JPH0466876B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention relates to a synthesizer for synthesizing nucleic acids such as DNA and RAN. More specifically, the present invention relates to a nucleic acid synthesizer capable of manually synthesizing a desired oligonucleotide such as DNA or RNA using the principle of solid-phase oligonucleotide synthesis. Prior Art In recent years, with the dramatic progress in genetic engineering,
In research on nucleic acids, the demand for synthetic oligonucleotides in particular is increasing, and the techniques for synthesizing them are also becoming more sophisticated. By the way, the diester method (Sciecn, 203 , 614,
(1979)), triester method (Nucleic Acids
Research, 8 , 2331 (1980), 8 , 5193 (1980),
8 , 5491 (1980)), solid phase method (Nature, 281 , 18
(1979), Biochemistry, 1980 , 6096 (1980)), liquid phase method (Proc, Natl, Acad, Scr.USA 75 , 5765
(1978)), Nucleic Acids
Research, 8 , 5753 (1980), the phosphite method (Tetrahedron Letters 22 , 1859 (1981)), or the currently frequently used triester method-solid phase method (Nucleic Acids Research, 8 , 5507).
(1980)), phosphite method-solid phase method (J.Am.
There are combination methods such as Chem.Soc. 103 , 3185-3191 (1981)), and fully automated nucleic acid synthesis devices have recently been devised based on these combination methods, and several models are already on sale. has reached. However, all fully automated acid nucleic synthesis apparatuses are extremely expensive and are currently difficult to purchase. On the other hand, the oligonucleotides currently required in the field of genetic engineering include primers, probes, and linkers, but most of these have chain lengths of 15 to 25, and can be used in a single genetic manipulation experiment. Only a few oligonucleotide chains are used, and the amount used is about 1 per oligonucleotide chain.
It is about 0.5OD. Therefore, there is a desire to develop an apparatus that can synthesize oligonucleotides easily and at low cost without purchasing an expensive automated nucleic acid synthesis apparatus. SUMMARY OF THE INVENTION The present invention aims to solve the above-mentioned problems, and achieves this purpose by providing a nucleic acid synthesizer that can easily manually synthesize oligonucleotides such as DNA and RNA. This is what we are trying to achieve. Therefore, the nucleic acid synthesizer for synthesizing nucleic acids by reaction on solid support particles according to the present invention comprises:
It is characterized by consisting of the following members. (A) A reaction chamber consisting of a cylinder having a volume sufficient to accommodate the solid support resin particles and carry out the reaction. (B) A mounting seat for attaching the solid support resin particle retention filter, which extends downward from the reaction chamber A and has an inner diameter smaller than the lower part of the reaction chamber A. (C) A filter for retaining the solid support resin particles, which is attached to the mounting seat B. (D) A liquid supply/drainage port which forms a downward extension of the reaction chamber A further below the mounting seat B and allows the liquid phase material involved in the reaction to enter and exit the reaction chamber A. (E) A supply/exhaust device installed at the upper part of the reaction chamber, which allows the liquid phase material to enter and exit the reaction chamber from the supply/drain port D using gas pressure. However, this supply/exhaust device E consists of a positive and negative pressure generation source Ea and a valve device Eb, and the positive and negative pressure generation source Ea communicates with the upper part of the reaction chamber A via the valve device Eb. Effects As described above, the present invention relates to a nucleic acid synthesizer that can be operated manually, and the nucleic acid synthesizer of the present invention can be used even by a person with no experience in organic synthesis if oligonucleotides are synthesized according to the operating manual. However, the desired oligonucleotide can be obtained reliably and in high yield with simple operations. Therefore, it goes without saying that the desired oligonucleotide can be obtained at low cost without using an expensive automated nucleic acid synthesis device. Furthermore, since the nucleic acid synthesizer of the present invention can easily synthesize primers, probes, linkers, etc. commonly used in the field of genetic engineering, it can be said to make a great contribution to research and development in this field. One of the features of the nucleic acid synthesizer of the present invention when viewed from the aspect of its components is that the cylindrical reactor has a filter attached to the bottom of the cylindrical reactor section to support a layer of resin particles necessary for solid phase synthesis. The diameter of the cylindrical extension is smaller than the diameter of the cylindrical extension. This feature is significant in that it reduces the scale of the reaction by concentrating the resin in a specific part of the reactor and improves the contact between the resin and the monomer nucleoside solution or solvent. That is, since the filter is provided in the cylindrical portion having a smaller diameter than the diameter of the cylindrical reactor, when resin particles are put into the reactor, the resin can be collected in a specific part of the reactor. In addition, the resin layer deposited on the filter becomes quite densely packed after the monomer nucleoside solution or solvent is flowed downward, but in this state, the next solution or solvent is flowed upward from below the filter. and the resin layer is cylindrical (i.e., the inner diameter is substantially the same in the vertical direction)
