FR2551443A1 - APPARATUS FOR SYNTHESIZING POLYNUCLEOTIDES - Google Patents

Abstract

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

Description

Apparatus for synthesizing polynucleotides.

  The present invention relates to an apparatus for

synthesize polynucleotides.

  A well-known process for the synthesis of polynucleotides, for example DNA (deoxyribonucleic acid), consists in using a support chemically combined with a nucleotide and in sequentially condensing the

  nucleotide using the triphosphate process, pro-ceded diphosphate, the phosphite process and the like.

  In this synthesis process, operations such as washing elimination of protection (deprotection) -) washing condensation reaction washing, etc., are repeated with a lower diversity of

  processing However, a large number of tediously repeated operations are necessary.

  Recently, various devices have been proposed for synthesizing deoxyribonucleic acid in order to eliminate

  the tedious nature of the synthesis operation.

  For example, there is known an apparatus for synthesizing polynucleotides which comprises a reactor, bottles which are loaded with reagents, solvents or other similar products necessary for the synthesis reaction of polynucleotides, taps which are available at the places of the passages. which cause the reagent bottles and the solvent bottles to communicate with the reactor, the reagents as well as the solvents and the like being successively sent to the reactor from the reagent and sol 30 bottles by opening and closing each tap to repeat the sequence washing> removal of protection -4 washing, etc. However, the above-mentioned apparatus has a large number of valves, so that there is a risk of operating error If such an operating error has place, it is necessary to start again all the operations from the beginning with consequently, the uselessness of all the previous operations Although one then replace the taps with solenoid valves to automate the device, the use of numerous solenoid valves causes difficulties such as a price of

  returns high and a complicated order.

  The authors of the present invention propose, to remedy the difficulties described in Japanese patent application 58-12 6249, an apparatus re10 presented in FIG. 1 In this figure, the reference 1 designates a nitrogen bottle; reference 2 a distributor; references 3 a to 3 i of passages for nitrogen gas; references 4, 5, 6, 8 of the reagent bottles; references 7 and 8 of the solvent bottles; reference 10 an eight-way switch valve; references llb to llg of the passages for reagents and solvents; reference 12 a two-way tap; reference 13 has a three-way valve; references 14 a to 14 h passages for reagents, solvents and nitrogen gas; reference 15 an eight-way valve; reference 16 of the reactors; and

  reference 17 for tanks for waste liquids.

  When a common 1 Oh port is brought into alignment with the lob port using an actuation button (not shown) on the eight-way switch valve 10, the reagent bottle 4 is put in communication with reactors 16 and 16 Under the effect of the pressure of the gaseous N 2 which was sent to the reagent bottle 4 from the nitrogen bottle through the intermediary of distributor 2 and passage 3 b , a reagent (deactivation agent) is sent to the reactor 16,16 from the reagent bottle 4 via a passage llb, an orifice lob, the common orifice h, the tap 12 to two-way, from tap 13 to three 35-way, passages 14 a and 14 b At this time, the eight-way tap is placed in the position shown in

  solid lines, i.e. the SUPPLY position.

  When the common port 10 h is brought into alignment with the port 10 c using the actuation button, the reagent bottle 5 is connected to the reactors 16, 16 Similarly, under the effect of the pressure of N 2 gas, a reagent (condensing agent / solvent solution II) is sent to the reactor 16,16 from the bottle 5 of reagent In addition, when the common port 10 h is put in communication with the orifice l Od, a reagent (auxiliary deactivation agent / solvent solution II) is sent to the reactors from the reagent bottles 6 When the common orifice 10 is placed in communication with orifice 10 f, a reagent ( removal of protection / solvent I) is sent to the reactors 16,16 from the reagent bottle 8 When the 10 o'clock orifice is placed in communication with the orifice Og, the solvent II is sent to reactants 16 and 16

  from bottle 9 of solvent.

  When it is necessary to discharge the liquid from the reactors 16 and 1 E, the eight-way valve 15 is placed in a position shown in broken lines, that is to say the

  UNLOADED position and the valves 16 a, 16 a are opened.

  As described above, the actuation of one of the 25 buttons makes it possible to connect the passages llb to llg. It is possible to reduce the operating errors compared to the conventional apparatus in which several valves are actuated in each passage for the open and

  close them, and automation of the apparatus is therefore easy.

  If we summarize the operation of the eight-way diverter valve 10 during a condensation operation, we see that the common orifice 1 Oh is connected successively in the order going from the orifices l Og, l Od, lob, 35 l Og, 1 Oe, lof, 10 e, 109 to l Oc Connection to the lob ports at l Og is made after the common lob port has been connected once to port 10 a In other words, it is necessary to repeat the rotation

  normal and the reverse rotation of the eight-way diverter valve 5 10 (i.e. its operating button).

  In addition, when the common orifice 10 h is connected to the orifices 10 b to l Og in the order going from Ob to l Og, it passes in front of two orifices 1 Oc and l Od When the common orifice 10 h is connected to the ports l Og to 10 e in order

  going from 10 g to 10 e, it passes in front of a single orifice 10 f.

  This is why the difficulty lies in the fact that the motor intended to drive the switch valve

eight-way is complicated.

  The objective of the present invention is therefore to

  create a new polynucleotide synthesis device.

  Another objective of the invention is to provide an apparatus for the synthesis of polynucleotides in which

  maneuvering errors can be reduced.

