Abstract:
The present invention provides a polymer synthesizer having a high efficiency production rate. The synthesis of the polymers, and more particularly of DNA and RNA, is done very quickly. Furthermore, it is possible to synthesize a plurality of polymers in the same batch without significantly increasing the time and the complexity of the process.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    There is no cross-reference to related applications. 
       FIELD OF THE INVENTION 
       [0002]    The present invention generally relates to polymer synthesis. More particularly, the present invention is an improved apparatus which is highly efficient. 
       BACKGROUND OF THE INVENTION 
       [0003]    To meet the increasing demand for nucleic acid synthesis, there has been an increase in the variety of designs, and the volume of production of nucleic acid synthesizers. Unfortunately, the currently available synthesizers are not designed to adequately meet the needs of the industry. In particular, available synthesizers are limited in their ability to efficiently synthesize large numbers of oligonucleotides. While synthesizers have been developed to simultaneously synthesize more than one oligonucleotide at a time, such machines are not efficient at the production of different types of nucleic acids simultaneously (e.g., different lengths of nucleic acids) and are unacceptably prone to performance failures and environmental contamination. Furthermore, available synthesizes are not suitably configured for use in large-scale nucleic acid production facilities or for automated nucleic acid synthesis. 
         [0004]    DNA synthesis is presently performed on automated instruments which are capable of concurrently producing multiple DNA segments. Frequently the apparatus uses reaction columns in which a support material for the reaction is positioned within the columns on top of inert, porous filters, referred to as frits. The support material generally has a starter material bound to the support onto which desired oligonucleotides may be synthesized. The reaction columns are placed within the automated apparatus and chemicals are added to the columns in sequence in appropriate amounts in an automated fashion. In order to address today&#39;s large demand for high throughput oligosynthesis, most automated apparatuses have a large footprint and take up a great deal of premium laboratory space. 
         [0005]    Most currently known automated synthesizers can produce only a few oligonucleotides at a time, which is limited by the number of reaction columns located within the machines. The number of reaction columns is limited as a practical matter by the increased complexity of the plumbing and valving network, as the number of columns increases. In addition, the system must be airtight to avoid contaminating the chemicals with air or water and to avoid human exposure to the chemicals. 
         [0006]    U.S. Pat. No. 5,368,823 issued Nov. 29, 1994, and U.S. Pat. No. 5,541,314 issued Jul. 30, 1996, address the need for producing a large number of oligonucleotides by disclosing a method and apparatus for oligonucleotide synthesis in which the plumbing and valving network is simplified. The patents disclose a system in which there is one supply line and one outlet located in the synthesis chamber for the delivery of reagents into the reaction columns. The outlet can be positioned above the inlet end of each of the columns so that nucleotide reagents, capping reagents, deblocking reagents, wash chemicals, etc. can be provided to each of the reaction columns. All of the reagents are located in a supply system which includes reservoirs and valving to connect the reservoirs with the supply line. A flush/prime column is also located within the chamber so that the supply line can be flushed and primed between each different chemical reagent addition. A vacuum source, located outside of the reaction chamber, is connected to the outlet end of the reaction columns to rapidly draw the chemicals from all columns simultaneously, thus leaving the columns dry and ready to receive the next reagent. 
         [0007]    The disclosed apparatus in these two patents provides multiple reaction columns, but the single supply line requires flushing and priming between the addition of each reagent. These steps are time consuming and waste reagents. Moreover, a large footprint is required to accommodate a reaction chamber encompassing the moving supply line and the reaction chambers as well as a vacuum source outside of the reaction chamber. The large footprint is a drawback to space-constrained laboratories. 
         [0008]    Another group of patents, U.S. Pat. No. 5,472,672 issued Dec. 5, 1995, U.S. Pat. No. 5,529,756 issued Jun. 25, 1996, and U.S. Pat. No. 5,837,858 issued Nov. 17, 1998, addresses the need for high throughput oligosynthesis by disclosing a polymer synthesis apparatus with many stationary supply lines. The patents disclose an apparatus with a head assembly with many nozzles, with each nozzle coupled to a reagent reservoir. Further, a base assembly has at least one reaction well but can utilize 96-well and other plates. A transport mechanism is coupled to the head assembly and/or base assembly to produce relative movement between the two. The transport mechanism moves horizontally to align a selected reaction well and a selected nozzle to deposit a selected liquid reagent into the reaction well for synthesis of a polymer chain. A sliding seal is positioned between the head assembly and the base assembly to form a common chamber that encloses both the reaction wells and nozzles therein. The seal is constantly being rubbed down by the movement of the metal piece back and forth to move the synthesis block. This wearing down of the seal results in a less efficient seal. 
         [0009]    Thus, the art is in need of polymer synthesizers that are efficient, flexible, and are amenable to large-scale production and automation for the synthesis of polymer, and more specifically of DNA. 
       OBJECTS OF THE INVENTION 
       [0010]    It is a first object of the present invention to provide an apparatus to synthesize polymers having high output efficiency. 
         [0011]    Another object of the present invention is to provide an apparatus to synthesize polymers which needs less inert gas during the synthesis. 
         [0012]    A further object of the present invention is to provide an apparatus to synthesize polymers which is capable of producing a plurality of polymers in the same batch. 
         [0013]    Other and further objects and advantages of the present invention will be obvious upon an understanding of the illustrative embodiments about to be described or will be indicated in the appended claims, and various advantages not referred to herein will occur to one skilled in the art upon employment of the invention in practice. 
