JPS60130597A - Nucleic acid synthesizer - Google Patents

Abstract

PURPOSE:A nucleic acid synthesizer that is provided with the reactor filled with the solid support of resin particles, the filter for holding the resin and the fixture therefor, outlet and inlet for feeding or exhausting liquid-phase materials and the device therefor, thus enabling simple and inexpensive synthesis of oligonucleotides by manual operations. CONSTITUTION:Oligonucleotides are obtained by manually operating the synthesizer which is composed of a cylindrical reactor A with a sufficient capacity to contain the solid support of resin particles, the fixture B for holding the filter C for supporting the resin particles, which is extended from the reactor A downward and has a smaller inner diameter than that of the lower part of the reactor A, the filter C fitted to the fixture B, the opening for feeding and exhausting liquid materials D, which is extended from the fixture B downward and the device E for feeding the liquid materials to the reactor A and exhausting it from the reactor A, which is provided with a positive and negative pressure generator Ea and a valve system Eb.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to a synthesizer 1 for synthesizing nucleic acids such as DNA and RNA. More specifically, the present invention relates to a nucleic acid synthesizer that can manually synthesize desired oligonucleotides such as DNA and RNA using the principles of solid-phase oligonucleotide synthesis.

PRIOR ART In recent years, with the rapid progress of genetic engineering, the demand for synthetic oligonucleotides has increased particularly in research on nucleic acids, and the synthesis technology has also become more sophisticated.

By the way, as a chemical synthesis method for oligonucleotides,
Diester method (5science, 203,614, (
(1979) ), triester method (Nttcleic
Ac1dsResearch, 8.2331 (1
980), IF? i8.5193 (1980), Mukami,
5491 (1980)), solid phase method (Nature
, 281.18 (1979), Biochemist
ry, 1980, 6096 (1980)), liquid phase method (P
roc, Natl, AcadlSci, USA 7
5.5765 (1978) Method using input enzyme (!Ju
cleic Ac1ds Re-5earch, 8
．． 5753, (1980), Phosphite method (Tetr
aheclron Letterg22.1859(1
981) ), or the triester method-solid phase method (Nucleic Ac1ds method), which is currently frequently used.
Re5search, 8.55o7 (1980))
, Phosphite Method - Solid Phase Method (J, Am, Ohem.

soa, 103, 3185-3191 (1981)
), etc., and recently a completely automated nucleic acid synthesis device has been devised based on a combination of these methods ゛° ζ ゛, and several models are already on sale. However, all fully automated acid nucleic synthesis devices are extremely expensive and currently difficult to purchase.

On the other hand, the oligonucleotides currently required in the field of genetic engineering include primers, gloves, and linkers, but most of these have chain lengths of 15 to 25, and are used in a single genetic manipulation experiment. The number of oligonucleotide chains is about several, and the amount used is about 0.50 D per oligonucleotide chain. Therefore, there is a desire to develop an apparatus that can synthesize oligonucleotides easily and at low cost without the need to purchase an expensive automated nucleic acid synthesis apparatus.

Summary of the invention Abstract.

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 K DNA and RNA.

Therefore, the nucleic acid synthesizer for synthesizing nucleic acids by reaction on solid support particles according to the present invention is characterized by comprising the following members.

(A) A cylindrical reaction chamber having a volume sufficient to accommodate the solid support resin particles and carry out the reaction.

(Bl A mounting seat for attaching the filter for retaining resin particles on the solid support, which has a downward extension part of the reaction chamber (i) and has an inner diameter smaller than the lower part of the reaction chamber (A).

(0) A filter for retaining resin particles on the solid support, which is attached to the mounting seat.

(D) A liquid supply/drain port for supplying and discharging the liquid phase material involved in the reaction into and out of the reaction chamber (A), which further forms a downward extension of the mounting seat (Bl) of the reaction chamber (Al).

(A supply/exhaust device installed at the upper part of the equalization reaction chamber, which allows the liquid phase material to enter and exit the reaction chamber from the supply/discharge liquid 0 (D) using gas pressure.
1 consists of a positive and negative pressure generation source (Ba) and a valve device (Eb), and the positive and negative pressure generation source (Ba) communicates with the upper part of the reaction chamber (A) via the valve device (Eb).