  Another object of the present invention is to provide an apparatus for the synthesis of polynucleotides

  which is controlled in a simple way if it is automated.

  According to the present invention, an apparatus for synthesizing polynucleotides comprising a reactor arrangement has been produced; a reagent and solvent bottle arrangement which is loaded with the reagents and solvents necessary for the polynucleotide synthesis reaction; a switch valve arrangement having at its inlet side several ports for reagents and solvents which are put in communication with said bottles of reagents and solvents and a common port put in communication with said reactor arrangement by the operation rotation of an operating button; and liquid supply means for supplying reagents and solvents from each reagent and solvent bottle to said reactor arrangement under the pressure of an inert gas, said ports for reagents and solvents being disposed in a sequence of liquid supply sequence by which a single rotation of said operating button causes the operation

  liquid supply during a condensation phase.

  We will now describe the present invention with reference to the accompanying drawings, in which: Figure 1 is a block diagram showing a conventional apparatus; Figure 2 is a perspective view showing an embodiment of the present invention; Figure 3 is a block diagram for Figure 2; Figure 4 is an enlarged block diagram showing the main part of the block diagram of Figure 3; Figures 5a and 5b are sectional views showing a 16-way switch valve; Figures 6a and 6b are sectional views showing a six-way valve; Figure 7 is a sectional view showing a reactor; Figure 8 is a sectional view showing an upper plug; Figure 9 is a partially broken away sectional view showing another embodiment of the reactor; Figure 10 is a sectional view showing another embodiment of a sixteen-way switch valve; Fig. 11 is a block diagram showing another embodiment in which a double two-way valve is used in place of the six-way valve; Figure 12a is a sectional view of a double two-way valve; Figure 12b is a plan view of the two-way double valve; Figure 13a is a sectional view showing another embodiment of the sixteen-way switch valve; Figure 13b is a plan view of the sixteen-way switch valve; Figure 14 is a block diagram showing another embodiment in which two eight-way turnout valves are used in place of the sixteen-way turnout valve; Figure 15a is a sectional view showing an eight-way switch valve used for the embodiment shown in Figure 14; Figure 15b is a plan view showing the eight-way turnout valve of Figure 15a; FIG. 16 is a block diagram showing another embodiment in which bubbling with N 2 gas can be carried out; Figure 17 is a sectional view of a six-way valve 20 used in the embodiment of Figure 16; FIG. 18 is a schematic view showing a twelve-way switch valve used in another embodiment in which two liquid supply operations are eliminated; Figure 19a is a plan view showing the twelve-way turnout valve of Figure 18; and Figure 19b is a sectional view of the valve

  twelve-way switch in Figure 19 a.

  We will first refer to Figure 2 which is a perspective view showing an embodiment of the synthesis apparatus according to the present invention and to Figure 3 which is a block diagram of this apparatus

of synthesis.

  A cavity Pl is formed in the upper part of one of the end sides (as can be seen on the left side of FIG. 2) of a front panel P of the body A of the apparatus Reactors 20,20 which are arranged in the cavity Pl are supported by arms 46 a of shaker devices 46 shown

in figure 3.

  An actuation button 22 for a sixteen-way switch valve 21 (see FIGS. 3, 4 and 5 a 10 and 5 b) corresponding to the eight-way switch valve 10 shown in FIG. 1 is arranged on the front panel P. Cavities A 1 and A 2 are formed in one of the sides of the body of the apparatus. A bottle 23 with reagent, which is loaded with a deactivation agent and with an auxiliary deactivation agent is disposed in the lower cavity A 1 A reagent bottle 24, which is loaded with a solution of protective agent (trityl group suppressant) / solvent I, a bottle 25 with solvent agent, loaded with solvent I, and a bottle

  26 solvent, loaded with solvent II, are arranged in the upper cavity A 2.

  In this embodiment, a reagent bottle 5 to 25 (condensing agent solution / solvent II) is removed so that the deactivation agent and the auxiliary deactivation agent are placed in

  the same reagent bottle 23, as described above.

  A small nitrogen bottle 27, a distributor 30 28, a six-way tap 29 and the sixteen-way switch valve 21, etc., are arranged inside the

body A of the device.

  The gaseous N 2 located in the nitrogen bottle 27 is sent via a passage 29 a to the distributor 28 where it is diverted The gas is then sent to the reagent bottles 23 and 24 and to the bottles 25 and 26 of solvents via passages 29 b, 29 c, 29 d and 29 e The reagent and then the solvent are sent 5 to the reactors 20.20 from the bottles 23 to 26 under

the pressure of N 2 gas.

  The switch valve 21 with sixteen channels is disposed between the bottles 23, 24 of reagents, the bottles 25, 26 of solvents and the reactors 20, 20.

  A description will be made with reference to FIGS. 4 and 5 a and b of the sixteen-way switching valve 21 which is a

  characteristic of the present invention.

  The sixteen-way switch valve 21 includes a

  valve body 30 and a rotor 31 which is rotatably mounted on the valve body 30.

  The valve body 30 includes inlet ports 30 a to 30 p and a common outlet port 30 q A peripheral recess 31 a and a longitudinal recess 31 b are formed on the outer periphery of the rotor 31. 20 Rotation of the rotor 31 has the effect of communicating the common orifice 30 q successively with the orifices 30 a to 30 p via the peripheral recess 31 a and the longitudinal recess 3 lb. The common orifice 30 q communicates with the upper part of the reactors 20, 20 via a passage 32 and a three-way valve 33 located

along the length of this passage.

