JPH0573758B2 - - Google Patents

Description

[Detailed Description of the Invention] (a) Industrial Application Field The present invention relates to a method 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.

On the other hand, as a method for separating nucleic acids related to the production of a specific protein from a mixture of various types of nucleic acids,
The base sequence of the nucleic acid is inferred from the amino acid sequence of the protein, DNA with a complementary base sequence to that base sequence is synthesized, this is marked and added to the mixture, and the synthesized DNA is bound to the target nucleic acid. There is a method of using the trace mark as a landmark and hoisting up the target nucleic acid.

However, in many cases there are multiple genetic codes for one amino acid in a protein, and as a result,
Numerous possibilities arise for DNA base sequences. In this case, all or some of the possible
Usually, DNA is synthesized and mixed.

However, with conventional devices of this type, it was not possible to synthesize multiple types of DNA at the same time, so multiple devices of this type were required, or one
Different DNAs must be synthesized one after another on the stand.
It was extremely inconvenient.

(c) Purpose of the invention The purpose of the present invention is to provide a nucleic acid synthesis method that can simultaneously synthesize many types of nucleic acids and also allow confirmation of what types of nucleic acids have been synthesized and how much. .

(d) Structure of the Invention This invention performs nucleic acid synthesis by supplying a plurality of nucleotide reagents blocked with a protecting group to a reactor according to a predetermined procedure and repeating a protecting group removal step, a synthetic condensation step, and a masking step. In the apparatus, an absorbance measuring means capable of measuring the absorbance of the waste liquid in the protecting group removal step at two or more different wavelengths is installed in the drain path of the reactor, and the absorbance measurement means is installed in the drain path of the reactor, and the absorbance of the waste liquid in the protecting group removal process is measured based on the output of the absorbance measuring means. A control means is provided on the output side of the absorbance measurement means to qualitatively characterize the removed protecting group contained in the protective group, quantify the concentration of the protecting group, and calculate the synthesis yield from the quantitative value of the concentration. Process and Synthesis In addition to repeating a synthesis cycle in which the condensation process and masking process are one cycle,
In the synthesis cycle, two or more nucleotide reagents are selected by a predetermined procedure from a plurality of nucleotide reagents blocked with different types of protecting groups and fed into the reactor substantially simultaneously, thereby simultaneously N(> 1) type of nucleic acid, and in parallel with the synthesis cycle, the absorbance of the waste liquid from the protecting group removal step is measured at different wavelengths at M (≧N) points using an absorbance measuring means, The control means derives and solves an M-element simultaneous equation for simultaneous determination of multiple components based on the output of the absorbance measuring means, calculates the concentration of each of the plurality of eliminated protecting groups mixed in the waste liquid, and Provided is a nucleic acid synthesis method characterized in that a synthesis yield is calculated from each concentration of a protecting group, and it is determined whether or not to continue the synthesis cycle based on the synthesis yield value.

There are basically four types of nucleotide reagents that differ in their bases, and the order in which they are used determines the base sequence of a nucleic acid. Therefore, by simultaneously using different types of nucleotide reagents, nucleic acids with different base sequences, that is, different types of nucleic acids, are simultaneously synthesized.

By attaching different types of protecting groups to different types of nucleotide reagents, when different types of nucleotide reagents are used simultaneously, different types of protecting groups can be mixed in the waste liquid from the protecting group removal step. . Therefore, by measuring the absorbance at at least as many different wavelengths as the number of types present, each protecting group can be quantified using the known multi-component simultaneous quantification method based on the data. Since quantification of each protecting group means quantification of each nucleotide reagent, it is possible to confirm how much nucleic acid of which base sequence has been synthesized.

To explain the above-mentioned known multi-component simultaneous quantification method, for example, when the absorbance of a sample containing two components X and Y is measured at wavelengths λ ₁ and λ ₂ , the data is E ₁ ,
When E _{2 , the} concentrations C _{X and CY} of _each component _X and _Y can _be calculated by solving _{E 1 = K} can get. However, K _X1 is _the extinction coefficient of _the X component at λ ₁ , _{K X2} is _the extinction coefficient of _the be.

By determining the extinction coefficient for each protecting group in advance, the above-mentioned known multi-component simultaneous quantification method can be easily applied.

(E) Example Reference numeral 1 shown in FIG. 1 is an example of an apparatus used to carry out the nucleic acid synthesis method of the present invention, and is an automatic DNA microsynthesis apparatus 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
is a material on which a support 9 such as polystyrene or silica beads can be mounted (not permeable), and is permeable to reagent solutions, solvents, and gases. The space above the filter 7 becomes the reaction section 10, which has a diameter of about 450μ.
It is a space of volume.

