GB2143240A

GB2143240A - Apparatus for synthesizing polynucleotide

Info

Publication number: GB2143240A
Application number: GB08417736A
Authority: GB
Inventors: Yasuyoshi Chino; Shigemi Miyamoto; Tatsuo Ohira; Yoshiki Nagahama
Original assignee: Nippon Zeon Co Ltd
Current assignee: Zeon Corp
Priority date: 1983-07-13
Filing date: 1984-07-12
Publication date: 1985-02-06
Also published as: GB8417736D0; FR2549477A1; DE3425763A1; GB2143240B; FR2549477B1

Abstract

An apparatus for synthesizing polynucleotide comprises reactor means mounted on a front panel of a body of the apparatus; receiving means at a visible position on the outside of a case of said body for detachably receiving bottles which are filled with liquids such as reagent and solvent required for a polynucleotide synthetic reaction; selector valve means along a passage between said bottles and said reactor means for selectively communicating one of said bottles with said reactor means; and supplying means for supplying said liquids to said reactor means.

Description

SPECIFICATION Apparatus for synthesizing polynucleotide BACKGROUND OF THE INVENTION This invention relates to a polynucleotide synthesizing apparatus, and more particularly to an apparatus for synthesizing polynucleotide which is easily operated.

A well-known method of synthesizing polynucleotide, for example DNA (deoxyribonucleic acid) includes a method in which a support chemically combined with nucleoside is used, and nucleotide is sequentially condensated by phosphotriester method, phosphodiester method, phosphite method and the like. In this synthesis method, processes such as washing deprotection > washing < condensation reaction < washing, etc. are repeated with less variety of process. However, repeated complicated operations are required many times.

Recently, various apparatus for synthesizing DNA have been proposed in order to simplify synthesis operation.

For instance, one of such known apparatus is an apparatus for synthesizing DNA having a manually operable valve which allows reagent and solvent required for synthesis reaction to be fed to a reactor where each operation such as washing ) deprotection < washing condensation reaction - washing. However, because it is necessary to operate many valves, there is possibility of erroneous operation. In case of occurrence of such erroneous operation, all the operations must be tried again from the start, resulting in waste of all the previous operations.

Further, since reagent bottles and solvent bottles are accommodated in a casing of the apparatus as mentioned above, confirmation of residual quantity in the bottles troublesomely requires opening of a side plate of the casing, and there is possibility of continuation of operation without confirmation of the residual quantity. Further, exchange of the reagent bottle and the solvent bottle is troublesome.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a novel apparatus for synthesizing polynucleotide.

It is another object of the present invention to provide an apparatus for synthesizing polynucleotide which is easily operated.

It is a further object of the invention to provide a polynucleotide synthesizing apparatus which may eliminate possibility of erroneous operation and additionally allow confirmation of residual quantity in the reagent bottle, etc. to be readily conducted.

A polynucleotide synthesizing device of the present invention comprises reactor means mounted on a front panel of a body of the apparatus; receiving means at a visible position on the outside of a case of said body for detachably receiving bottles which are filled with liquids such as a reagent and solvent required for a polynucleotide synthetic reaction; selector valve means along a passage between said bottles and said reactor means for selectively communicating on of said bottles with said reactor means; and supplying means for supplying said liquids to said reactor means.

Consequently, according to the present invention, the residual quantity of the liquid in the reagent solvent bottles may be readily confirmed, and liquid may be readily supplemented into the bottles. In addition, operational performance is improved and there is no possibility of erroneous operation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view showing an embodiment of the synthesizing apparatus according to the present invention; Figure 2 is a flow sheet of Fig. 1; Figure 3 is a detailed sectional view showing the upper portion of the reactor; Figure 4 is a side view of the reactor with the heater mounted thereon; Figure 5 is a cross sectional view of Fig. 4; Figure 6 is a flow sheet showing another embodiment of the synthesizing apparatus including two reactors; Figure 7 is a perspective view showing the synthesizing apparatus of Fig. 6; and Figure 8 is a perspective view showing a further embodiment of the synthesizing apparatus.

DETAILED DESCRIPTION OF THE EMBODIMENTS A preferred embodiment of the present invention will now be described with reference to drawings.

Referring to Figs. 1 and 2, there are shown an embodiment of the synthesizing apparatus according to the present invention, wherein reference numerais A, B and 1 designate a body of the device, a casing and a front panel, respectively.

There is provided a recess 1 a at an end portion (on a left side in Fig. 1) of the front panel 1, and there is disposed a reactor 2 in the recess 1 a, which reactor 2 is supported by a support arm 3 of a vibrator (not shown) incorporated in the casing B.