  Among the ports 30 a to 30 p, the ports 30 a, c, 30 e, 30 i, 30 k, 30 m, 300, for reagents and solvents communicate with the bottles 23, 24 of reagents and with the bottles 25 and 26 of solvents The orifices 30 b, d, 30 f, 30 h, 30 d, 301, 30 n, 30 p for the gas communicate with the distributor 28 via a passage 34 e. The orifices 30 a, 30 c, 30 e, for reagents and solvents are arranged in a liquid supply operation order. Between these orifices, the orifices 30 b, 30 d and 30 f are arranged for the gas as well as a part

  r liquid supply shutdown.

  Consequently, a single rotation of the rotor 31 results in a liquid supply operation in the order: supply of solution for removal of protective agent / solvent I from the bottle 24 of reagent, supply of gaseous N 2, liquid supply stop, supply of solvent I from the solvent bottle 25, supply of gaseous N 2 and stop supply of liquid In other words,

  a single rotation of the rotor 31 completes the operation of supplying liquid and discharging liquid during a single phase of condensation.

  The position corresponding to the stopping of the supply of liquid has the same function as the two-way valve 12 shown in FIG. 1 The two-way valve 12 is eliminated in the present invention. 20 The 30 g port communicates with a vacuum pump via a passag 34 f When the q port is connected to the 30 g port, the reactors 20,20

  communicate with the vacuum pump 35 to simultaneously boil and dehydrate.

  If you want to automate the device, you can

  connecting the rotor 31 to a stepping motor or to a servomotor and the motor is controlled sequentially according to a program stored in a control device.

  In this case, the passage over one or two orifices, as is done by the apparatus of FIG. 1, does not take place, since the orifices 30 a, 30 c, 30 e for reagents and solvents are arranged in an order corresponding to a liquid supply operation On

  can use a drum type sequencer to drive the motor.

  The rotor 31 is conical so that it has a reduced diameter at one of its ends (as seen on the left side of FIG. 5 b) A spring 36 is disposed between the valve body 30 and the rotor 31 to push this rotor towards one end. Therefore, it does not want to form an interval

  between the valve body 30 and the rotor 31 The orifices 30 a to 30 p do not communicate with the common orifice 30 q, except through the peripheral recess 31 a 10 and the longitudinal recess 31 b.

  A latching mechanism 37 is arranged at one end of the valve body 30 The latching mechanism 37 comprises a disc 38 which is fixed to one of the ends of the rotor 31 and which is in sliding contact with the face d end of the valve body 30 and with a ball 40 which is disposed in a hole 30 'formed in one of the end faces of the valve body 30 and which is pushed against the sliding face of the disc 38 by the pushing force of a spring 39 The sliding face of the disc 38 has a recess 38 a corresponding to the orifices 30 a to 30 p and to the position 30 r of liquid supply stop The positioning of the rotor 31 is obtained by embedding

  of the ball 40 in the recess 38 a.

  The above-mentioned six-way valve comprises a valve body 41 and a rotor 42 which is mounted

  rotatably on the body 41 of the tap.

  The body 41 of the tap is provided with orifices 41 a to 41 f The rotor 42 is provided with peripheral recesses 30 42 a to 42 d intended to communicate the orifices 41 a

at 41 f with each other.

  The combination of connection of the orifices 41 a to 41 f with the reactors 20,20 is shown in FIG. 3 The orifices 41 a and 41 d communicate with the upper part of the reactors 20,20 via passages 43 a and 43 a The ports 41 c and 41 f communicate with the lower part of the reactors 20, 20 via passages 43 b, 43 b The ports 41 b and 41 e

  communicate with the reservoirs 44, 44 of residual liquid via passages 43 c and 43 c.

  The orifices 41 a and 41 d communicate with the orifices 41 b and 41 e via peripheral recesses 42 a and 42 c and the orifices 41 c and 41 f are closed in the position represented by a line in solid lines on FIG. 3, that is to say in the supply condition When the rotor 42 is rotated to bring the peripheral recesses 42 a to 42 f to the position represented by a dashed line on the Figure 3, the orifices 41 b and 41 a are placed in communication with the orifices 41 c 15 and 41 f and the orifices 41 a and 41 d pass into a state

  closed to establish a DISCHARGE conditions.

  The six-way tap 29 is also provided with a latching mechanism 45 used for positioning the peripheral recesses 42 a to 42 d in the 20 positions represented by solid lines and broken lines in FIG. 3 and is also used for placing the recesses in intermediate positions at the positions represented by the solid lines and dashed lines. When the recesses are in their intermediate position, the openings 41 a and 41 f are completely closed. The six-way valve 29 works as a

  combination of the six-way valve 15 shown in FIG. 1 and of the valves 16 a and 16 of the reactors 16, 16.

  Consequently, the reactors 20, 20 do not have a valve. The rotor 42 of the six-way valve 29 can be connected to a stepping motor as in the case of a sixteen-way switching valve. au35 tomatisation by driving the motor according to a program

  stored in a control system.

  On the front panel P, there is an actuation button 41 for the six-way valve 29, an actuation button 48 for the three-way valve 33, an actuation button 49 for a timer (not shown) of the shaker device 46, a pressure gauge 50 for N 2 gas connected to the distributor 28, and a supply switch 51, etc. Fixing means 23 a to 26 a for the necks of the bottles 23 to 26 which are not shown at 10 details are provided on the upper wall of the cavities

A 1 and A 2.