Furthermore, the reaction tube for a nucleic acid synthesizer for synthesizing nucleic acids by reaction on solid support particles according to the present invention is characterized by comprising the following members.

(A) A cylindrical reaction chamber 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 has a downward extension of the reaction chamber tA+ and has an inner diameter smaller than the lower part of the reaction chamber (i).

(0) A filter for retaining the solid support resin particles, which is attached to the mounting seat (B).

(2) A liquid supply/drain port for supplying and discharging the liquid phase material involved in the reaction into and out of the reaction chamber (Al), which forms a downward extension of the reaction chamber (A) and the mounting seat (B).

Effects As described above, the present invention relates to a nucleic acid synthesizer that can be operated manually, but 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. The desired oligonucleotide can be obtained reliably and in high yield through 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, gloves, 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 feature 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 provides good contact pressure 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 introduced into the reactor, it is possible to avoid collecting the resin in a specific portion 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. If the resin layer is present in a cylindrical portion (i.e., the inner diameter is substantially the same in the upper and lower directions), the resin layer remains intact, i.e., without decomposing into individual resin particles, Therefore, the contact between the resin particles and the solution (and solvent) is insufficient.In such a situation, according to the present invention, the reaction chamber containing the resin layer moves the resin above the resin layer. Since the diameter is larger than the layer part, the resin layer is decomposed into individual resin particles by the solution or solvent flowing from below, resulting in good contact between the resin ladle and the solution or solvent.Solid phase method These features improve nucleic acid synthesis efficiency (including washing efficiency) since they target reactions on the surface of resin particles.

The apparatus of the present invention is also characterized in that the liquid phase material is moved in and out of 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 compared to the case where the liquid phase material is directly pressurized, the device for achieving the purpose of liquid phase material inflow and outflow is It may be simple (for example, one consisting of a piston and a cylinder like Ba in Figures 1 and 1), and therefore the reaction chamber and resin can be easily and reliably cleaned during liquid phase material conversion. This is because when press-fitting a liquid phase material, it is 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. Further, as the equipment is changed, the cleaning operation required each time the liquid phase material is changed becomes complicated.

Nucleic Acid Synthesizer Overview of the Synthesizer The nucleic acid synthesizer according to the present invention is an IT used for synthesizing nucleic acids by reaction on solid support resin particles, that is, by an in-phase method, and comprises the aforementioned members A-, It consists of B. The movable part of the device of the present invention is mainly the air supply and exhaust system (El), but although it is of course possible to drive this part electrically or to perform the combined operation under the control of a heater, it is also possible for the experimenter to operate it manually. It is most preferable to use the device of the present invention.

Considering that it can be easily used manually, the positive and negative pressure generating device ff1 (Flta) in the device of the present invention is preferably a syringe consisting of a cylinder and a piston, and the positive and negative pressure generating device (Ea) and the valve device ( El)) and between the valve device fii (El)) and the reaction chamber (A), especially between the valve device (Kb) and the reaction chamber (A), may be detachably connected. preferable.

A specific example of a synthesizer according to the present invention is shown in FIG.

Reaction Chamber (A) The 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 approximately one circle, and whose length is sufficiently larger than the diameter (or equivalent diameter if the cross section is not a perfect circle). Same thing) means). In addition, the shape or size of the cross section does not have to be the same in the length direction, and if necessary, it may have a protrusion inside. A small-diameter connector Aa is usually provided to connect to one port of the three-way cock (details will be described later) of fF? (Eb).The connector Aa also serves as a lid on the top of the reaction chamber. ing.

In the specific example shown in FIG. 1, the reaction chamber (Al) and the top structure Aa are connected by a tape or tube Ab.

On the other hand, a filter mounting seat (Bl) having a smaller diameter 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 it allows you to easily check the state of the reaction and the state of the resin. Furthermore, it is more preferable to coat the inner wall of the reactor with silicon 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 10 mrng degrees.