Further, there is also provided a recess 1 b at a central portion to the other end portion (on a right side in Fig. 1) of the front panel 1, which recess 1 b serves as a receiving portion of a reagent and solvent bottles. The recess 1 b fo-med at the central portion receives small capacity reagent bottles 4, 5 and 6 of deactivator, condensating agent/solvent II solution, deactivation auxiliary/solvent II solution and the like. Further, the recess 1 b at the other end receives large capacity reagent and solvent bottles 7, 8 and 9 of solvent I, deprotection agent/solvent I, solvent II and the like. Since, the solvent I which inhibits condensation reaction is frequently used, the solvent Il which does not inhibit condensation reaction should be used in condensation process.Further, there are provided mountings 1 c-I h at a ceiling portion of the recess 1 b, although such mountings being not shown in detail, and the mountings serve to detachably mount openings of the bottles 4-9.

Further, there are arranged at the central portion of the front panel 1 an operation knob 11 for an eight-way selector valve, an operation knob 13 for a two-way valve 12, an operation knob 1 5 for a four-way valve 14, an operation knob 16 for a timer (not shown) of a vibrator, a power switch and the like.

The casing B incorporates a portable and small capacity nitrogen cylinder 18 and a distributor 1 9 in addition to the eight-way selector valve 10, the two-way valve 12, the four-way valve 14, the vibrator (not shown) and the like as above. As is above described, since the nitrogen cylinder 18 is incorporated in the casing B, it is not necessary in use to carry a large nitrogen cylinder and the like near the body A of the apparatus, or carry the body A of the apparatus to a site where the nitrogen cylinder is situated. Further, it is not necessary to connect the nitrogen cylinder with the distributor 1 9.

The nitrogen cylinder 18 is connected through a tube 21a to an inlet port 19a of the distributor 19, and when a valve 20 of the nitrogen cylinder 18 is opened, N2 gas is supplied to the port 19a. The N2 gas is distributed from the port 1 9a to each outlet port 19b-19i of the distributor 1 9.

The ports 19b-19g are connected through tubes 21b-21g to the mountings 1c-1h, and N2 gas is fed to the bottles 4-9 mounted to the mountings 1 c-1 h. The port 1 9h is connected through a tube 21h to an inlet port 10a of the eight-way selector valve 10, and the port 19i is connected through a tube 21 i to a port 1 4a of the four-way valve 14.

The other inlet ports 1 Ob- 10g of the eight-way selector valve 10 are connected through the tubes 22b-22g to the mountings 1c-1h, and when N2 gas is fed to the bottles 4-9, liquids such as reagent and solvent in the bottles 4-9 are fed out to the ports 10b-10g by the pressure of the N2 gas.

An outlet common port 1 Oh of the eight-way selector valve 10 is connected through 221 to the two-way valve 1 2, and an outlet of the two-way valve 12 is connected through a tube 22h to an upper inlet 2a of the reactor 2. For instance, when the common port 1 Oh is brough into communication with the port lOb by the operation of the operation knob 11 as shown in Fig. 2, deactivator in.the bottle 4 is fed to the reactor 2 by the pressure of N2 gas. Although the eightway selector valve 10 is employed in accordance with the number of the bottles 4-9 in this embodiment, it is required to use a selector valve having more number of selection in the case that the reagent includes polynucleotide raw material.

The other port 1 4b of the four-way valve 14 is connected through a tube 22i to the upper inlet 2a of the reactor 2, and a port 1 4c is connected through a tube 22j to a bottom outlet 2b of the reactor 2. A port 1 4d is a discharge port and is connected to a tube 22k. Under the condition as shown in Fig. 2 (when the operation knob 1 5 is set at BLOW), N2 gas is supplied through the ports 1 4a and 14b, and the tube 22i to the upper inlet 2a of the reactor 2, and after passing through the interior of the reactor 2, it flows from the bottom outlet 2b through the tube 22j and the ports 1 4c and 14d, and is discharged through the tube 22k.When the operation knob is set to FEED, the ports 1 4a is brought into communication with the port 14d, thereby allowing the N2 gas to flow from the bottom to the top of the reactor 2.

Referring now to Fig. 3, there is shown the upper portion of the reactor 2 in detail. In Fig. 3, there is provided a substantially cross-like raw maierial injection pipe 23 at the upper inlet 2a of the reactor 2. When raw material such as nuc;eotide is injected, a cap 24 only is required to be removed without necessity of moving a plug 25 fitted to the upper inlet 2a. A branch portion 23a of the raw material injection pipe 23 is connected with the tube 221, and a branch 23b of said pipe 23 is connected with the tube 22i.