  Figure 7 is an enlarged sectional view showing the reactor 20 The reactor 20 includes a reactor body 55 and upper and lower plugs 56 and 57. The upper and lower plugs 56 and 57 are fixed to the reactor body 55 upright using a fixing means such as a clamp, the collars 56 a and 57 a on the flanges 55 a and 55 b formed at the ends

  upper and lower body 55 of the reactor.

  The body 55 of the reactor is made of glass and has shoulder parts 55 c and 55 d in the respective upper and lower openings The end pieces 56 b and 57 b of the upper and lower plugs are introduced into

  the body until they come into contact with shoulder parts 55a and 55d, respectively.

  The upper and lower plugs 56 and 57 are formed of a material which does not react with the reagent and the solvent, etc., mentioned above, and which is elastically deformable, such as for example fluorosilicone rubber, fluorinated rubber, copolymer

  propylene hexafluoride and ethylene tetrafluoride, and the like.

  The plugs 56b and 57b have an outside diameter slightly larger than the inside diameter of the upper and lower openings so that the outer surface of the plugs 56a and 57a are in close contact with the inner surface of the upper and lower openings of so as to maintain watertightness

vis-à-vis fluids.

  The upper plug 56 is provided with a tap 58 through which a raw material is loaded.

  plugs are provided with passages 56 c and 56 d which communicate respectively with passages 32 and 43 a.

  The lower plug 57 has a passage 57 c

  which communicates with a passage 43 b.

  O-rings can be placed in peripheral grooves 56 b 'and 57 b' formed on the end caps

56 b and 57 b respectively.

  In the present invention, a filter can be placed on the side of the liquid supply part of the reactor where the reagent and the solvent as well as similar products are introduced. The liquid supply part can be provided with a passage. intake serving for the introduction of the reagent and the solvent and other similar products into this part of liquid supply via the filter and a purge passage for purging the gas from the reactor via the filter

(pressure purge passage).

  In this way, the reagent and the like can be prevented from being purged from the production passage directly via the purge passage and the valves can be protected along the formed passages.

in the body of the device.

  We will describe with reference to Figure 9 the

present embodiment.

  Filters 124 are sandwiched between the shoulder parts 121a, 121a of the body 55 and the upper and lower plugs 56 and 57 Filters 124 are made of a material such as tetrafluoroethylene, polypropylene, polyethylene and the like, which can 35 capture the support loaded in the body 55 and which is insoluble in the solvent mentioned above and is permeable towards the solvent One of the end parts of the upper plug 56 has a diameter slightly larger than the internal diameter of the body 55 A peripheral recess 52 a is formed on the external periphery of the end part The end phase comprises a hemispherical recess 122 b so that the end part is elastically deformed in the direction of the internal diameter of the body 55 to come into close contact with the inner periphery of the body so as to maintain the airtightness with the body 55. Inlet and drain passages 122 c and 122 d extending d ans an axial direction are formed in the upper plug 56 in the vicinity of each other The

  intake and drain passages 122 c and 122 d are connected to the 5120 a and 120 bh passages respectively.

  A tube 122 e extending through the filter 124 is connected to one of the ends of the inlet passage 122 c so that an inlet inlet is located inside the filter 124 On the other hand, the inlet of the purge passage 122 d is located in the recess 122 b, that is to say outside of the filter 124 Consequently, although the intake and purge passages 122 c, 122 d are adjacent l one from the other, the solvent and the like which have been introduced into the intake passage 122 c penetrate into the body 55 without directly entering the purge passage 122 d since the inlets of the intake passages and of

  purges are separated from each other.

  The N 2 gas and the solvent and other similar products which overflow from the body 55 when the pressure is purged are discharged from the purge passage 122 d into the passage 120 b by means of the filter 124 Par

  Consequently, it may happen that the tap in step 120b is out of service by foreign bodies.

  The end part of the lower plug 57 which is introduced into the body 55 has a peripheral recess 123 a and a recess 123 b The end part comes into close contact with the internal periphery of the body 55 due to the elastic deformation of so as to maintain airtightness towards the air A purge passage 123 c extending axially is formed in the lower plug 57 and communicates

with the passage 120 c.

  Collar parts 121 d and 121 b are present at opposite ends of the body Collar parts 122 f and 123 d are formed at a midpoint of the length of the upper and lower plugs 56 and 57 The upper and lower plugs can be fixed15 laughing 56 and 57 to the body 55 by connecting the collar parts 121 b and 121 b to the collar parts 122 f and 123 d respectively The presence of a taper at the inner periphery of the ends of the body 55 facilitates the insertion of the upper plugs inferior

56 and 57.

  The reactors 20 can be arranged in a cavity Pl formed on the housing of the device so that they can be seen directly from the outside The reactors 20 are provided with heaters (not shown) The heater is in the form a C in section and includes a hot plate as well

  than a temperature detector embedded in a cylindrical body.

  The cylindrical body is made of silicone rubber and has an axially extending slot The reactors can be seen through the slot so as to observe the liquid supply A coloring of the cylindrical body and the presence of an aluminum foil on the inner surface facilitate the observation 35 of the liquid supply to the reactor 20 (made of glass so that one can observe its interior).

  The heating plate is formed of a resistor coated with silicone rubber The plate is embedded in the vicinity of the inner peripheral surface of the cylindrical body and along this surface to surround the reactor 20 The heating plate has a dimension such that it can effectively heat the area

reaction of reactor 20.