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',4' trimethoxytrityl group (TMTr), and the nucleotide reagent solution 13 having guanine as a base is blocked with 4,4'-dimethoxytrityl group. The nucleotide reagent solution 14 having thymine as a base is blocked with a trityl 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 inhaled and supplied to the reactor 2 via a mixing means consisting of a liquid reservoir 56 and a mixing coil 57.

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 supplies nitrogen gas directly 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 plunger control mechanisms 26 to 30 as described above, the control circuit 34 also controls the switching mechanism 16.
-20, valves 42-48, and valves 50-55 are controlled. The user also interacts with the operator via the console 35.

The control circuit 34 also contains amino acids in the DNA.
It has a table 34a for converting into a DNA base sequence, and has a function of converting an amino acid sequence input from the operation console 35 into a DNA base sequence. The contents of the table 34a are as shown in FIG. However, in Figure 2, the letter “A”,
"C", "G" and "U" represent the bases "adenine", "cytosine", "guanine" and "uracil", respectively, and the amino acids are represented by the abbreviations shown in the table below. Amino acid (AA) Abbreviation alanine ala arginine arg asparagine asn aspartic acid asp cysteine cys glutamine gln glutamic acid glu glycine gly histidine his isoleucine ileu leucine leu lysine lys methionine met phenylalanine phe proline pro serine ser threonine thr tryptophan trp tyrosine tyr valine val this According to Table 34a, the amino acid sequence is first converted into an mRNA base sequence, and the mRNA base sequence is changed to "G" ← → "C", "A" → "T", "U".
→ Perform “A” conversion to obtain the cDNA base sequence. The base sequence of this cDNA represents the base sequence of probe DNA for raising mRNA. Probe DNA for lifting cDNA
The nucleotide sequence is “C”←→“G”, “A” in the nucleotide sequence of the probe DNA for lifting mRNA.
It is obtained by applying the transformation ←→“T”.

The console 35 has alphabetic and numerical keys, by which an amino acid sequence can be input.

The switching cock 82 switches and connects the drain flow path 83 of the reactor 2 to the waste flow path 84 or the measurement flow path 85, and its operation is controlled by the control circuit 34.

The dilution container 86 is a cylindrical container with an internal capacity of about 1200 ml, and its head is connected to the measurement channel 85.
The bottom part is connected to the absorbance measuring unit 6 via the measuring valve 87.
8 flow cell 69.

An optical level sensor 8 is installed on the side wall of the dilution container 86.
8 is attached, and the output of the level sensor is connected to the control circuit 34.

The dilution vessel 86 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 86 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 87 is controlled by a control circuit 34.

The absorbance measuring unit 68 converts the light emitted from the white light source 70 into a single wavelength light using an interference filter, and detects the light with a light receiver 78 through a flow cell 69 . As shown in FIG. 3, 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. do. By rotating the holder 71 around the rotating shaft 71a, any one of these interference filters 72 to 75 can be selected and used. FIG. 4 shows the absorbance measurement unit 68 as viewed from a direction perpendicular to FIG. 77 is a reference cell, and 79 is a light receiver. The outputs of the light receivers 79, 79 are connected to the control circuit 34.
4 performs qualitative and quantitative determination of the removed protecting group and calculation of the yield based on the output signals of the photodetectors 78 and 79.

Next, the nucleic acid synthesis method of the present invention using this apparatus 1 will be explained. For convenience of explanation, for example,
An example of synthesis of probe DNA for raising mRNA corresponding to the amino acid sequence "aspartic acid-lysine-glutamine-tyrosine" will be explained.

First, on the console 35, “1-asp, 2-lys,
3-gln, 4-try/mRNA/cDNA/probe
(mRNA). ” and press the key.

Then, the control circuit 34 refers to the table 34a, displays a screen as shown in FIG. 5 on the display of the console 35, and displays the converted mRAN,
Inform the operator of the base sequence of cDNA and probe DNA. The display 100 at the top of this screen is
This is the input amino acid sequence, indicating that it is a sequence from the NH ₂ side. Next stage display 101
is the base sequence of mRNA converted from table 34a of the contents of Fig. 2, where the left side of the screen is the 5' side,
This indicates that the right side is the 3′ side. Display 102
was obtained based on the base sequence of the mRNA above.
This is the base sequence of cDNA. Display 103 is the above
This is the base sequence of probe DNA for lifting mRNA.