As shown in Figs. 4 and 5, the reactor 2 is provided with, for example, a clip-type heater 26.

The heater 26 includes a heater 26a consisting of flexible piate-like heating material bonded on its inner surface for receiving electric current through a lead wire 26b so as to heat the reactor 2 and accelerate reaction. Although winding of trie heater 2b around the reactor may be considered for heating the reactor 2 while imparting vibration to the same, there is a possibility that the heater 26 may be disconnected with the reactor 2 due to vibration. It is necessary to wind the heater 26 around the reactor 2 and to firmly secure the heater 26 to the reactor 2 for avoiding the separation of the heater therefrom.

However the heater 26 is unnecessary when the condensation reaction is carried out while it is necessary when washing and deprotection is carried out. If the heater 26 is secured to the reactor 2, it is disadvantage that supply condition of liquid such as solvent can not be viewed.

Thus in the present invention, a heater including a portion for nipping the reactor by fastening means and a heating incorporated in the nipping portion is removably attached to the reactor.

Accordingly the heater may be heated while being vibrated. The heater may not be readily detached from the heater. Furthermore the liquid supply condition may be visually seen by separating the heater from the reactor when washing or deprotection is carried out.

Operation for synthesizing DNA with use of the above synthesizing apparatus will be described: The operation knob 1 5 of the four-way valve 14 is set to FEED, and the valve 2c of the reactor 2 is closed. Then, support (e.g. silica gel) combined with nucleotide is filled in the reactor 2, and the reagent bottles 4, 5, 6 and 8, and the solvent bottles 7 and 9 are mounted to the mountings 1c-lh. (I) Then, the common port 1 Oh is brought into communication with the port 10g by operating the operation knob 11, and the two-way valve 1 2 is opened. As a result, solvent lt in the bottle 9 is fed to the reactor 2 under the pressure of N2 gas.Although the two-way valve 1 2 is operated in connection with selecting operation of the eight-way selector valve 10, subsequent explanation of operation of the two-way valve 12 will be omitted for purposes of simplification of explanation. When washing by the solvent II is completed, the operation knob 1 5 of the fourway valve 14 is switched to BLOW, and the valve 2c of the reactor 2 is opened to discharge the liquid. Then, the valve 2c is closed, and the operation knob 1 5 is switched to FEED. The operation knob 11 is operated to communicate the common port 1 Oh with the 1 Od, and in turn communcate the common port 1 Oh with port lOb. As a result, the deactivation auxiliary/solvent II solution in the bottle 6 and the deactivator in the bottle 4 are sequentially fed to the reactor 2 by the pressure of N2 gas.After standstill for some minutes, the operation knob 1 5 is switched to BLOW, and the cock 2c of the reactor 2 is opened to discharge the liquid. (II) Next, the operation knob 1 5 is switched to FEED, and the operation knob 11 is operated again to communicate the common port 1 Oh with the port 10g and feed the solvent II to the reactor 2 for washing.Then, the liquid is discharged in the same manner as the above. (III) The operation knob 11 is operated to communicate the common port 1 Oh with the port 1 Oe and feed the solvent I to the reactor 2 for washing, then discharging the liquid. (IV) Thereafter, the common port 1 Oh is communicating with the port 10f to feed deprotection agent/solvent I solution to the reactor 2, and some minutes later the liquid is discharged. (V) Such an operation is repeated several times.

Thereafter, the common port 10h is again communicated with the port 1 Oe to feed the solvent I to the reactor 2 for washing, then discharging the liquid. (VI) Then, the common port 10h is communicated with the port 10g to wash the reactor 2 with the solvent II, and then discharge the liquid. (VII) The reactor 2 is purged with N2 gas for several minutes (VIII), and solution of monomer salt as represented by the following structural formula dissolved in the solvent II is injected through the raw material injection pipe 23 into the reactor 2. Then, the common port 1 Oh is communicated with the port 10c to feed condensation agent/solvent II solution to the reactor 2.

(IX) The solution of the monomer salt dissolved in the solvent II may be charged in the reagent bottle, and fed to the reactor 2 by the pressure of N2 gas.

Where R1 is a protective group (e.g. benzoyl); R2 is alkyl; B is nucleic acid base such as adenine (A), guanine (G), cytosine (C) and thymine (T).

After standstill for 40-50 minutes, the chemical in the reactor 2 is discharged (X), and the above operation (II) and the subsequent operations are repeated. Thus, nucleotide chain is sequentially condensated.

Dimer salt or trimer salt may be substituted for the monomer salt.