  The thermal detector is formed by a platinum resistor and is arranged in the vicinity of the heating plate 10 to detect the temperature of this plate.

  We can use a chrome-aluminum thermocouple and

  a thermistor instead of a platinum resistor.

  The internal diameter of the cylindrical body is slightly larger than the external diameter of the reactor 20 The cylindrical body is fixed to the reactor 20 in the bent state, that is to say that an elastic recovery force acts The cylindrical body is therefore brought into close contact with reactor 10 to increase

thermal conductivity.

  We will describe the DNA synthesis operation

  using the device mentioned above.

  We load a support which is combined with nucleotides in reactors 20 and 20 We place in the bindings 23 a-26 a the bottles 23 and 24 of reagents 25 and the bottles 25 and 26 of solvents The support then swells under the effect solvent I. The support can be an organic material, such as polystyrene, polyester, polyamide, polyurethane, polypeptide, and an inorganic material, such as silica, aluminosilicane, borosilicate Polystyrene and silica

  are typically used as a support.

  After having finished the preparation in this way, the trityl groups are eliminated. During this phase of elimination of the trityl groups, the common orifice 30 q of the switching valve 21 to sixteen

  is connected to port 30 a to transfer the solu-

  protection agent / solvent I from the reagent bottle 24 to the reactors 20,20 When the transferred solution reaches a given quantity, the orifice 30 q is connected to the orifice 30 b The N 2 gas expels the residual solution protective agent / solvent I

  remaining in passage 32 to reactors 20,20.

  The valve 21 is switched to position 30 r to stop the supply of liquid. The shaker device 46 is then put into action if necessary After the flow of a predetermined period of time, the six-way tap 29 is switched in the DOWNLOAD position (position represented by a dashed line in Figure 3) The valve 21 is switched to the orifice 30 b so that the solution of protection agent / solvent I is discharged from the reactors 20,20 in the reservoirs 44, 44 of residual liquid via the passage 43 b, the orifices 41 c, 41 b, 41 e, 41 f and the passage 43 c under the effect of the pressure of the gas N 2 When the discharge of the waste liquid 20 is completed, port 30 q is in communication with port 30 c and the six-way valve 29 is

  placed on the SUPPLY position (position represented by a dashed line in Figure 3).

  The protective suppressing agent can be a protonic acid, such as benzene sulfonic acid, dichloracetic acid, trichloracetic acid,

  trifluoroacetic acid and Lewis acid such as zinc bromide, aluminum chloride, titanium tetrachloride.

  Solvent I can be dichloroethane, chloroform, acetonitrile, nitromethane, dioxane, tetrahydrofuran. If necessary, solvent I can be used in conjunction with an alcohol such as methanol, ethanol, isopropanol. , n35 butanol Then, a washing is carried out with solvent I. During this washing phase, the switching valve 21 with sixteen channels is switched in the order going from the orifices 30 c, 30 d to 30 r The solvent I is transferred from the bottle 25 of solvent to the reactor 20, 20 without any remaining in the passage 32 After the shaker device 46 has been operating for a given period of time, if this has proved necessary, the six-way tap 29 is placed in the DISCHARGE position The sixteen-way switch valve 21 is switched in the order from port 30 d to port 30 r so as to discharge the residual solvent I Then, the six-way valve 29 is placed in the position

FOOD.

  The groupestrityl removal phase (I) and the washing phase (II) can be repeated several times

times if need be.

  Then washing with solvent II is carried out.

  During the washing phase (III), the sixteen-way switch valve 21 is switched to the orifices in the order ranging from 30 e, 30 f to 30 r to transfer the solvent II from the bottle 26 of solvent to the reactors 20,20 without solvent II remaining in the passage 32 After the shaker device 46 has been operating for a predetermined period of time, the six-way valve 25 29 is placed in the DISCHARGE position The valve d switch 21 to sixteen channels is switched to ports 30 f to 30 r The six-way valve 29 is placed in the SUPPLY position after the discharge of solvent II is complete Solvent II can include pyridines, such as pyridine , picoline, lutidine and quinoline, isoquinoline,

  oxazole and it can be appropriately used in conjunction with ethers such as tetrahydrofuran and dioxane.

  The washing phase (III) can be repeated several times if necessary.

  After this, the charge of raw material is injected During this phase (IV) of charge injection, the valve 58 (see FIG. 7) is open. The solution of the charge in which a monomer salt having the above structure afterwards is dissolved using solvent II and is injected through the upper part of the

reactors 20.20.

  DM Tr (R 1) d BOPOH (R 23 O 9 Cl o Rl represents a protective group (benzoyl group and the like); R 2 represents an alkyl group; and B represents a nucleic acid motif such as adenine (A) , guanine (G), cytosine (C), thiamine (T) A Dimer salt can be used

instead of a monomer salt.

  The load is then simultaneously subjected to boiling and dehydration under reduced pressure to remove the water content. During this dehydration phase (V), the six-way tap 29 is fully open The diverter valve 21 sixteen channels are switched to the 30 g port and the pump

  35 is put into action. This evacuates the reactors 20, 20 so as to eliminate by boiling and simultaneous dehydration the water which, from the feed of raw materials, has been dissolved in solvent II.

  When dehydration is complete, valve 21 is

  switched to the 30 h orifice to charge the N 2 gas.