, the resin layer remains as it is,
That is, the resin particles do not decompose into individual resin particles, but only move in the cylindrical portion, resulting in insufficient contact between the resin particles and the solution or solvent. In such a situation, according to the present invention, the diameter of the reaction chamber that accommodates the resin layer is larger above the resin layer than in the resin layer portion, so that the resin layer can be separated into individual parts by the solution or solvent flowing from below. Decomposes into resin particles,
As a result, contact between the resin particles and the solution or solvent becomes better. Since the solid-phase method targets reactions on the surface of resin particles, these features improve nucleic acid synthesis efficiency (including washing efficiency). The apparatus of the present invention is also characterized in that the liquid phase material is introduced into and removed from the reaction chamber by gas pressure. In other words, the liquid phase material is moved in and out of the reaction chamber by creating a positive pressure condition or a negative pressure condition in the reaction chamber, so it is easier to move the liquid phase material in and out than to directly pressurize the liquid phase material. The device may be simple (for example, the one in Figure 1).
(like Ea, which consists of a piston and a cylinder), therefore cleaning the reaction chamber and resin during liquid phase material conversion is easy and reliable. This is because when press-fitting a liquid phase material, it becomes necessary to use new equipment to achieve the purpose of injection, or to change the device to make it easier to press-fit the liquid phase material. This is because the cleaning operation required each time the liquid phase material is changed becomes complicated. Specific Description of the Invention Nucleic Acid Synthesizer Overview of the Synthesizer The nucleic acid synthesizer according to the present invention is a device used to synthesize nucleic acids by reaction on solid support resin particles, that is, by a solid phase method. And,
It consists of the above-mentioned members A to E. The movable part of the device of the present invention is mainly the supply/exhaust device E.
Although it is of course possible to drive this part electrically or to perform the synthesis operation under computer control, it is most preferable for the experimenter to operate it manually when using the apparatus of the present invention. Considering that it is easy to use manually,
It is preferable that the positive and negative pressure generating device Ea in the device of the present invention is a syringe consisting of a cylinder and a piston. valve device
It is preferable that Eb and the reaction chamber A be removably connected. A specific example of a synthesizer according to the present invention is shown in FIG. Reaction Chamber A Reaction chamber A consists of a cylinder having a volume sufficient to accommodate the solid support resin particles for solid phase synthesis and carry out the reaction. Here, a "cylindrical cylinder" is one whose cross section is substantially circular and whose length is sufficiently larger than its diameter (or equivalent diameter if the cross section is not a perfect circle) (in particular, a cylinder whose length is at least equal to the diameter). means something that is). Further, the shape or size of the cross section may not be the same in the length direction, and the protrusion may be provided inside as necessary. A small-diameter connector Aa is usually provided at the top of the reaction chamber A so that it can be connected to one port of a three-way socket (details will be described later) of the valve device Eb. The connector Aa also serves as a lid at the top of the reaction chamber. In the specific example shown in Figure 1, the reaction chamber A and the top structure
Aa is connected with tape or tube Ab. On the other hand, a filter mounting seat B having a diameter smaller than the inner diameter of the reaction chamber cylinder is provided at the lower part of the reaction chamber A (details will be described later). The reaction chamber may be made of any material as long as it is resistant to organic solvents, and suitable materials include stainless steel, Teflon, and glass. Glass is preferable because the state of the reaction and the state of the resin can be easily confirmed. Also,
More preferably, the inner wall of the reactor is coated with silicone to prevent the resin used in the nucleotide condensation reaction from adhering to it. In addition,