As can be seen from the screen in Figure 5, there are 16 types of probe DNAs in total, since each amino acid has two types of base sequences corresponding to each other. Here, the operator can use the "cursor movement" key and the "delete" key on the console 35 to move the cursor 104 under the displayed base that is deemed unnecessary and delete it. In other words, this allows the number of types to be synthesized to be reduced.

The operator should synthesize from the final display
Learn the chlorine sequence of DNA. For example, from the screen shown in FIG. 5, one learns that the synthesis will start from a support to which a nucleotide having the base cytosine is bound, removes the stopper 5, and places this support into the reactor 2. This amount is preferably 10 mg to 50 mg in total when the support is polystyrene powder, for example.
After the stopper 5 is returned to its original position, the amount of support inserted and the like are input to the control circuit 34 via the console 35, and then a start command is input.

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

The 5' hydroxyl group of the nucleotide bound to the support 9 has been protected in advance by attaching a protecting group, for example, a dimethoxytrityl group (DMTr), but the above operation removes the protecting group at that site. .

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

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

Since the nucleotide to be bound next has thymine as a base, the control circuit 34 operates the switching knob 19 and the plunger drive mechanism 29,
Also, the exhaust valve 54 is operated to supply the nucleotide reagent solution 14 to the liquid reservoir 56 . Generally, a nucleotide reagent solution corresponding to the base of the nucleotide to be bound next is selected from among 11 to 14, and the corresponding switching cock, plunger drive mechanism and exhaust valve 54 are operated to release the nucleotide reagent solution. The liquid is supplied to the liquid reservoir 56. What should be noted is that when there are multiple types of nucleotide bases to be bonded next, all nucleotide reagent solutions corresponding to these bases should be supplied. In other words, as shown in the display 103 at the bottom of FIG. 5, the second synthesis cycle requires a nucleotide having an adenine as a base and a nucleotide having a guanine as a base. Nucleotide reagent solutions 11 and 13 are both supplied to reservoir 56 .

Once the necessary nucleotide reagent solution is supplied to the reservoir 56, the valves 51, 52, and 55 are operated to supply the nucleotide reagent solution in the reservoir 56 to the reactor 2. Even if there are multiple types of nucleotide reagent solutions, they will be supplied after being uniformly mixed by the mixing coil 57.

Simultaneously with the supply of the nucleotide reagent solution, the control circuit 34 operates the switching pot 20 and the plunger drive mechanism 30 to supply the condensing agent solution 15 to the reactor 2 through the syringe pump 25 .

The amount supplied is controlled so that the total amount of nucleotide reagent solution and condensing agent is the minimum amount sufficient to swell the support 9. Specifically, for example, when the support 9 is a polystyrene powder, the amount of the nucleotide reagent and the condensing agent is about 3 ml and about 2 ml per gram of the support, respectively. Needless to say, it is necessary to adjust the concentration so that the minimum amount of the reagent solution contains a sufficient amount of the reagent.

By the above operation, a new nucleotide having a desired base is linked to a predetermined site of the nucleotide that was bound to the support 9. When a mixture of multiple types of nucleotide reagent solutions is supplied, nucleotides with different bases linked together will coexist.

After a certain period of time, the valves 50 to 55 are operated to wash the liquid reservoir 56, the mixing coil 57, and the inside of the reactor 2 with pyridine 37.

When support 9 is polystyrene powder, support 1
Approximately 0.1 mmol of the terminal portions of DNA molecules are attached per gram, and new nucleotides are attached to most of them as described above. However, more than a few percent remains unreacted. For this reason, the control circuit 34 masks unreacted reactive groups as follows.

That is, the valves 46, 47, and 51 are operated to supply the masking reagent solution 40 and the masking condensing agent solution 41 to the reactor 2.

After masking, the control circuit 34 controls the valves 43, 48,
50 and 51 to fill the reactor 2 with pyridine 37.
Wash at

Through the operations up to this point, one cycle of newly linking a nucleotide to a nucleotide that has been previously bound to the support 9 is completed.

The control circuit 34 operates the valves 44, 48, 50, 51 to supply a mixed solvent 38 of isopropanol and methylene chloride, repeating the synthesis cycle from the protecting group removal operation to the masking operation to synthesize the target DNA. .