  Then, the valve 21 is switched to the position 30 r for stopping the supply of liquid and the six-way tap 29 is placed in the SUPPLY position. Then, a condensation is carried out During this condensation phase (VI), a solution of condensing agent / solvent II is injected through the upper part of the reactors 20,20 If necessary, the shaker device 46 is put into action and the reactors 20,20 are heated by a heater (not shown) When the condensation reaction is finished, the shaker 46 and the heater are switched off The six-way tap 29 to 5 is placed in the DOWNLOAD position and the sixteen-way switch valve 21 is switched to the ports 30 h and 30 r successively When the liquid discharge is complete, the tap 29 to six

  channels is placed on the SUPPLY position The condensing agent can be for example 2,4,6-tri-isopropylebenzènesulfonyltétrazoride, pnitrobenzènesulfonyltriazoride, benzenesulfonyltriazoride, 2,4,6trimethylbenzènesulfonyltriazoride, 2,4,6trimethylbenzènesulfonylnitrotriaz benzenesulfonyltetrazoride, 2,4,6-tri -isopropylebenzènesulfonylchloride, 2,4,6-trimethylbenzènesulfonylchloride.

  Then washing with solvent II is carried out During the washing phase (VII), the sixteen-way switch valve 21 is switched in order

  going from orifices 30 i, 30 j up to 30 r successively.

  The operation is similar to the washing phase - (III).

  The washing phase can be repeated several times

  times if necessary A '' styling is then performed.

  During this styling phase, the sixteen-way switch valve 21 is switched in the order going from the orifices 30 k, 301 to 30 r successively so that the solution of deactivation agent / auxiliary agent of deactivation / solvent II is transferred to the reactor 20,20 via the passage 32 from the reagent bottle 23 After the shaker device 46 has been operating for a given period of time, the six-way tap 29 is placed on the UNLOADED position and the switch valve 35 21 to sixteen channels is switched in the order going from the orifices 301 to 30 r successively to discharge the residual liquid When the residual liquid has been discharged, the tap 29 to six channels is placed on

the POWER position.

  The deactivating agent can include acetic anhydride, phenylisocyanate, diethylemonotriazorophosphite and, if necessary, a base such as N, N-dimethylaminopyridine, N, N-diethylaminopyridine, N, N-dimethylaminopicoline can be used

as a reaction promoter.

  Washing with solvent I is then carried out.

  During this washing phase (IX), the sixteen-way switch valve 21 is switched in order

  going from orifices 30 m, 30 n, up to 30 r successively.

  The operation is similar to that of phase (III).

  Washing with the solvent (I) is then carried out During this washing phase (X), the valve

  switch 21 to sixteen channels is switched in the order from orifices 30 o, 30 p to 30 r successively.

  The operation is similar to that of phase (III). The washing phases (IX) and (X) can be

  repeated several times if necessary.

  The previously mentioned phases (I), (II), (III), (IV), (VII), (IX) and (X) are repeated several times if necessary. During this time, it is possible to actuate only the six-way tap 29 For example, if phase (II) is repeated, the six-way tap 29 is communicated successively in the SUPPLY, UNLOAD, SUPPLY and DISCHARGE positions,

  while the valve 21 is adjusted to the orifice 30 c. This allows a reduction in the number of actuations.

  A repetition of phases (I) to (X) results in a

  successive condensation of nucleotide chains.

  It is possible to reduce the number of valve actuations since the six-way switch valve 21 21 and the six-way valve 29 also play the role, in the present embodiment, of the two-way valve 12 and taps 16 a, 16 has

  reactors 16 and 16 shown in Figure 1.

  For example, to wash with solvent II in the apparatus shown in Figure 1, (1) the eight-way diverter valve 10 is set to port g, (2) the two-way valve 12 is closed, open then closed, (3) the valve is set to port l Oa, (4) the two-way valve 12 is closed, open 10 then closed, (5) the eight-way valve 15 is placed the SUPPLY position to the DOWNLOAD position, (6) the valve 16a is closed, open and then closed, (7) the eight-way valve 15 is placed from the position

  UNLOADED in the POWER position.

  On the other hand, in the present embodiment, (1) the sixteen-way switch valve 21 is switched in the order going from the orifices 30 e, 30 f to 30 r, (2) the tap 29 to six tracks is placed from the SUPPLY position to the DOWNLOAD position, (3) the valve is switched in the order from the orifices 30 f to

  r, (4) the six-way valve 29 is placed from the SUPPLY position to the DOWNLOAD position.

  This amounts to saying that, in the present mode of

  embodiment, the number of valve actuations can be reduced by three times.

  FIG. 10 shows another embodiment of the switch valve 21 with sixteen channels In the present embodiment, the orifices 30 a to 30 p are placed in communication with the common orifice 30 q only when the recess longitudinal 3 lb is aligned with the holes 30 a to 30 p by exerting a pressure on the rotor 31 in the direction of the arrow B in FIG. 10 against the thrust force of the spring 52 In other words, the reagent, the solvent, the gas 35 N 2, can be supplied to the reactors 20,20 when the

rotor 31 is pushed.

  FIG. 11 shows another embodiment in which a double two-way valve 54 is used in place of the six-way valve 29 In the two-way double valve, the ports 54 a and 54 b are connected to the bottom of the reactors 20.20 via the passages 43 b, 43 b respectively and the orifices 54 c, 54 d are connected to the reservoirs 44.44 of residual liquid via the passages 43 c 10 and 43 c The line in solid lines represents the SUPPLY position and the dashed line

represents the DOWNLOAD position.