When the reagent and the solvent remaining in the bottles 4-9 is substantially lost, the bottles 4-9 are exchanged, or liquid is supplemented. In this case, as the mountings 1 c-I h are mounted to the body, exchange of the bottles and supplementation of the liquid can be readily operated.

According to the above synthesizing apparatus, since the reactor 2 does not project from the front panel 1, there is no possibility that the operator erroneously touches the cock 2c in operation, and the reactor 2 is damaged. In order to accelerate the reaction, the reactor 2 may be vibrated by the vibrator, or heated by the heater 26. Further, it may be heated by the heater 26 while being vibrated by the vibrator. Operation time of the vibrator may be set by a timer device.

Referring now to Figs. 6 and 7, there is shown another embodiment of the present invention in which two reactors are provided. Reference numerals A, B and 1 designate a body of the device, a casing and a front panel, respectively.

There is provided a recess 1 a on the front panel 1, and there are two reactors 2 in the recess la, which reactors 2 are supported by support arms 3 of a vibrator (not shown) in the casing B.

With this arrangement, there is no possibility of erroneously touch with cocks 2c of the reactors 2 in operation, and further there is no possibility of damage of the reactors 2 by the other instruments.

Further, there are also provided recesses 30a and 30b on one side of the casing B, which recesses serve as a receiving portion of a reagent and solvent bottles. The lower recess 30a receives small capacity reagent bottles 4, 5, and 6 of deactivator, condensating agent/solvent II solution, deactivation auxiliary/solvent II solution and the like. The upper recess 30b receives large capacity reagent and solvent bottles 7, 8 and 9 of solvent I, deprotection agent/solvent I solution, solvent II and the like. Accordingly, residual quantity in the bottles 4-9 is visually confirmed from outside of the casing.

There are provided mounting 1c-1b at ceiling portions of the recess 30a and the recess 30b, although such mounting being not shown in detail, and the mounting serve to detachably mount openings of the bottles 4-9.

Further, there are arranged at a central portion of the front panel respective operation knobs 11, 13, 15, 27 and 1 6 of an eight-way selector valve 10, a two-way valve 12, a three-way valve 27 (See Fig. 6) and a timer (not shown) of the vibrator, and a heater switch 1 7.

The casing B incorporates a portable and small capacity nitrogen cylinder 18 and a distributor 19 in addition to the eight-way selector valve 10, the two-way valve 12, the three-way valve 27, the four-way valve 14, the vibrator (not shown) and the like as above. The nitrogen cylinder 18 is not necessarily installed in the casing B, however, when installed in the case B, it is not necessary in use to carry the nitrogen cylinder near the casing B, and it is not necessary to connect same with the distributor 1 9.

The nitrogen cylinder 18 is connected through a tube 21 a to an inlet port 1 9a of the distributor 19, and when a valve 20 of the nitrogen cylinder 18 is opened, N2 gas is fed to the port 19. The N2 gas is distributed from the port 1 9a to outlet ports 19b-19i of the distributor 19.

The ports 19b-19g are connected through tubes 21b-21g to the mountings 1c-1h respectively, and N2 gas is fed to the bottles 4-9 mounted to the mountings 1c-1h. Further, the port 1 9h is connected through a tube 21 h to an inlet port 1 Oa of the eight-way selector valve 10, and the port 19i is connected through a tube 21i to port 14a of the four-way valve 14.

Inlet ports 1 Ob-10g of the eight-way selector valve 10 are connected through tubes 22b-22g to the mountings 1b-1h, and when N2 gas is fed to the bottles 4-9, reagent and solvent are fed from the bottles 4-9 through the tubes 22b-22g to the port 1 Ob-10g by the pressure of the N2 gas.

The two-way valve 12 is connected through a tube 22 to an outlet common port 1 Oh of the eight-way selector valve 10, and a discharge port of the two-way valve 1 2 is connected to a port 27a of the three-way valve 27.

The other ports 27b and 27c of the three-way valve 27 are connected through tubes 22h to upper inlets 29 of the reactors 2. For example, when the operation knobs 1 2 and 16 are operated to set the eight-way selector valve 10 and the three-way valve 27 under the condition as shown in Fig. 6, and open the two-way valve 12, the deactivator is fed from the bottle 4 to the reactors 2 by the pressure of N2 gas.