In parallel with the above synthesis cycle, the control circuit 34 performs quantitative determination of the protecting group. That is, when discharging the waste liquid from the reactor 2 several times in the deprotection step, the switching pot 82 is switched to the measurement flow path 85 side and the waste liquid is fractionated into the dilution container 86. Further, the waste liquid from washing with the mixed solution 38 of isoprobanol and methylene chloride is similarly collected into the dilution container 86. In this way, all of the removed protecting groups are collected in the dilution container 86. Thereafter, the control circuit 34 operates as follows in parallel with returning the switching pot 82 to the waste channel 84 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 88.
As a result, the waste liquid to be measured is diluted to an appropriate constant concentration. Next, the valve 66 is activated to bubble nitrogen gas to stir the inside of the dilution container 86, and the valves 87 and 65 are activated to discharge the gas through the flow cell 69.

At this time, some of the filters 72 to 75 are appropriately selected and absorbance measurements are performed at different wavelengths equal to the number of types of nucleotide reagents used in the previous synthesis cycle. For example, when measuring the absorbance of the waste liquid from the third protecting group removal step, since two types of nucleotide reagents were used in the second synthesis cycle as described above, the absorbance can be measured using, for example, interference filters 72 and 73. Take measurements. FIG. 6 shows an example of data obtained thereby, in which the absorbance at a wavelength of 478 nm is 0.90 and the absorbance at a wavelength of 503 nm is 0.52. The nucleotide reagent solutions used in the second synthesis cycle were 11 and 13, and their protecting groups were MMTr and DMTr.
By the way, as shown in Figure 7A, the concentration
The absorbance of 13 μmol/MMTr is at a wavelength of 478 nm.
0.87, and 0.16 at a wavelength of 503 nm, so the extinction coefficient at a wavelength of 478 nm is
67,000, and the extinction coefficient at a wavelength of 503 nm is set in the control circuit 34 to be 12,000. In addition, as shown in Figure 7B, it has been previously measured that the absorbance of DMTr at a concentration of 13 μmol/is 0.37 at a wavelength of 478 nm and 1.18 at a wavelength of 503 nm.
The extinction coefficient of is 28000, and the extinction coefficient of wavelength 503nm is
It is set in the control circuit 34 to be 9000. Therefore, the control circuit 34 derives the following two-dimensional simultaneous equations.

0.90=67000C _M +28000C _D 0.52=12000C _M +90000C _D (1) Here, C _M is the concentration of MMTr, and C _D is the concentration of DMTr. The control circuit 34 solves this equation (1),
C _M = 11.7 μmol/, C _D = 4.2 μmol/ are obtained.

In this way, the concentration of the protecting group is quantified,
It thus represents the condensation amount of each of the nucleotide reagents used in the previous synthesis cycle. Therefore, it is possible to confirm which nucleotide reagent has been condensed and how much.

Another modification of the above embodiment is to measure the absorbance at four wavelengths using all of the filters 72 to 75, derive a four-dimensional simultaneous equation, and solve it to calculate the concentration for all protecting groups. can be mentioned. In this case, only the concentration of protecting groups corresponding to the nucleotide reagents used in the previous synthesis cycle should be obtained, so if the concentration of protecting groups corresponding to the nucleotide reagents not used in the previous synthesis cycle is If substantially obtained, it means that the nucleotide reagent was used incorrectly. Then, the synthesis cycle is stopped, and an alarm lamp is lit on the console 35 to issue an alarm to the operator.

Another modification is to further calculate the yield from the obtained quantitative value of the protecting group concentration. The yield is calculated by dividing the quantitative value in the synthesis cycle by the stored quantitative value in the previous synthesis cycle and multiplying the result 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. Also, calculate the overall yield by multiplying all the yields of the synthesis cycles up to that point, and calculate it on the console.
Displayed at 5. Furthermore, the control circuit 34 controls whether the yield of a certain synthesis cycle is a predetermined value (for example, 80%).
and whether or not the overall yield is above a predetermined value (for example, 30%). The current synthesis cycle is stopped, and an alarm lamp is lit on the console 35 to issue an alarm to the operator. A flowchart of the operation in such a modified example is shown in FIG.

Modifications of the synthesis section include those in which the reactor 2 is funnel-shaped, barrel-shaped, and in which the volume of the reaction section is varied between 80 and 800 microns. Examples of solid supports include those using Kel-F.g-styrene, silica gel, polyacrylic morpholide, and the like. These supports have particle size
It is preferably about 30 to 300 microns.