It is usually sufficient for the inner diameter of the cylinder to be about 10 mm. Filter mounting seat B Mounting seat B for mounting the solid support resin particle holding filter (details will be described later) is a member that extends downward from the reaction chamber A and has a smaller diameter than the lower part of the reaction chamber A. . Here, "the inner diameter is smaller than the lower part of reaction chamber A" means that the part where this filter is attached needs to be smaller than the inner diameter of reaction chamber A at the part where this filter mounting seat B is connected. It means that. Therefore, this does not exclude the possibility that there is a portion with a smaller diameter above the reaction chamber A, and the inner diameter of the filter mounting seat B continues downward as shown in Fig. 1. It also includes a case where the diameter of the filter mounting seat B is increased below the filter mounting location. A specific example of the filter mounting seat B is funnel-shaped as shown in the figure. Although the illustrated one has a relatively small vertical dimension compared to the diameter, it is of course possible to have a funnel shape that is longer than the illustrated one. Further, the inner diameter of the funnel may be decreased continuously (downward), but the inner diameter may be decreased in stages. Filter C Filter C may be of any shape, material, or other material as long as it has a structure that supports the solid support resin particles for solid phase synthesis and allows passage of the liquid phase material. The most typical filter is a so-called glass filter, which has a multilayer structure. Ceramics can also be considered as filter members. The pore size of the filter is preferably about 40 to 50 microns (about G-2 standard glass filter) so that the support resin particles do not deviate. Another example of a filter is glass wool. The filter C is attached to the mounting seat B, and the term "attached" here includes both cases where it is removable from the mounting seat and cases where it is not removable. If it is removable, consideration should be given to how to attach it so that it is not pushed upward by the liquid phase material for synthesis reaction that enters from below. Supply/Exhaust Port D The supply/discharge port exists as an extension of the reaction chamber A further below the mounting seat B. In addition to being a simple opening, this may be in the form of a liquid supply/drainage pipe Da as shown in FIG. Supply/Exhaust Device E The supply/exhaust device E is used to create positive pressure and negative pressure in the reaction chamber as necessary, and to use the pressure to move liquid phase material into and out of the reaction chamber. Therefore, this device is a positive and negative pressure generator.
The positive and negative pressure generation source Ea communicates with the upper part of the reaction chamber A via the valve device Eb. The positive and negative pressure generator Ea can be any suitable pump device. However, from the viewpoint of ease of device and operation, it is preferable to use a cylinder and a piston as shown in FIG. The valve device Eb usually consists of a three-way valve. In that case, the first port communicates with the upper part of the reaction chamber A, and the second port communicates with the positive and negative source Ea.
Preferably, the conduit is configured such that it communicates with the cylinder (if it consists of a cylinder and a piston), and the third port is closed. The third port can be conveniently used to introduce an inert gas (eg, nitrogen) for drying the resin particles for solid-phase methods or for forming an atmosphere in the reaction system. The above-mentioned preferred structure of the three-way socket Eb in FIG. 1 has a conduit shown by a dotted line in its rotating portion. Operation Manual The operation of the nucleic acid synthesizer of the present invention, that is, the synthesis of oligonucleotides by the nucleic acid synthesizer of the present invention, is usually performed according to the following manual. It should be noted that since this operating manual describes a preferred specific example, it goes without saying that operating conditions other than those described below may also be used. Outline of Nucleic Acid Synthesis Nucleic acids such as DNA and RNA are synthesized by condensing nucleotides by a solid phase method using the nucleic acid synthesizer of the present invention. The synthesizer according to the present invention was designed primarily for the synthesis of oligonucleotides such as DNA and RNA, and methods for synthesizing oligonucleotides include sequentially condensing monomers with nucleotides fixed on a resin, and nucleotide blocks ( One possible method is to obtain the desired oligonucleotide by repeatedly selecting a monomer, dimer, trimer, etc., and repeating condensation, but which method is used depends on the preparation of the raw nucleotide block, the number of reactions, and the average yield of condensation. It may be selected as appropriate, taking into consideration the separation and purification of the target product, etc. For a specific method of synthesizing oligonucleotides using a solid phase method, please refer to Examples and the literature (Nucleic Acids Rcsearch 8 , 5507 (1980)) described later. Introduction of 3 ¹ -terminal nucleotide into resin The 3 ¹ -terminal nucleoside of the desired oligonucleotide is immobilized on the resin used for oligonucleotide synthesis. Various methods of bonding resins and nucleosides can be considered, but condensation of resins with amino groups with succinic acid derivatives of nucleosides (Nucleic Acids
Research, 8 , 5473 (1980)). In addition, as a quick method, a reagent for nucleic acid synthesis in which a nucleoside with a protected 5 ¹ -hydroxyl group is immobilized on a resin is commercially available.