The mounting seat (B) for attaching the solid support resin particle retention filter (details will be described later) is a member that forms a downward extension of the reaction chamber (Al) 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 the reaction chamber (A)" means that the point where this filter is attached is the same as that of the reaction chamber (A) at the point where this filter attachment v (B) is connected. ).This means that it is sufficient that the inner diameter is smaller than the inner diameter of the reaction chamber (A).Therefore, this does not exclude the case where there is an even smaller diameter portion above the reaction chamber (A). In this case, the inner diameter of the filter mounting seat (B) decreases continuously downward.
This also includes the finished part 1 whose diameter is enlarged below the filter attachment point of 1.

A specific example of the filter mounting seat (B) is a funnel-shaped one 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 vertically 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.

The filter (0) 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 of e-recess construction, known as a 19'rNil glass filter.

Ceramic is also considered to be a member of the filter.

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 (q is installed on the mounting seat (B),
Here, the term "attached" includes both cases where it is removable from the mounting seat and cases where it is not removable. If it is removable, care should be taken when attaching it so that it is not pushed upward by the liquid phase material for synthesis reaction that enters from below.

Liquid supply/drainage port (D) The liquid supply/drainage port exists as a downward extension of the reaction chamber (Al) and the mounting seat (Bl). Supply and drain pipe (Da) as shown in
It may be in the form of

The supply/exhaust device (I!+) 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 consists of a positive and negative pressure generator (Ba) and a valve device (
mb), and a positive and negative pressure source (Fia)
communicates with the upper part of the reaction chamber (Al) via a valve device (lung).

The positive and negative pressure source (Ka) can be any suitable pumping 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 (xb) usually consists of a three-way cock. In that case, the first port communicates with the upper part of the reaction chamber (Al), and the second port communicates with the positive/negative source (Ra) (if this consists of a cylinder and a piston,
It is preferable that the conduit is configured such that it communicates with the cylinder) and the third port is closed. The third port can be conveniently used for drying the resin particles for the in-phase process or for introducing an inert gas (eg, nitrogen) for forming the reaction atmosphere. The preferred three-way cock structure described above has the three-way cock (ILb) in FIG. 1 having 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. Please note that this operating manual describes preferred specific examples.
It goes without saying that operating conditions other than those described below are also possible.

Nucleic acids such as DNA and RNA are synthesized by condensing nucleotides by the in-phase method using the nucleic acid synthesizer of the present invention. The synthesizer according to the invention has a DN
The main purpose of this method is to synthesize oligonucleotides such as A and RNA.
The desired oligonucleotide can be obtained by sequentially condensing nucleotide blocks (monomers, dimers, trimers, etc.) or by repeatedly condensing nucleotide blocks (monomers, dimers, trimers, etc.), but which method is used depends on the preparation of the raw nucleotide blocks. , may be appropriately selected in consideration of the average yield of 9 reactions, separation and purification of the target product, etc. - Specific methods for synthesizing oligonucleotides by solid-phase method are described in Examples and literature (Nucleic Acls Re5
Please refer to 8.5507 (]-980) ) etc.

Introduction of the 31-terminal nucleoside into the resin The 3'-terminal nucleoside of the desired oligonucleotide is immobilized on the resin used for oligonucleotide synthesis. There are various ways of bonding resins and nucleosides, but condensation of resins with amino groups with succinic acid derivatives of nucleosides (Nu
cleic Ac1dsResearch, B, 54
73 (1980)). In addition,
As a quick and easy reagent for nucleic acid synthesis, 51-
Products in which nucleosides with protected hydroxyl groups are immobilized on resins are commercially available, such as aminomethyl polystyrene resin (0.10-015 mmol/g, % divinylbenzey 100-200 mesh) [Biosearch Co., Ltd.]. In the case of the present invention, this commercially available product is used, and 20 mg of it is packed into a reactor.

This is a common operation when synthesizing oligonucleotides, etc. by the resin swelling 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 (Fia) and the reaction chamber (Al
This is done by leaving it 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 (Ha) to create positive pressure inside the reaction chamber.