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 number of types of protecting groups to be qualitatively determined. Further examples include those that measure spectra with spectrophotometers that use gratings or prisms. If the nucleotide reagents are monomers with about four types, it may be possible to change the wavelength by switching several interference filters, but if the nucleotide reagents are dimers or more, the number of types of nucleotide reagents increases significantly, so it is recommended to use gratings or prisms. preferable.

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.

Still other modified embodiments include one in which multiple amino acid sequences can be input.

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 continues 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.

Now, according to the above embodiment, by simply inputting the amino acid sequence of a peptide, it becomes possible to automatically synthesize the corresponding DNA, that is, the DNA for producing the peptide and/or the DNA complementary thereto. Moreover, it becomes possible to simultaneously synthesize multiple types of DNA. Furthermore, it has the following advantages:

In the above-mentioned apparatus 1, the reaction part 10 of the reactor 2 is miniaturized, and the support 9 is placed on the filter 7, and the reagent solutions 11 to 15 are supplied from above and drained from the bottom. It constitutes the container 2. 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 for linking a new nucleotide, the inside of the reactor 2 is washed and dried with THF 36 and blown with dry gas, so that the inside of the reactor 2 can be completely dried in a short time. Moisture that inhibits the condensation reaction can be completely removed, so the reaction efficiency is not reduced and no extra reagents are 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 As understood from the above explanation, according to the nucleic acid synthesis method of the present invention, different types of nucleic acids can be synthesized simultaneously. Further, even when different types of protecting groups are mixed in the waste liquid from the protecting group removal step, each protecting group can be quantified. Therefore, by changing the type of protecting group for each different type of nucleotide reagent, it is possible to confirm which nucleotide reagent is condensed and how much of it is synthesized in the synthesis cycle based on the quantitative determination of the protecting group. You can check whether it is.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the configuration of one embodiment of the nucleic acid synthesis apparatus used in carrying out the present invention, FIG. 2 is an explanatory diagram showing the contents of a conversion table possessed by the apparatus shown in FIG. 1, and FIG. FIG. 4 is an explanatory diagram of the structure of the interference filter portion of the apparatus shown in FIG. 1. FIG. 5 is an explanatory diagram of the structure of the absorbance measurement unit of the apparatus shown in FIG. Figure 6 shows an example of absorbance measurement data for wastewater containing a mixture of MMTr and DMTr, and Figure 7
A diagram showing an example of absorbance measurement data regarding MMTr, FIG. 7B is a diagram showing an example of absorbance measurement data regarding DMTr, and FIG. It is a chart diagram. 1...DNA micro-automatic synthesizer, 2...Reactor,
11-14... Nucleotide reagent solution, 15,3
9-41... Reaction reagent solution, 16-20... Switching pot, 21-25... Syringe pump, 26-
30...Plunger drive mechanism, 34...Control circuit, 36-38...Solvent, 42-48, 50-5
5... Valve, 56... Liquid reservoir, 57... Mixing coil, 68... Absorbance measurement unit, 69... Flow cell, 72-75... Interference filter, 77...
Reference cell, 78, 79... Light receiver, 82
...Switching is easy.

Claims (1)

[Claims] 1. 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 synthetic condensation step, and a masking step, An absorbance measuring means capable of measuring the absorbance of the waste liquid in the protecting group removal step at two or more different wavelengths is provided in the drain path of the reactor, and based on the output of the absorbance measuring means, the absorbance of the waste liquid is determined based on the output of the absorbance measuring means. A control means is provided on the output side of the absorbance measuring means to qualitatively characterize the removed protecting group, quantify the concentration of the protecting group, and calculate the synthesis yield from the quantitative value of the concentration, thereby controlling the protecting group removal step and the synthesis. A synthesis cycle consisting of a condensation step and a masking step is repeated, and in the synthesis cycle, two or more nucleotide reagents are selected according to a predetermined procedure from among multiple nucleotide reagents blocked with different types of protecting groups. and substantially simultaneously to the reactor, thereby simultaneously supplying N (a natural number >1) to the reactor.
Synthesis of various types of nucleic acids is carried out, and in parallel with the synthesis cycle, the absorbance of the waste liquid from the protecting group removal step is measured at different wavelengths at M (≧N) points by the absorbance measuring means, and by the controlling means, Based on the output of the absorbance measurement means, an M-element simultaneous equation for simultaneous determination of multiple components is derived and solved to calculate the concentration of each of the multiple eliminated protecting groups mixed in the waste liquid, and each of the protecting groups is 1. A method for synthesizing a nucleic acid, comprising calculating a synthesis yield from a concentration, and determining whether or not to continue a synthesis cycle based on the value of the synthesis yield.