JPH0465838B2 - - Google Patents

Description

[Detailed Description of the Invention] (a) Industrial Application Field This invention relates to a device for synthesizing nucleic acids such as DNA and RNA.

(b) Prior art As a nucleic acid synthesis method, the so-called diester method,
Improvements and developments have been made to the triester method and the phosphite method, and the solid support method, which utilizes these methods and uses a solid support, has come to be widely used because it has various advantages. Various apparatuses for synthesizing nucleic acids using these methods have also been proposed.

Each device basically consists of a reactor, a nucleotide reagent supply means capable of supplying multiple types of nucleotide reagents blocked with protecting groups to the reactor in a predetermined procedure, a protecting group removing agent, and a condensing agent for synthesis. A reaction reagent supply means capable of supplying a masking agent and a masking condensation agent to the reactor, respectively, and a synthetic condensation step with a protecting group removal step and masking by controlling the nucleotide reagent supply means and the reaction reagent supply means. The apparatus is equipped with a control means for repeating the condensation step to accomplish the acid synthesis.

By the way, there are basically four types of nucleotide reagents depending on their bases, and unless the procedure for using these reagents is correct, it is not possible to synthesize a nucleic acid with a desired base sequence.

However, in conventional nucleic acid synthesizers, it is not possible to check whether the nucleotide reagents are used correctly in a given procedure, so mistakes are often made, for example, by incorrectly setting the nucleotide reagents in the device. There was a case where a nucleic acid with a base sequence was synthesized.

(c) Purpose of the Invention The purpose of the present invention is to provide a nucleic acid synthesis apparatus capable of checking whether nucleotide reagents are being used correctly in a predetermined procedure during nucleic acid synthesis.

(d) Structure of the Invention The present invention provides an apparatus for synthesizing nucleic acids by supplying a plurality of nucleotide reagents blocked with protecting groups to a reactor according to a predetermined procedure and repeating a protecting group removal step, a condensation step, and a masking step. In this case, the absorbance of the waste liquid from the protecting group removal step is set to 2 in the drain path of the reactor.
In addition to providing an absorbance measuring means capable of measuring at different wavelengths as mentioned above, a qualitative/quantitative calculation means is provided on the output side of the absorbance measuring means for qualitatively and quantitatively determining the eliminated protecting group contained in the waste liquid based on the output thereof. A nucleic acid synthesis device is provided.

By attaching different types of protecting groups to different types of nucleotide reagents and qualitatively characterizing the removed protecting groups, it is possible to know which nucleotide reagent was used, thereby ensuring that the nucleotide reagents are not used in a given procedure. You can check whether it is set or not.

The apparatus of the present invention can be easily achieved by attaching the absorbance measurement means and the qualitative/quantitative calculation means to a conventionally known nucleic acid synthesis apparatus.

(E) Embodiment (1) shown in FIG. 1 is an embodiment of the present invention, which is an automatic DNA microsynthesis device using the phosphotriester method.

First, the configuration of the synthesis section will be explained.

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-like flange 4 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 volume of about 450 μl.

There are 8 types of reagent solutions in total.

11 to 14 are various nucleotide reagent solutions,
The bases are adenine, cytosine, and guanine, respectively.
Contains thymine. The 5' hydroxyl group of the nucleotide reagent solution 11 having adenine as a base is blocked using a 4-monomethoxytrityl group (MMTr) as a protecting group. Similarly, the nucleotide reagent solution 12 having cytosine as a base is blocked with 4,4'-dimethoxytrityl group (DMTr), and the nucleotide reagent solution 13 having guanine as a base is blocked with 4,4',4''-trimethoxytrityl group (DMTr). The nucleotide reagent solution 14 having thymine as a base is blocked with a trimethyl group (Tr).

15 is a condensing agent, which is a pyridine solution of 2-4-6-trimethylbenzenesulfonyl-3-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 condensing agent for masking, 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 absorbed 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. 36 of these solvents
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 is for directly supplying nitrogen gas to the reactor 2 in order to blow the inside of the reactor 2 with nitrogen gas. The nitrogen gas is dried with a desiccant 49 such as calcium chloride.

In addition to controlling the planshaft 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.

The switching cock 52 switches and connects the drain flow path 53 of the reactor 2 to the waste flow path 54 or the measurement flow path 55, and its operation is controlled by the control circuit 34.

The dilution container 56 is a cylindrical container with an internal capacity of about 1200 ml, and its head is connected to the measurement channel 55.
The bottom part is connected to the absorbance measuring unit 6 via the measuring valve 57.
8 flow cell 69.

An optical level sensor 5 is installed on the side wall of the dilution container 56.
8 is attached, and the output of the level sensor is connected to the control circuit 34.