For example, aminomethyl polystyrene resin (0.10~
0.15mmol/g, 1% divinylbenzene 100~
200mesh) [Biosearch Co., Ltd.] etc. In the case of the present invention, this commercially available product is used, and 20 mg of this is packed into a reactor. Swelling of the resin This is a common operation when synthesizing oligonucleotides and the like using the solid phase method, and the resin is usually swollen with the solvent used for deprotection (methylene chloride in the present invention). In the present invention, methylene chloride is absorbed with a syringe Ea, and this is left in the reaction chamber A for a certain period of time. The swollen solvent is discharged to the outside of the reaction chamber by pushing the piston of the syringe Ea to create positive pressure inside the reaction chamber. Deprotection operation To extend the nucleotide chain, first deprotect the ⁵¹ -terminal hydroxyl group of the nucleoside immobilized on the resin, then perform a dehydration condensation reaction between this and the phosphate residue of the nucleotide block. unresponsive
5 ¹ -Protection of the hydroxyl group is performed by repeating the same series of operations. Usually, the protecting group for 5 ¹ -hydroxyl group is dimethoxytrityl group, so benzenesulfonic acid, ZnBr ₂ (Nucleic Acids
Research, 10 , 1755 (1982)), deprotect with TCA (trichloroacetic acid), etc. In the present invention, this is carried out by absorbing TCA-containing methylene chloride and leaving it in the reaction chamber for a certain period of time. The solvent containing the removed protecting group is discharged from the reaction chamber by operating the syringe Ea, and if necessary, the resin is washed by repeating the operation of absorbing the solvent into the reaction chamber A and then discharging it. do. Condensation reaction The condensation reaction is carried out by dehydration condensation of the 5 ¹ -hydroxyl group on the resin and the phosphoric acid group of the nucleotide block (the 5 ¹ -hydroxyl group is protected with, for example, a dimethoxytriethyl group) in the presence of a condensing agent. Let's do it. Various triazolide and tetrazolide derivatives of allenesulfonic acid have been developed as condensing agents, including 2,4,6-trimethylbenzenesulfonyl-3-nitrotriazolide (hereinafter referred to as MSNT) and triisopropylbenzenesulfonyl. Tetrazolide (referred to as TPSTe) is a typical example.
In the synthesizer of the present invention, a pyridine solution in which a condensing agent and a nucleotide block are dissolved is sucked,
The condensation reaction is carried out by leaving this in the reaction chamber for at least 30 minutes. After condensation, excess reactants and the like are discharged, and the resin is washed in this manner as necessary. Protection of unreacted 5 ¹ -hydroxyl group Since the condensation reaction does not necessarily proceed quantitatively, it is inevitable that in the above condensation reaction, products that do not reach the specified chain length will be produced as by-products. If the condensation reaction is further carried out with long chain products mixed together, short chain products will be produced as by-products at each stage of the condensation reaction, so that after a certain condensation step, products with various chain lengths will be mixed together. In order to avoid such a situation, after one condensation step, short chain products that do not undergo condensation and the 5 ¹ -hydroxyl group remains deprotected are removed so that they do not participate in the subsequent condensation reaction. It is desirable to protect the 5 ¹ -hydroxyl group. Protection of the 5 ¹ -hydroxyl group of such short chains is usually carried out by acetylation or the like. In the present invention, pyridine-containing dimethylaminopyridine
The unreacted ⁵¹ -hydroxyl groups are protected by acetylation by aspirating acetic anhydride (9:1 (v/v)) and then standing for at least 5 minutes. After blocking the reactivity of short-chain long substances in this way,
Deprotect, condense, and unreact above as many times as desired
A desired oligonucleotide can be obtained by repeating a series of operations for protecting the ⁵¹ -hydroxyl group. Note that the yield of the condensation reaction in the case of the present invention is determined by colorimetrically quantifying the trityl group liberated by the above-mentioned deprotection operation. Recovery and purification of oligonucleotides Recovery and purification of oligonucleotides usually involves the release of oligonucleotides immobilized on resin and 5 ¹
The method consists of deprotecting the 5 ¹ -hydroxy group of the terminal nucleotide other than the protecting group, then deprotecting the 5 ¹ -hydroxy group, and then performing chromatography, electrophoresis, etc. In one embodiment, a solution of tetramethylguanidine-pyridine-2-carbalaloximate in dioxane-water (9:1, v/v) is sucked into the reaction chamber, and the solution is left at room temperature for at least one day. , drain and collect this solution. A conceivable method is to add concentrated ammonia water to this solution, leave it at 55°C, remove tritylation (for example, by treating with 80% acetic acid), desalt, and purify by HPLC. In addition, in this embodiment, before releasing the oligonucleotide from the resin, the resin in the reactor is suspended in pyridine, this suspension is taken out from the reactor into a pressure-resistant container, and the pyridine is distilled off. Release of the nucleotide from the resin followed by deprotection may be performed in the same manner as described above. For detailed operations, etc., please refer to Examples described later. Confirmation of Base Sequence The nucleic acid taken out from the synthesizer as described above should have a predetermined base sequence, but this can be confirmed if necessary. The nucleotide sequence can be confirmed using the nucleotide sequencing method of Lei Wu et al. (Methods in Enzymology 65 , Nucleic Acids).