A port 1 4b of the four-way valve 14 is connected through a tube 22i to the upper inlets 2a of the reactors 2, and a port 1 4c is connected through a tube 22j to lower outlets 2b of the reactors 2. Further, a port 14d serving as a discharge port is connected with a tube 22k. Under the condition as shown in Fig. 6 (when the operation knob 1 5 is switched to BLOW), N2 gas is supplied through the ports 1 4a and 14b, and the tube 22i to the upper inlets 2a of the reactors 2. Thereafter, it passes through the interior of the reactors 2, and is fed out from the lower outlets 2b through the tube 22j to the ports 1 4c and 14d, and is discharged through the tube 22k. When the operation knob 1 5 is switched to FEED, the ports 1 4a and 14c are brought into communication with each other, and the ports 14b and 14d are also brought into communication with each other, thereby allowing N2 gas to flow from the lower portion to the upper portion of the reactors 2.

Operation for synthesizing DNA with use of the above synthesizing apparatus is similar to that of the embodiment described with reference to Figs. 1 to 5. The reagent and the solvent may be supplied to each reactor 2, thereby achieving simultaneous operation of both the reactors 2 by a single operation. Further, in the case that different raw materials are injected (for example, A monomer salt is injected into one reactor, while G monomer salt is injected into the other reactor), it is possible to carry out two kinds of synthetic reaction at a time. Further, at least three reactors 2 may be installed to simultaneously operate all the reactors 2, or carry out more than three kinds of synthetic reactions, thus attaining remarkable reduction of synthesis time.In this embodiment, the bottles 4-9 are installed in recesses 30a and 30b formed on one side of the case B rather than the front panel 1. Reference numeral 31 in Fig. 7 designates a manometer of N2 gas.

Fig. 8 shows a further embodiment of the present invention. In this embodiment, there is provided a receiving portion 29 partitioned by a partition plate 28 on the rear side of the casing B for receiving the bottles 4-9 as well as a nitrogen cylinder 10. With this arrangement, the nitrogen cylinder 18 may be readily exchanged.

Although all the previous embodiments disclose use of N2 gas as a liquid feeding means, it will be understood that the liquid feeding means is not limited to N2 gas and may employ a liquid feeding pump or utilize head.

Furthermore, it will be understood that the synthesizing apparatus of the present invention is not limited to application to synthesis of DNA, but is also useful for synthesis of RNA (ribonucleic acid).

As is described above, according to the present invention, since the receiving portion (the recesses 11 b, 30a and 30b, and the receiving portion 29) of the reagent and solvent bottles required for polynucleotide synthesizing reaction, which the bottles are detachably mounted in the receiving portion, is provided at a visible area on the outside of the case, it is possible to visually confirm residual quantity of the reagent and the like, and readily exchange the bottles.

Moreover, since the selector valve for selecting conduit connecting the reagent and solvent bottles with the reactor by the operation of a single operation dial arranged on the front panel of the case is provided on the way of the conduit, it is possible to remarkably reduce erroneous operation.

Further, since the reactor is mounted on the front panel, it is possible to operate the reactor while visually confirming such an operation as washing deprotection -, washing, etc.

Furthermore, since there is provided a distributor having plural ports on the outlet side of the two-way valve 13, and each reactor is connected to each port of the distributor to feed reagent and solvent to each reactor at a time, it is possible to synthesize DNA having different base number or having different base arrangement by a single operation, thereby remarkably improving the efficiency of sythesizing operation.

Claims

1. An apparatus for synthesizing polynucleotide comprising (a) reactor means mounted on a front panel of a body of the apparatus; (b) receiving means at a visible portion on the outside of a case of said body for detachably receiving bottles which are filled with liquids such as reagent and solvent required for a polynucleotide synthetic reaction; (c) selector valve means along a passage between said bottles and said reactor means for selectively communicating one of said bottles with said reactor means; and (d) supplying means for supplying said liquids to said reactor means.

2. The apparatus as defined in claim 1, in which said reactor means includes a plurality of reactors, each of which being communicated with said valve means via a distributor having an inlet connected with said valve means and a piurality of outlets, each connected with respective reactors.

3. The apparatus as defined in claim 1, in which a heater including a nipping portion for nipping said reactor by fastening means and a heating element incorporated in the nipping portion is detachably mounted on the reactor means.

4. The apparatus as defined in claim 1, in which said receiving means includes a recess.

5. The apparatus as defined in claim 1, in which said selector valve means is actuated by a knob on the front panel of the body of the apparatus.

6. The apparatus as defined in claim 1, in which said supplying means includes a pressurized gas source.

7. The apparatus as defined in claim 6, in which said pressurized gas is N2 gas.

8. The apparatus as defined in claim 1, further including valve means in a passage between said distributor and said reactor for changing the flow direction of the gas in said reactor supplied from said supplying means.

9. The apparatus as defined in claim 8, in which said valve means is actuated by a knob on the front panel of the body of the apparatus.