  In the present embodiment, it is

  possible to further reduce the number of valve actuations 15 compared to the previous embodiment.

  For example, when washing with solvent II, (1) the sixteen-way switch valve 21 is switched in the order going from orifices 30 e, 30 f, to r, (2) the tap double 54 two-way is placed from the SUPPLY position, to the DISCHARGE position and then to the SUPPLY position Consequently, it is possible to reduce the number of valve actuations twice compared to the previous embodiment

  and it is possible to reduce this number by five times 25 compared to the apparatus represented in FIG. 5.

  The time of the synthesis operation can therefore be reduced because, in the present embodiment, the internal pressure prevailing in the reactors 20.20 takes the value of 1 kg / cm 2 which is substantially equal to that of N 2 gas propelling the liquid when half of the reactors 20,20 is filled with liquid, the liquid supply is automatically stopped by the internal pressure without switching to the position 30 r of liquid supply stop thanks to which the number of valve actuations

can be further reduced.

  The double tap 54 two-way mentioned above comprises a body 60, a rotor 61, a mechanism

  snap-fastening 62, as can be seen in FIGS. 12 a and 12 b The body 60 is provided with orifices 54 a to 54 d.

  Grooves 61 a and 6 lb which communicate the orifices 54 a to 54 d with each other are formed on the

periphery of the rotor 61.

  The switch valve 21 with sixteen channels can be produced as shown in FIGS. 13 a and 13 b. In this case, a common orifice 30 q is formed in the center of the end face of the valve body 30 The orifices 30 a, 30 c for the reagent and the solvent are formed so as to surround the orifices The orifices b, 30 d for the gas are formed radially on the periphery of the valve body 30 The rotor 31 has a common groove 31 c intended to make the common orifice 30 q communicate with the orifices 30 a to 30 p, so that this groove extends from the end face

  to the periphery of the rotor 31.

  FIG. 14 shows a case in which two eight-way diverter valves 63,64 are used in place of the sixteen-way diverter valve The eight-way diverter valve 63 comprises the first half of the orifices 30 a at 30 h among the aforementioned orifices 25 30 a to 30 p for the supply of liquid and the other eight-way switch valve 64 has the second half of the orifices 30 i to 30 p for the supply in liquid Each of the eight-way valves 63, 64 to

  eight lanes has a common port 30 q.

  In this embodiment, after phases (I) to (VI) have been carried out by switching the eight-way diverter valve 63, phases (VII) to

  (X) are performed by switching the eight-way diverter valve 64.

  You can increase the spacing between the holes

  a at 30 p by assigning the orifices to two air valves

  eight-way guiding 63 and 64 so that the sealing properties are easily ensured and

  that the reliability of the valve can be increased.

  Figures 15 a and 15 b show the eight-way gate valves 63.64 with eight channels The basic structure is identical to that of the gate valve 21 with sixteen channels, while the spacing of the orifices 30 a, 30 p

are bigger.

  FIG. 16 shows a case in which an eight-way valve 65 is used in place of the six-way valve 29 so that bubbling with N 2 gas can be carried out As shown in FIG. 16, the orifices 65 g at 65 h are connected to a bifurcation passage 66 which branches off from passage 34 e. 15 The bifurcation passage 66 is provided with an orifice

  67 for the passage of a necessary quantity of N 2 gas.

  A capillary or needle valve can be used in place of the orifice The orifices 65 a to 65 f are connected to the reactors 20,20 and to the reservoirs 44 and 20 44 of residual liquid as in the case of the tap

29 to six lanes.

  In the present embodiment, a small amount of N 2 gas which has passed through the orifice 67 is blown to the bottom of the reactors 20, 20 to effect bubbling when the eight-way valve 65 is placed in the SUPPLY position. , the shaker device 46 can be omitted, which makes it possible to obtain a light and compact device and a reduction

of the cost price.

  Bifurcation passage 66 can be provided

  a push valve for bubbling while pressing the valve push button.

  In the present invention, the passages for supplying reagents, solvents and the like, which are connected to the upper part of the reactors, can be connected, as shown in FIG. 16, to the lower part of the reactors, and a inert gas can be sent to the lower part of the reactors via a flow regulator which regulates the gas flow to a value necessary for bubbling. In this way, the introduction of reagent to the bottom of the reactors causes agitation simultaneously with the supply of liquid. Consequently, sufficient contact of the reagent and other similar products with the support can be obtained and the time of the operation can be reduced compared to the case in which the reagent and other similar products arrive at the top of the reactors The reagent and other similar products are not likely to overflow the reactors when sparging is carried out and it

there is no waste of time.

  FIG. 17 shows the eight-way valve 65, mentioned previously. As can be seen in this FIG. 17, the orifices 65 a to 65 h are arranged in the body 68 in a radial manner. The rotor 69 is arranged in a rotary manner at the center of the orifices The rotor 69 has peripheral grooves 69 a to 69 d to make the orifices 65 a to 65 h communicate with each other.

others.