Part, 620, (1980)) or Maxam
This can be easily carried out by the Gilbert method (ibid., 499, (1980)). Experimental Example Experimental Example 1 GTTCT (G is guanine, T is thymine, C is cytosine, and in the following experimental examples, A is adenine,
The nucleotides contained in each are shown) were synthesized. (1) Swelling of resin Fill a reaction chamber with 30 mg of resin immobilized with dimethoxytritylthymidine (commercial product), and add methylene chloride (hereinafter referred to as CH ₂ Cl ₂ ) to a container CH-3.
(CH ₂ Cl ₂ was prepared in three containers, and each container was designated as CH-1, CH-2, and CH-3). The purpose is to ensure that the resin is homogeneously suspended). Furthermore, the operation of suctioning and discharging CH ₂ Cl ₂ was repeated twice, and the third time
The CH ₂ Cl ₂ was left under vacuum for 30 minutes. (2) Detritylation ○A After discharging the CH ₂ Cl ₂ sucked from the container CH-1, 1 ml of CH ₂ Cl ₂ containing 3% TCA was sucked up (in this case, the solution in the reaction chamber was orange), After suctioning an additional 1 ml of air, the solution was allowed to stand for at least 1 minute, and then the solution was drained (this solution was saved to determine the recovery rate). ○B Next, 1 ml of CH ₂ Cl ₂ was suctioned from the container CH-2, and after 1 ml of air was suctioned, it was immediately discharged. Note that this operation was performed three times (to wash away the tritylation agent). ○C CH ₂ Cl ₂ containing 3% TCA (hereinafter referred to as 3% TCA/
CH ₂ Cl ₂ (abbreviated as CH 2 Cl 2 ), the operations in ○a and ○b were repeated until no coloration (orange to yellow) was observed (confirmation of detritylation). What should be noted here is that 3% TCB/CH ₂ Cl ₂ after the second
The processing time is limited to less than 10 seconds (because
Although the detritylation reaction is almost certainly carried out in one go, we carry out the operation in ○c for confirmation. Therefore, the detritylated
Furthermore, since a detritylating agent is added, if the treatment is carried out for a long period of time, there is a risk that debridement, which is a side reaction, may occur and the base may be eliminated. ○D 1 ml of CH ₂ Cl ₂ was suctioned from the container CH-3, and after 1 ml of air was suctioned, it was immediately discharged (washing). This operation was performed three times in total. (3) Condensation reaction ○a Anhydrous pyridine (hereinafter anhydrous pyridine will be referred to as Py, prepare it in three containers and fill each container with
Py-1, Py-2, Py-3) are containers Py
-1, 1 ml of air was suctioned, and then 1 ml of air was suctioned and immediately discharged. This operation was then repeated twice. ○B 1 ml of Py was sucked up from the container Py-2, and the same operation as ○B was repeated a total of 3 times. ○C The same operations as ○B and ○B above were repeated twice using container Py-3. The operations in ○a to ○c were carried out for the purpose of converting the solution. ○D After closing only the piston side with a three-way lock, nitrogen gas was passed through the gas inlet at a flow rate of 300 ml/min for 10 minutes. (Drying of resin). ○E Close only the gas inlet with a three-way screw, and close the Py
After suctioning 0.3 ml of a solution of MSNT (20 mg) and nucleotide (TC dimer 20 mg) dissolved in the reaction chamber into the reaction chamber, seal the solution inlet in the reactor part and leave it at room temperature for 30 minutes, then drain the solution. did. ○F In order to wash away the condensing agent and the reactive nucleotide, the operations ○I to ○C were repeated. (4) Protection (masking) of unreacted 5 ¹ -hydroxyl group ○A Py-acetic anhydride (9:1 (v/v)) containing a catalytic amount (approximately 10 mg) of dimethylaminopyridine
After suctioning 1 ml of the solution and further suctioning 1 ml of air, the solution suction port of the reactor was sealed and left for 5 minutes, and then the solution was discharged. ○B During the operation in (3) above, the same operations as ○B and ○B were performed (washing). ○C 1 ml of CH ₂ Cl ₂ was suctioned from the container CH-1, then 1 ml of air was suctioned, and the solution was immediately discharged. This operation was performed three times in total. ○D Using CH ₂ Cl ₂ from container CH-3, the same operation as in ○C above was repeated three times (○C and ○D above were washed). At this stage, it is a trinucleotide
TCT was synthesized, and then the same operations as 2 to 4 above were performed using the dimer (GT) to finally obtain the pentamer (GTTCT). The condensation yield was 93% on average as a result of colorimetric determination (OD504) of the detritylated solution collected in step (2) (see the table below).