The dilution vessel 56 can be supplied with a dilution and washing liquid via a valve 63, for example methylene dichloride 60 containing 4% trichloroacetic acid. Further, a valve 6 is inserted into the dilution container 56 from the top and bottom.
Nitrogen gas can be supplied via 5 and 66. The operation of these valves 63, 65, 66, exhaust valve 67, and measurement valve 57 is controlled by a control circuit 34.

The absorbance measurement unit 68 converts the light emitted from the colored light source 70 into a single wavelength using an interference filter, and detects the light with a light receiver 78 through a flow cell 69 . As shown in FIG. 2, there are four types of interference filters, 72 to 75. The interference filter 72 transmits light with a wavelength of approximately 500 nm, the interference filter 73 transmits light with a wavelength of approximately 480 nm, the interference filter 74 transmits light with a wavelength of approximately 440 nm, and the interference filter 75 transmits light with a wavelength of approximately 420 nm. . By rotating the holder 71 around the rotating shaft 71a, any one of these interference filters 72 to 75 can be selected and used. 1st
Absorbance measurement unit 6 viewed from a direction perpendicular to the figure.
8 is shown in FIG. 77 is reference cell,
79 is a light receiver. The outputs of the photodetectors 78 and 79 are connected to a control circuit 34, and the control circuit 34 qualitatively and quantitatively determines the removed protecting group and calculates the yield based on the output signals of the photodetectors 78 and 79. .

For DNA synthesis, first, the stopper 5 is removed and a support 9 to which only the terminal portions of DMA 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. Note that the 5' hydroxyl group at the end of the DNA molecule is blocked with, for example, DMTr. Stopper 5
After returning the support body 9 to its original position, the amount of the support 9 put in is inputted to the control circuit 34 via the operation console 35, and then a start command is inputted.

Then, the control circuit 34 switches between the switching switches 16 to 2.
0, plunger drive circuit 26-30, valve 42-4
8, 50, and 51 to execute the synthesis cycle shown in FIG. And at the same time, the second
Perform qualitative, quantitative, and yield measurements as shown in Figure B.

That is, when discharging the waste liquid from the reactor 2 several times in the deprotection step, the switching pot 52 is switched to the measurement flow path 55 side and the liquid is collected into the dilution container 56. Further, the waste liquid from washing with the mixed solution 38 of isopropanol and methylene chloride is similarly collected into the dilution container 56. In this way, all of the removed protecting groups are collected in the dilution container 56. Thereafter, the control circuit 34 operates as follows in parallel with returning the switching pot 52 to the waste channel 54 and continuing the synthesis cycle.

First, the valve 63 is operated to inject the diluent 60 so that the liquid level is at the level sensor 58.
As a result, the waste liquid to be measured is diluted to an appropriate constant concentration. Next, valve 66 is activated to bubble nitrogen gas into the dilution container 56, and valves 57 and 65 are activated to discharge nitrogen gas through flow cell 69. At this time, filters 72, 73, 7
Absorbance is measured at different wavelengths using each of Nos. 4 and 75, and then washed with diluent 60.

Figure 5 A, B, C, and D are MMTr, respectively.
It is a diagram of absorbance spectra of DMTr, TMTr, and Tr. As can be easily understood from the figure, 420nm,
By obtaining the absorbance at 440nm, 480nm and 500nm, their relative patterns indicate that the protecting group is
It is possible to identify whether it is MMTr, DMTr, TMTr, or Tr. After the removed protecting groups are thus qualitatively determined, it is checked whether the removed protecting groups are in the predetermined order. If it is a different protecting group, it means that the nucleotide reagent for the prescribed procedure was not used, so the synthesis cycle is stopped and the operator is notified by lighting an alarm lamp on the console 35, etc. Issue an alarm. On the other hand, if the qualitatively determined protecting groups are in a predetermined order, this means that the nucleotide reagent is used in a predetermined procedure, and therefore quantification is performed next.

Since the type of protecting group is qualitatively classified and the dilution of the waste liquid is an appropriate constant concentration, quantitative determination can be easily performed by comparing with preset calibration values. After quantitative determination, calculation of yield is performed.

The calculation of yield is performed by dividing the quantitative value in the synthesis cycle by the memorized quantitative value in the previous synthesis cycle and multiplying by 100.
The yield obtained is that of the previous synthesis cycle. In other words, the yield of the first synthesis cycle is obtained in the middle of the second synthesis cycle. The yield is displayed on the console 35.

The control circuit 34 calculates the yield of a certain synthesis cycle as described above, and calculates the overall yield by multiplying all the yields of the previous synthesis cycles.
It is displayed on the console 35.