  FIG. 18 shows the case in which a twelve-way switch valve 70 is used in place of the sixteen-way switch valve 21. In the present embodiment, the abovementioned orifices 30 c, d, 30 m, 30 N have been eliminated because the protective suppressant 30 used during the elimination phase of trityl groups and the deactivation agent as well as the auxiliary deactivation agent used during the styling phase are dissolved in the solvents I and II respectively In the first embodiment, there takes place a washing phase during which at 551443 the reactor is washed with solvent I to remove the protective suppressing agent (agent for suppressing trityl groups ) remaining in the reactors after the phase (I) of removal of trityl groups while the washing phase (II) is followed by a washing phase (III) using solvent II in the present embodiment Consequently, the agent

  protective removal can be dissolved and removed during the washing phase if washing phase 10 (II) is removed.

  The styling phase (VII) is followed by the washing phase (IX) during which the reactors are washed with solvent II for the removal of the auxiliary deactivation agent remaining in the reactors 20, Because the washing phase (IX) is followed by a washing phase (X) intended to wash the reactors with solvent I, the deactivating agent

  and the auxiliary deactivation agent can be dissolved and removed during phase (X) even if the washing phase (IX) is removed.

  FIGS. 19 a and 19 b show the aforementioned twelve-way switch valve 70 The basic structure of this valve is identical to that of the sixteen-way switch valve 21 shown in FIGS. 12 a and 12 b , except that the spacings of the holes 30 a to 30 p increase in length insofar as the holes 30 c, 30 d, 30 m, 30 N have been

  deleted It is easy to obtain sealing at the orifices 30 a to 3 Op and the reliability of the valve can be increased.

  In the embodiments mentioned above, two reactors 20, 20 are present The invention is not limited to this case It is possible to use a single reactor or more than two reactors The structure 35 of the tap 29 with six channels and of the eight-way valve 65 is different from those of the embodiments

  previously mentioned when using a single reactor or when using more than two reactors.

  A case has been described in which the charge of raw material is introduced into the reactor by opening the valve 58 The solution comprising the raw material dissolved in a solvent introduced into a bottle can be sent to the reactor under the effect

N 2 gas pressure.

  Two or three solvents are conventionally used during each washing phase and the reactors are washed successively in a repeated manner. This implies a complicated operation and an extended period of operating time. There is also a drawback in that the The apparatus cannot be compact due to the increase in the number of solvents. The present invention overcomes this disadvantage by the use of a single solvent which is capable of dissolving the impurities existing in a product obtained during the previous phase that can be used during the phase

  next In other words, a sufficient effect can be obtained by washing with a single solvent.

  The synthesis apparatus of the present invention can be used for the synthesis of RNA (acid

  ribonucleic) as well as for DNA.

  According to the present invention, the reagent and solvent inlet ports of the switch valve with which the passages are changed making the reagent and solvent bottles react with the reactor by rotation of the actuation button are arranged in a order corresponding to the liquid supply operation A rotation of the actuation button completes a liquid supply and liquid discharge operation Consequently, the operation is very simple and it is possible to reduce

  the risk of an operating error.

  Automation of the device does not require a large number of solenoid valves For example, a stepper motor or a servo motor is sufficient to automate the device 5 Its control is simple It is easy to

  transform the device for automation.

Claims (7)

  1 Apparatus for synthesizing polynucleotides comprising: reactor means; reagent and solvent bottles which are loaded with the reagents and solvents necessary for the synthesis reaction of polynucleotides; a means forming a switch valve comprising at its inlet side a plurality of orifices for reagents and solvents which co-connect with said bottles of reagents and solvents and a common orifice communicating with said means forming a reactor at its outlet side, said switch valve means being adapted to modify the passages in order to communicate each of the reagent and solvent bottles with said reactor means when a button is turned; and a liquid supply means for supplying reagents and solvents, from the bottles of reagents and solvents, said means forming a reactor under the effect of the pressure of an inert gas, said orifices for reagents and solvents being arranged in an order corresponding to a liquid supply sequence by which a turn of the actuation button 25 causes the execution of a supply operation in   liquid during a condensation phase.   2 Apparatus according to claim 1, characterized in that a passage for introducing said inert gas into said reactor means is provided with a bifurcation passage comprising a flow reduction means through which the inert gas s' flows   at a rate necessary for bubbling.   3 Apparatus according to claim 1, characterized in that a liquid supply passage is connected with the lower part of said reactor means so that said inert gas arrives, from the bottom of said reactor means, by through said one-way flow control means   flow required for bubbling.   4 Apparatus according to claim 1, characterized in that a filter is arranged in the liquid supply part of said reactor means and an inlet passage for introducing the reagents and solvents into the means 10 reactor through said filter and a purge passage serving to evacuate outside the reactor means by means of the filter, the gases and other similar products generated in the forming means   reactor are present in the liquid supply part.   Apparatus according to claim 1, characterized in that said reactor means is arranged so that it can be seen and that a heating device comprising a heating element is embedded in the main body of the reactor means of such so that this heating element surrounds the reactor means, said main body comprising a slot through which one can directly observe said reactor means and said body being made so that one can grasp said reactor means. 6; Apparatus according to claim 1, characterized in that said means forming a switching valve and serving to change the passages communicating the bottles of reagents and solvents with the means   forming a reactor when the actuation button is turned comprises a plurality of valves.   7 Apparatus according to claim 1, characterized   in that said reactor means comprises a plurality of reactors.   8 Apparatus according to claim 1, characterized in that said reactor means comprises a single reactor.   9 Apparatus according to claim 1, characterized in that it uses a single solvent which, during each phase, can dissolve the impurities of the product obtained during the previous phase and which can be   used in the next phase.