[Table] (5) Recovery and purification of oligonucleotides After completing the above condensation, dry the resin, add 300μ of a solution of 0.5M picolinaldoximutetramethylguanidine in pyridine-water (9:1), and heat at 37°C. I left it overnight. Next, concentrated aqueous ammonia (4 ml) was added, and after standing at 55°C overnight, the resin was removed by filtration, the liquid was dissolved in water, and extracted with ether (deprotection). Next, gel filtration was performed using Sephadex G-50 (eluent: 50 mM triethylammonium bicarbonate, pH 7.5). At this time, the absorption at 260 nm of each fraction was measured, and the first eluted peak was collected and subjected to high performance liquid chromatography (HPLC), followed by 2 ml of 8% acetic acid.
The mixture was reacted for 10 minutes at room temperature, and the desired synthetic fragment was fractionated as a ⁵¹ -hydroxyl base. The column used was μBondapak C18 (reverse phase). The base sequence of the synthesized oligonucleotide was confirmed using the method of Lei Wu et al.
It was confirmed that it was GTTCT. Experimental example 2 Same as experimental example 1, chain length 13
(GCGATGCTGCGTG) was synthesized, recovered and purified. Also,
As a result of confirming the base sequence using the method of Lei Wu et al., as shown in Figure 3.
It turned out to be GCGATGCTGCGTG. The base sequence is shown vertically at the right end of FIG. Experimental Example 3 An oligonucleotide with a chain length of 20 was synthesized using the same procedure as in Experimental Examples 1 and 2. The nucleotide sequence is GA-CA-AAA-GC-TT-GG-GG AA-TT-C, and 25 mg of dimer and 30 mg of trimer are used as nucleotide blocks, and the synthesis is repeated sequentially to achieve the desired chain length. The yield was 35%. The purification and recovery were performed in the same manner as in Experimental Example 1, and the base sequence was confirmed by the Maxam-Gilbert method.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing a specific example of the DNA synthesis device of the present invention. A...Reaction chamber, B...Filter mounting seat, C...
...filter, D...supply/exhaust liquid port, E...supply/exhaust device. FIG. 2 is a reproduction of an autoradiogram obtained by Lei Wu et al.'s base sequencing method.

Claims (1)

[Scope of Claims] 1. A nucleic acid synthesizer for synthesizing nucleic acids by reaction on solid support resin particles, characterized by comprising the following members: (A) A reaction chamber consisting of a cylinder having a volume sufficient to accommodate the solid support resin particles and carry out the reaction. (B) A mounting seat for attaching the solid support resin particle retention filter, which extends downward from the reaction chamber A and has an inner diameter smaller than the lower part of the reaction chamber A. (C) A filter for retaining the solid support resin particles, which is attached to the mounting seat B. (D) A liquid supply/drainage port which forms a downward extension of the reaction chamber A further below the mounting seat B and allows the liquid phase material involved in the reaction to enter and exit the reaction chamber A. (E) A supply/exhaust device installed at the upper part of the reaction chamber, which allows the liquid phase material to enter and exit the reaction chamber from the supply/drain port D using gas pressure. However, this supply/exhaust device E consists of a positive and negative pressure generation source Ea and a valve device Eb, and the positive and negative pressure generation source Ea communicates with the upper part of the reaction chamber A via the valve device Eb. 2. The positive and negative pressure generation source Ea consists of a cylinder and a piston, and the valve device Eb consists of a three-way socket, the first port of which is connected to the reaction chamber A.
The pipe is configured such that the second port is connected to the cylinder of the positive and negative pressure source Ea, and the third port is closed. A nucleic acid synthesizer according to claim 1.