JPS6241677B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a DNA synthesizer, and more particularly, to a DNA synthesizer capable of simultaneously synthesizing different types of DNA.

DNA synthesis methods have been improved and developed into the so-called phosphodiester method, phosphotriester method, and phosphite method, and solid-phase synthesis methods using solid supports that utilize these methods are widely used due to their various advantages. It has reached the point where it will be done. Various apparatuses for synthesizing DNA 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 base sequence complementary to that base sequence is synthesized, this is marked and added to a mixture of nucleic acids, and the synthesized DNA is used as the target nucleic acid. After binding, there is a method of using the mark as a guide to lift 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.

As a result of various studies, the inventor of this invention discovered that the known
Succeeded in improving the DNA synthesis device.

Thus, according to the present invention, the input means for inputting the amino acid sequence of a peptide, and the input means for inputting the input amino acid sequence to one or more types of DNA corresponding thereto.
DNA comprising a conversion means for converting into a base sequence, and a reagent supply means capable of selecting a reagent for DNA synthesis based on each of the one or more types of base sequences obtained and supplying the reagents to a reactor substantially simultaneously. A synthesizer is provided.

The main features of the device of this invention are that it is a system that can automatically synthesize the corresponding DNA by inputting an amino acid sequence;
When there is more than one type of DNA, the system is capable of simultaneously synthesizing multiple types of DNA.

Hereinafter, this invention will be explained in detail based on embodiments shown in the drawings. However, this invention is not limited thereby.

1 shown in FIG. 1 is an automatic DNA synthesis device using the phosphotriester method to which the present invention is applied.

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 dovetail 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 serves as a 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 diameter of approximately 450μ.
is a space with a volume of

There are 8 types of reagent solutions in total. 12 to 15 are various nucleotide reagents, each having adenine, cytosine, guanine, and thymine as bases, and are
A protected nucleotide for synthesis. 11 is a condensing agent, which is a pyridine solution of mesitylenesulfonyl-3-nitrotriazolide (MSNT). 3
9 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 masking reagent, 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 sucked in 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. These solvents 36
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. Note that 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, controls the operation of valves 42~48, 50~55. 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 bases of the letters "A", "C", "G", and "U" are "adenine", "cytosine", "guanine", and "uracil", respectively.
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 ile 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 cDNA represents the base sequence of probe DNA for raising mRNA.
The base sequence of the probe DNA for elevating cDNA is the same as that of the probe for elevating mRNA.
“C”←→“G”, “A”←→ in DNA base sequence
It is obtained by applying the “T” transformation.

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

Next, the operation of this device 1 will be explained.
This will be explained using an example of synthesizing probe DNA for lifting mRNA.

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

Then, the control circuit 34 refers to the table 34a, displays a screen as shown in FIG. 3 on the display of the console 35, and displays the converted corresponding
Inform the operator of the base sequences of mRNA, cDNA, and probe DNA. Display 10 at the top of this screen
0 is the input amino acid sequence, indicating that it is a sequence from the NH ₂ side. Next stage display 10
0 is the base sequence of mRNA converted from the table 34a of the contents of FIG. 2, and 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. displayed here
For cDNA, the left side of the screen is the 3' side and the right side is the 5' side. Display 103 is the base sequence of the probe DNA, with the left and right sides of display 102 swapped.

As can be seen from the screen in Figure 3, there are 16 possible types of probe DNA since each amino acid has two types of base sequences. Here, the operator "moves the cursor" on the console 35.
Cursor 10 using the key and “delete” key
You can delete it by moving 4 below the base display that you think is unnecessary. In other words, this allows the number of types to be synthesized to be reduced.

Operators should synthesize from the final display
Learn the base sequence of DNA. For example, from the screen shown in FIG. 3, one learns that the synthesis will start from a support bound to a nucleotide having cytosine as the base, 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 valves 48 and 50 are operated to drain the liquid.

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

The control circuit 34 then again controls the valves 44, 48, 5.
0.51 to supply a mixed solution 38 of isopropanol and methylene chloride to the reactor 2 and wash the support 9. Furthermore, valves 43, 48, 50, 51
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,
Activate 50 and 51 and blow inside 2 with nitrogen gas 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 20 and the plunger drive mechanism 30,
Also, the exhaust valve 54 is operated to supply the nucleotide reagent solution 15 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 12 to 15, 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. 3, in the second synthesis cycle, a nucleotide having an adenine as a base and a nucleotide having a guanine as a base are required. Solutions 12 and 14 are supplied together 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 16 and the plunger drive mechanism 26 to supply the condensing agent solution 11 to the reactor 2 through the syringe pump 21 .

