and lower plugs 22 and 23 are made of a material that does not react with the aforementioned solvents, reagents, etc. and is elastically deformable, such as fluorosilicone rubber,
It is made of fluorine comb, hexafluoropropylene-tetrafluoroethylene copolymer, polypropylene, polyethylene, etc., and is fitted into both ends of the main body 21.

Filters 24 are interposed between the step portions 21a, 21a of the main body 21 and the upper and lower plug bodies 22.degree. 23, respectively. This filter 24 can capture the support filled in the main body 21, does not dissolve in the above-mentioned solvent, and can pass through the solvent, and is made of, for example, tetrafluoroethylene, polypropylene, polyethylene, etc. ing.

One end of the upper plug body 22 that is fitted into the main body 21 is connected to the main body 21.
It is formed slightly larger than the inner diameter of.

A circumferential groove 22a is formed on the outer circumferential surface of this one end, and a substantially hemispherical recess 22b is formed on the end surface.
1 and maintains airtightness with the book 7*21.

Inside the upper plug body 22, an introduction passage 22c and an outlet passage 22d extending in the axial direction are provided close to the pressure valve due to space limitations. These introduction passage 22c and outlet passage 22d are the aforementioned flow passage 20a.

20b.

A tube 22e passing through the filter 24 is connected to one end of the introduction passage 2c, and the introduction port is connected to the filter 24.
It is located inside. On the other hand, the outlet of the outlet passage 22d is located within the four portions 22b, that is, on the outside of the filter 24. Therefore, even if the introduction passage 22c and the outlet passage 22d are provided close to each other, since the introduction port and the outlet are separated, the solvent etc. that flowed from the introduction passage 22C do not enter the main body 29 and directly exit the outlet passage 22d. There is no risk of it getting inside.

Furthermore, N2 gas and the like that come out of the main body 21 when the pressure is released and solvent that overflows from the main body 21 pass through the filter 24 and are discharged from the outlet passage 22d to the flow passage 20b. Therefore, there is no fear that the four-way cocks 14 and 15 provided in the flow path 20b will be damaged by foreign objects or the like.

The upper plug body 2 is also attached to one end of the lower plug body 23 that is inserted into the main body 21.
2, a circumferential groove 23a and a recess 23b are formed,
Due to elastic deformation, it comes into close contact with the inner circumferential surface of the main body 21 to maintain airtightness. A discharge passage 23c is provided inside the lower plug body 23, penetrating in the axial direction, and this discharge passage 23c is provided.
3c is connected to the aforementioned flow path 20c.

Note that flange portions 21b are provided at both ends of the main body 21, respectively.
, 21b are provided, and flange portions 22f and 23d are also provided at the intermediate portions of the upper and lower plug bodies 22 and 23. The upper and lower plug bodies 22, 23 can be fixed to the main body 21 by connecting these flange parts 21b, 21b and 22f, 23d with a clip 25 (see FIG. 1). In addition,
If the inner peripheral surfaces of both ends of the main body 21 are tapered, the upper and lower plug bodies 22 and 23 can be easily inserted.

FIG. 4 to FIG. 6 show the external appearance of the above-mentioned synthesis apparatus.

A recessed mounting portion 27 that opens at the side and top surfaces of the case 26 is provided on the front surface of the aus 26 of the device, and the aforementioned reactors 11 and 11 are mounted on this mounting portion 27 via a support member 28. There is.

Since the mounting portion 27 is open not only on the front side but also on the side and top surfaces, the reactor 11.11 has good operability. When not in use, a transparent or translucent cover is attached to the attachment portion 27.

Further, on the front surface of the case 26, there are operating knobs 29, 30, 31, 32 for the above-mentioned pressure gauge 18, the above-mentioned switching valves 9, 10, three-way cocks 12, 13, four-way cocks 14, 15, and an illuminated switch 33. 34.35 and a timer (not shown)
An operation knob 36 and temperature adjustment operation knobs 37 and 38 for the heater 47 are provided.

Further, one side of the case 26 is provided with recesses 39 and 40 (see FIG. 6), the above-mentioned bottles 3 and 4.5 are installed in the upper recess 39, and the bottles 3 and 4.5 are installed in the lower recess 40. is equipped with the aforementioned bottle 6 and reserve tank 7.8. The ceiling portions of the recesses 39 and 40 have lid members 41 to 46 for airtightly mounting the bottles 3 to 6 and the reserve tank 7.8.
is provided. Note that the recesses 39 and 40 are closed by transparent covers.

In the above embodiment, the case is shown in which the present invention is applied to a type of synthesis apparatus in which N2 gas is used as the liquid supply means and the flow paths are switched by two switching valves 9 and 10, but the present invention is not limited thereto. There are types that use a liquid supply pump as the liquid supply means, and a type that has a cock in each flow path.

It can also be applied to types that switch the flow path. That is, the main body of the apparatus is equipped with a reactor and a plurality of storage sections each filled with reagents, solvents, etc. necessary for polynucleotide synthesis reaction, and the reagents, solvents, etc. are reacted from each storage section using a liquid means. It can be widely applied to a synthesizer configured to sequentially feed into a container.

As explained above, according to the present invention, a filter is provided on the liquid supply part (upper plug body 22) side of the reactor into which solvent etc. are fed, and the reagent, solvent etc. are passed through the filter into the liquid supply part and reacted. Introduction passage into the vessel (introduction passage 22c1 tube 22
e) and a discharge passage for guiding gas, etc. coming out of the reactor from inside the reactor through a filter, so even if the introduction passage and the discharge passage are close to each other, the solvent, etc. will not flow into the reactor. This eliminates the possibility that the liquid is discharged directly through the outlet passage without entering the main body of the liquid, and as a result, the liquid supply section can be made compact. In addition, the valve in the flow path can be protected by a filter on the liquid supply section side.

[Brief explanation of drawings]

The drawings show one embodiment of the present invention; Fig. 1 is an overall flow sheet, Fig. 2 is a partially omitted sectional view of a reactor, and Fig. 3 is a partially omitted sectional view of a reactor.
1! + Enlarged sectional view of main parts, Figure 4 is a front view of the device, Figure 5
The figure is a plan view of the same, and FIG. 6 is a side view of the same. DESCRIPTION OF SYMBOLS 1...Liquid supply means (to nitrogen bomb), 3-8...Storage part, 9.10...Switching valve, 11...Reactor, 21...
・Main body, 22...Liquid supply part (upper plug body), 22c, 2
2e...Introduction passage, 22d...Output passage, 24...
·filter. Patent applicant Zeon Corporation

Claims (1)

[Claims] The main body of the apparatus is equipped with a reactor and a plurality of storage sections each filled with reagents, solvents, etc. necessary for polynucleotide synthesis reaction, and reagents, solvents, etc. are supplied from each storage section to the reactor using a liquid supply means. In a polynucleotide synthesis apparatus configured to sequentially feed the reagents, solvents, etc., a filter is provided on the liquid supply section side of the reactor into which the reagents, solvents, etc. are fed, and the reagents, solvents, etc. are passed through the filter into the liquid supply section and reacted. 1. A polynucleotide synthesis apparatus, comprising: an introduction passageway for introducing the inside of the reactor, and an outlet passageway for guiding gas, etc. exiting from the inside of the reactor through a filter to the outside of the reactor.