Deprotection operation To extend the nucleotide chain, first, the 51-terminal hydroxyl group of the nucleoside immobilized on the resin is deprotected, and then,
This is carried out by carrying out a dehydration condensation reaction with the phosphoric acid residue of the nucleotide block, then protecting the unreacted 5I-hydroxyl group, and repeating the same series of operations. Usually, the binding group for 51-hydroxyl group is dimethoxytrityl group, so benzenesulfonic acid, ZnBr, (Nuc
leic Ac1dsResearch, 10, L
755 (1982)), TOA (trichloroacetic acid), or the like. In the present invention, TOA-containing methylene chloride is absorbed and left in the reaction chamber for a certain period of time. The solvent containing the removed protecting group is a syringe (crazy)
The resin is washed by repeating the operation 1- to remove the solvent from the reaction chamber and, if necessary, absorb the solvent into the reaction chamber (Al) and then remove the solvent.

Condensation Reaction The condensation reaction is carried out by dehydration condensation between the 51-hydroxyl group on the resin and the phosphoric acid group of the nucleotide block (the 51-hydroxyl group is protected with, for example, a dimethoxytrityl group) in the presence of a condensing agent. As the condensed culm], various triazolido and tetrazolide derivatives of are/sulfonic acids have been developed. M.S.
Typical examples include NT) and noisopropylbenzenesulfonyl tetrazolide (TPSTe).

In the present invention σ] Synthesis '4>, a pyridine solution in which a condensing agent and a nucleotide block are dissolved is aspirated and left in a reaction chamber for at least 30 minutes to carry out a condensation reaction. After the condensation, excess reactants and the like are discharged, and the resin is washed in this manner as necessary.

The protection condensation reaction of the unreacted 51-hydroxyl group does not necessarily proceed quantitatively. , such a short chain long car was mixed ~
If the condensation reaction is further carried out, short chain length products will be produced as by-products at each stage of the condensation reaction, so that after a certain condensation step, products of various chain lengths will coexist. In order to avoid such a situation, after carrying out the condensation step - times,
Condensation force: It is desirable to protect the 51-hydroxyl group so that the 51-hydroxyl group remains deprotected and the short-chain product does not participate in the subsequent condensation reaction. The protection of the 51-hydroxyl group of long chains is usually carried out by acetylation, etc. In the present invention, pyridine-acetic anhydride (9:
1 (v/v)) and then allowed to stand for at least 5 minutes to carry out acetylation, thereby protecting unreacted 5-hydroxyl groups.

After blocking the reactivity of short-chain molecules in this way, the desired oligonucleotide can be obtained by repeating the above series of deprotection, condensation, and protection of unreacted 51-hydroxyl groups as many times as desired. I can do it. In the case of the present invention, the condensation reaction σ) yield %-1 is carried out by colorimetrically quantifying the trityl group liberated in the above deprotection operation.

The recovery and purification of oligonucleotides and 8′l￥ oligonucleotides are usually carried out by releasing the oligonucleotide immobilized on the resin and releasing the 51-terminal nucleotide.
After deprotection of groups other than the protecting group of '-hydroxyl group, 5
The process consists of deretaining the 1-hydroxyl group and then performing chromatography, electrophoresis, etc.

In one embodiment, a solution of tetramethylguanidine-pyridine-2-carbalaloximate in dioxane-water (9: i, ”/v) is sucked into the reaction chamber, and this is left at room temperature for at least one day. This solution is drained and collected. Concentrated aqueous ammonia is added to this solution and left at 55°C, followed by detritylation (for example, 80% acetic acid treatment), desalting, and purification using HPLO. In addition, in this embodiment, before releasing the oligonucleotide from the resin, the resin in the reactor is suspended in pyridine, and this suspension is taken out from the reactor into a pressure-resistant container. , after distilling off pyridine, the same nucleotide release from the resin as above and subsequent deprotection operation may be performed.For detailed operations, etc., refer to the experimental example below (So. The nucleic acid extracted from the synthesizer should have a predetermined base sequence, but it can be confirmed if necessary.The base sequence can be confirmed using the base sequencing method (Methoasin) of Lei Wu et al. mnzymo
logic Omi, Nucleic Ac1ds Part
I, 6! O.