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, about 3 ml of the nucleotide reagent and about 3 ml of the condensing agent are used per 1 g of the support. 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 bonded to the support 9. When a mixture of multiple types of nucleotide reagent solutions is supplied, nucleotides with different bases linked together will coexist. Note that the 5' hydroxyl group of the newly linked nucleotide has been blocked in advance with the protecting group DMTr.

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 g, and new nucleotides are attached to most of them as described above. However, a few percent
It may remain unreacted to some extent. 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 reagent 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. .

Now, according to the automatic DNA synthesizer 1 of the above embodiment, just by inputting the amino acid sequence of a peptide, it is possible to automatically synthesize the corresponding DNA, that is, the DNA for producing the peptide and/or the DNA complementary thereto. Become. 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 discharged from the bottom of the reactor. 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 will swell it, as well as the reaction section 1 above the filter 7.
It stays within 0 and does not fall downward. Therefore, all the reagent solutions supplied will participate in the reaction and nothing will accumulate in the dead space. As a result, DNA
It has become possible to synthesize small quantities of , the minimum amount of supply (about 5 to 7 times the support volume) is sufficient, and mixing and contact operations such as shaking the reactor to promote the reaction are no longer necessary. There is. In addition, this device 1 is equipped with a reactor 2 before the reaction for linking a new nucleotide.
It is constructed so that the inside of the reactor 2 can be completely dried in a short time by cleaning and drying the inside with THF 36 and blowing with dry gas. As a result, water that inhibits the condensation reaction can be completely removed, so the reaction efficiency does not decrease and no extra reagents are required.

In the end, the DNA micro-automatic synthesizer 1 of the above example
is a useful device from many points of view.

A modified example is one in which a plurality of amino acid sequences can be input.

Other examples include a funnel-shaped reactor, a barrel-shaped reactor, and a reactor with a volume of 80 μm.
Examples include those that vary between ~800μ.
Further examples include those in which the present invention is applied to DNA synthesis devices using the phosphomonotriazolide method, the phosphite method, or the phosphodiester method.

Other examples of solid supports include Kel-F.g styrene, silica gel, polyacrylic morpholide, and the like. These supports preferably have a particle size of about 30 to 300 microns.

As understood from the above explanation, the present invention
According to the DNA synthesizer, it becomes possible to simultaneously synthesize different types of DNA. As mentioned above, this is very useful when separating a specific nucleic acid from a mixture of many types of nucleic acids.

In addition, as an application of the device of the present invention, if a manual means for directly inputting the base sequence of one or more types of DNA is provided and the reagent supply means is operated based on the base sequence, multiple types of DNA can be directly input. DNA can be synthesized simultaneously. Furthermore, by providing an input means for inputting the base sequence of one or more types of RNA, changing the reagent solution as appropriate, and operating the reagent solution means based on the base sequence of the RNA, multiple types of RNA can be simultaneously synthesized. It is also possible to do so.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram of the configuration of an automatic DNA synthesis apparatus which is an embodiment of the present invention, Fig. 2 is an explanatory diagram showing the contents of a conversion table possessed by the apparatus, and Fig. 3 is an example of the display screen of the apparatus. FIG. 1... DNA automatic synthesizer, 2... Reactor, 1
2-15... Nucleotide reagent solution, 21-25
...Syringe pump, 34...Control circuit, 34a
...Conversion table, 35...Operation console, 56...Liquid reservoir, 57...Mixing coil.

Claims (1)

[Claims] 1. An input means for inputting the amino acid sequence of a peptide, a conversion means for converting the input amino acid sequence into one or more corresponding DNA base sequences, and an input means for each of the one or more base sequences obtained. comprising a reagent supply means capable of selecting reagents for DNA synthesis based on the method and supplying them to the reactor substantially simultaneously;
A DNA synthesis device is characterized in that it is thereby capable of simultaneously synthesizing one or more types of DNA.