Furthermore, the control circuit 34 determines whether the yield of a certain synthesis cycle is equal to or higher than a predetermined value (for example, 80%) and determines whether the overall yield is equal to or higher than a predetermined value (for example, 30%). If either one of them is not equal to or greater than a predetermined value, the synthesis cycle that is being executed in parallel is stopped, and an alarm lamp is lit on the console 35 to issue a warning to the operator.

As a modification of the synthesis section, reactor 2 may be made into a funnel shape, a barrel shape, or the volume of the reaction section may be reduced.
Examples include those varying between 80μ and 800μ. In addition, kel-F・g styrene was used as a solid support.
Examples include those using silica gel, polyacrylic morpholide, etc. These supports have a particle size of 30~
A thickness of about 300 microns is preferable.

A variation of the method for quantifying deprotecting groups is to collect the waste fluid discharged several times during the deprotection process and then dilute it. The total amount of protecting groups may also be determined.

A modification of the absorbance measurement unit 68 includes one in which the number of interference filters (ie, the number of measurement wavelengths) is increased or decreased depending on the type of protecting group to be qualitatively determined. Further examples include those that measure spectra with spectrophotometers that use gratings or prisms.

As the protecting group, a trityl group optionally substituted with a lower alkoxy group other than those listed in the above examples may be used.

Further, instead of the diluting liquid, a diluted coloring liquid such as a mixed liquid of ethanol and perchloric acid may be added.

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 diester method. Another example is one in which one deprotecting group quantitative determination/yield calculation/automatic stop control section is associated with a plurality of synthesis sections. In this case, the synthesis operation is continued in the synthesis section with a high yield, and the synthesis operation is stopped in the synthesis section with a low yield.

Note that even if the synthesis operation is stopped due to a decrease in yield, it is preferable to continue the synthesis operation again according to instructions from the operator.

According to the DNA synthesis apparatus 1 of the above embodiment, the reaction section 10 of the reactor 2 is miniaturized, the support 9 is placed on the filter 7, and the reagent solutions 11 to 15 are supplied from above, The reactor 2 is configured so that liquid is drained from the bottom. Therefore, 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 amount of feed (approximately 5 to 7 times the volume of the support) is sufficient, and mixing and contacting operations such as shaking the reactor to promote the reaction are no longer necessary. In addition, before the reaction to link new nucleotides, the inside of reactor 2 was heated with THF36.
The reactor 2 is configured to be washed and dried with water and blown with dry gas to completely dry the inside of the reactor 2 in a short time.As a result, water that inhibits the condensation reaction can be completely removed, so the reaction efficiency does not decrease. No extra reagents required. Furthermore, it is very convenient to easily know the reaction yield during synthesis. This is because it is extremely easy to check whether the synthesis is being performed properly. Furthermore, since the synthesis operation is automatically stopped when the yield falls below a predetermined level, waste of reagents and time is avoided, and unattended operation is also possible, which is practical.

(F) Effects of the Invention According to the nucleic acid synthesis apparatus of the present invention, it is possible to qualitatively characterize the protecting group removed in the protecting group removal step. Therefore, for example, if the type of protecting group is changed for each different type of nucleotide reagent, the type of nucleotide reagent used in the synthesis can be estimated by qualitatively characterizing the protecting group, and the nucleotide reagent can be used in a predetermined procedure. The reliability of the device is improved because it can be confirmed whether the device is present or not.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the configuration of a micro-DNA synthesis device which is an embodiment of the nucleic acid synthesis device of the present invention, FIG. 2 is an explanatory diagram of the configuration of the interference filter portion of the device shown in FIG. 1, and FIG. 4 is a flowchart of the operation of the device shown in FIG. 1, and FIG.
It is a spectrum diagram of the absorbance of. 1...DNA micro-automatic synthesizer, 2...Reactor,
11-14... Nucleotide reagent solution, 15,3
9-41... Reagent solution, 34... Control circuit, 35
...Operation console, 52...Switching knob, 53...Drainage channel, 55...Measurement channel, 56...Dilution container, 5
8... Level sensor, 60... Diluent, 68...
Absorbance measurement unit, 69...flow cell, 7
2, 73, 74, 75...interference filter, 77...
...Reference cell, 78, 79... Light receiver.

Claims (1)

[Scope of Claims] 1. In an apparatus that performs nucleic acid synthesis by supplying a plurality of nucleotide reagents blocked with protecting groups to a reactor according to a predetermined procedure and repeating a protecting group removal step, a condensation step, and a masking step, a reaction is performed. An absorbance measuring means capable of measuring the absorbance of the effluent in the protecting group removal step at two or more different wavelengths is provided in the drainage path of the device, and the output side of the absorbance measuring means determines the amount of water contained in the effluent based on the output. 1. A nucleic acid synthesis apparatus comprising qualitative and quantitative calculation means for qualitatively and quantitatively determining the removed protecting group.