(1980) ), or the Maxam-Gilbert method (
499, (1980)) i0 Experimental Example Experimental Example 1 Follow the procedure 1 below to perform GTTOT (where G is guanine, T is thymine, C is cytosine, and the following experimental example In the above, A indicates adenine, and the nucleotides containing the respective nucleotides were synthesized.

(1) Swelling of resin Fill ao mg of resin (commercially available) with immobilized dimethoxytrityl thymidine into a reaction chamber, and prepare methylene chloride (hereinafter referred to as CH2Cl2) in container CH-3 (0H2ON□ in three containers). 0H-1, O
H-2, 0H-3) and then 1 m of air.
1 was suctioned (hereinafter, this operation is performed for the purpose of uniformly suspending the resin).

The operation of suctioning and discharging 0H2ON□ was repeated twice, and for the third time, the tube was left for 30 minutes while CH2Cl2 was being sucked.

(2) Detritylation■ After discharging the 0H2C12 sucked from the container 0H-1, sucked up 121ml of 0H20 containing 3% T'OA (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 (note that this solution was saved to increase the recovery rate).

(2) Next, 1 ml of CH2Cl2 was suctioned from the container 0H-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 tritylating agent).

θ 3% TOA-containing C! H2O1□ (hereinafter 3%
TOAlo) I2C12) colored (orange to yellow)
The operations of ① and 0 were repeated until no more was observed (confirmation of removal of trityl). What should be noted here is 2.
The time for 3% TOB10H20U2 processing after the first time is 10
(This is because the detritylation reaction is almost certain to occur once, but the θ operation is performed for confirmation. Therefore, the detritylation reaction is carried out in one step. Therefore, the detritylation reaction is carried out once. This is because if the treatment is carried out for a long time, a side reaction called depurination may occur, causing the base to drop out).

1 ml of @0H2O12 was suctioned from the container CH-3, and after sucking in one ounce of air, it was immediately discharged (washing).

This operation was performed three times in total.

(3) Condensation reaction ■ Anhydrous pyridine (hereinafter referred to as anhydrous pyridine P7, prepare three containers, each containing P7-1, py-2,
1 ml of P7-3) was suctioned from the volume &1tPy-1, and after 1 ml of air was suctioned, it was immediately discharged. This operation was then repeated twice.

(2) 1 ml of Py was sucked up from the container Py-2, and the same operation as (2) was repeated three times in total.

θ Using container Py-3, perform the same operations as 0 and ■ above in 2.
The repeated operations from ■ to θ were performed for the purpose of converting the solution.

G After closing only the piston side with a three-way cock, nitrogen gas was passed through the gas inlet at a flow rate of 300 ml/17 min for 10 minutes (drying the resin).

■ Close only the gas inlet with a three-way cock, and suck 0.3 ml of a solution of ysmT (20 mg) and nucleotide (TO dimer, 20 mg) dissolved in water into the reaction chamber, and then open the solution inlet in the reactor section. 3. Seal and store at room temperature.
After standing for 0 minutes, the solution was drained.

To wash away the O condensing agent and reactive nucleotides, ■
The operations from ~θ were repeated.

(4,) Preservation (masking) of unreacted 51-hydroxyl groups■
1 ml of Py-acetic anhydride (9:1 (v/v)) solution containing a catalytic amount (approximately 1 o mg) of dimethylaminopyridine
After suctioning 1 ml of air, the solution suction port of the reactor was sealed, and the reactor was left to stand for 5 minutes, and then the solution was discharged.

(2) During the operation (3) above, the same operations as (1) and (0) were performed (washing).

○ 0H201,! is aspirated from container CH-1,
Then, 1 ml of air was suctioned and the solution was immediately discharged.

This operation was performed a total of three times.

○ Using 0H2012 from container 0H-3, the same operation as in the above θ was repeated three times (6.O is washing).

TOT, which is a trinucleotide, has been synthesized in the steps up to this point, and then by performing the same operations as 2 to 4 above using a dimer (GT), the pentamer (GT) is finally synthesized.
GTTOT) was obtained.

The condensation yield was 93% on average as a result of colorimetric determination (OI) 504) of the detritylated solution recovered in step (2) (see the table below).

(5) Recovery and purification of oligonucleotides After the completion of the above condensation, the resin is dried, and a solution of 0.5M picolinaldoxime-tetramethylguanidine in pyridine-water (9:1) 3
00 μm was added and left at 37° C. overnight. Next, add concentrated ammonia water (4 ml) and leave it at 55°C overnight, then r
The resin was removed by filtration, the r liquid was dissolved in water, and extracted with ether (deprotection). Next, gel filtration was performed using Sephadex 0G-50 (eluate was 50-related triethylammonium bicarbohydrate, pH 7.5). At this time, the absorption of each fraxilon at 260 nm was measured, and the first eluted peak was collected and subjected to high performance liquid chromatography (HPLO).Then, the 15'-hydroxyl group was reacted in 2 ml of 8% acetic acid at room temperature for 10 minutes. The desired synthetic fragment was fractionated. Note that the column used was p Bondapak O18 (reverse phase). Confirmation of the base sequence of the synthesized oligonucleotide is done by Ray Ray.
It was confirmed to be GTTCT by the method of Wu et al.

Experimental Example 2 The chain length was 13 (GOGATGOTG) in the same manner as Experimental Example 1.
CGTG) oligonucleotide was synthesized, recovered and purified. Furthermore, the base sequence was confirmed by the method of Lei Wu et al., and as a result, it was found to be GOGATGCTGOGTG, as shown in FIG.

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 Example 1.2. Its base sequence is GA-OA, -A
AA-GO-TT-GG-GGAA-TT-0,
As the nucleotide block, 25 rng of dimer and 35 mg of trimer were used, and the synthesis was carried out sequentially (total condensation yield was 35% when the desired chain length was reached. The purification and recovery were the same as in Experimental Example 1. The base sequence was confirmed by the Maxam-Gilbert method.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing a specific example of the DNA synthesis apparatus of the present invention. A... Reaction chamber B... Filter mounting seat C... Filter D... Liquid supply/drainage port E... Supply/exhaust device Figure 2 shows the base sequence determination method of Lei Wu et al. This is a copy of the autoradiogram from that time. Applicant's agent Kiyoshi Inomata i Figure 62

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 reaction chamber consisting of a cylinder having a volume sufficient to accommodate the solid support resin particles and carry out the reaction. (B) A reaction chamber tA' having a downward extension of the reaction chamber 1A). A mounting seat for attaching the solid support resin particle retention filter, which has an inner diameter smaller than the lower part of the solid support. (C) A filter for retaining the solid support resin particles, which is attached to the mounting seat (B). CD) Reaction chamber (Al), which forms a downward extension of the mounting seat (B), and a liquid supply/drainage port for supplying and discharging liquid phase materials involved in the nuclear reaction to and from the reaction chamber + Al. (B) Reaction chamber The supply/exhaust device (E) is installed on the top of the device to allow liquid phase material to enter and exit the reaction chamber from the supply/drain port (D%) using gas pressure. Consists of a generation source (Fia) and a valve device (Eb),
Further, the positive and negative pressure generation # (Ea) communicates with the upper part of the reaction chamber (A) via the valve device f (B'b). 2. The positive and negative pressure generation source (Ka) consists of a cylinder and a piston, and the valve system R (Eb) consists of a three-way cock, whose first port is detachably connected to the upper part of the reaction chamber (Al). The pipe line 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 item 1. 3. A reaction tube for a nucleic acid synthesizer for synthesizing nucleic acids by reaction on solid support resin particles, characterized by comprising the following members: μ) A reaction chamber consisting of a cylinder having a volume sufficient to accommodate the solid support resin particles and carry out the reaction. (Bl A mounting seat for attaching the solid support resin particle retention filter, which has a downward extension of the reaction chamber (A) and has an inner diameter smaller than the lower part of the reaction chamber +A). (A filter for retaining the solid support resin particles attached to the mounting seat (Bl). Φ) A filter for the reaction chamber (Al) that forms a downward extension of the mounting seat (of the reaction chamber rA). A liquid supply/drainage port for entering and exiting the liquid phase material involved.