       SUMMARY OF THE INVENTION 
       [0014]    A polymer synthesizer having an airtight cabinet with a lid, synthesis columns having a bottom extremity, and connected to a plurality of reagent containers through reagent dispense lines and wherein the reagents are dispensed with tip dispensers into the synthesis columns, comprising a block having a plurality of waste columns disposed on a horizontal plane having x and y axes, waste channels to drain synthesis columns and means to displace the block along both y axis and x axis to dispose at least one synthesis column under a particular tip dispenser. 
         [0015]    The polymer synthesizer may be used to synthesize polymers with a flow-through process, such as, for example, oligonucleotides, peptide nucleic acids (PNA), polypeptides, nucleic acids, DNA or RNA. The containers may also contain the solutions used in the reactions, such as deblocking agent, washing solution, coupling activator, capping agent, oxidizer or other solutions required for a reaction, which are also designated by the term “reagent” in the present document. 
         [0016]    The synthesis columns are sealingly connected into the waste columns of the block to make an airtight fit with sealing means such as, for example, pressure fit, a flat mat or an o-ring. However, any other suitable sealing means may be used for this purpose. The synthesis of a polymer occurs in the synthesis column in a derivatized Controlled Porosity Glass (further referred as CPG), which is a solid support for covalent attachment of biological molecule, or parent molecule, as known in the art. The solid support can be derivatized CPG, polystyrene or any other convenient material having a large surface area and capable of being derivatized (during the process of derivitization, a protected nucleotide or linker is attached to which the growing DNA chain is attached). The CPG is a white powder held between two porous plastic frits. Other solid phase supports may also be held between frits or imbedded in a solid frit. The reagents are dispensed at the top of the synthesis column and left for an appropriate time, and then drained through waste column by overpressure. The sealed box in which all of this occurs is continuously pressurized. When a valve is opened to provide an exit pathway then the reagents on the columns will flow out the exit pathway provided. The next step of the synthesis is then carried out as others reagents are dispensed into the column until the polymer is completed. The reagents are dispensed into the synthesis columns by tip dispensers, which are controlled by valves, connected to the corresponding pressurized reagent containers. The number of tip dispensers installed on an apparatus is variable and adapted to the occurring synthesis. Furthermore, it is possible to synthesize different polymers in the different synthesis columns in the same batch or the same block. To further improve the efficiency of the apparatus, the less reactive reagent may be dispensed first into the corresponding synthesis column to save time and then the other reagents are dispensed. Usually the reagent containers (and order of dispensing) are alphabetically ordered with the name of the reagent but in the present invention, the reagent containers are decreasingly ordered depending of the reagent reactivity increasing again the efficiency of the apparatus. To improve the displacement, it is also possible to for the synthesis column to be optically identifying prior the delivering of a reagent. 
         [0017]    The airtight cabinet has a lid on the top to place or remove the block and the synthesis columns. The tip dispensers are sealingly fixed in the lid and a translucent window may be installed in the lid. They may be threaded inside the lid or insert in a hole and sealed with sealing means. The lid configuration may be interchangeable to allow a different number or configuration of the tip dispensers. 
         [0018]    To displace the block, two linear slides, controlled by a servomotor or step motor, are used in both the y and x axes in a horizontal plane. The block may be adapted to be disposed directly on the slides or on a movable plate connected on the slides and adapted to receive a block. This block is moved so as to place the appropriate synthesis column under one of the dispense tips so that a specific reagent can be dispensed into that synthesis column. An optical device may be used to identify a synthesis column prior to the displacement. A plurality of synthesis column may be filled by a plurality of tip dispensers at the same time with different reagents. The motion of the slides is controlled by a computer which sends the appropriate instructions to the valves and motion controllers. The computer can be either on-board or outside the apparatus. Several polymer synthesizer can be controlled by one computer. 
         [0019]    The block is used to hold the synthesis columns by their bottom extremity and to drain the waste, or used reagents, to a waste container. The block is a structure having a two dimensional matrix of vertical holes, or waste columns, that are connected to a waste container through waste channels. The waste channels may drain the waste of a plurality of synthesis columns or only one synthesis column. The evacuation of a plurality of waste column or a sole waste column is controlled by a valve installed on the waste line that is preferably controlled by a computer. The reagent is allowed to stay in the synthesis column for a defined amount of time and is drained through the synthesis column to waste with overpressure when the waste valve is opened. Because a plurality of waste column or a sole column may be controlled independently from the others, there is no need to wait for other longer reactions to drain wastes, increasing the efficiency of the apparatus. A purge waste port is also provided to drain and/or to prime a dispense line. 
         [0020]    The block may be composed by only one part or by a plurality or parts. For example, the block may be composed by two parts; a spacer block, or top part, and an o-ring block or bottom part. The idea is to fix the sealing means, in this case o-rings, in the waste columns of the bottom part. When the synthesis columns are inserted in the two part block, the pressure tight seal is made by the pressure of the o-ring on the column. This allows for a more effective seal as columns of slightly different size seal. In addition, the pressure required to insert or remove the columns is much less than in a design with a pressure fit to fix the synthesis column into the waste columns. In this example the sealing means are inserted into the bottom part but they may be inserted at the bottom extremity of the spacer or any other suitable positions. 
         [0021]    A support may be use to hold a plurality of synthesis column. The support allows the columns to be inserted all at once and is then inserted and removed as a unit into the block. Using a dimensionally slightly modified version of this same block, multiwell plates may also be used. 
         [0022]    The present invention provides also a method to synthesize one or more polymer by simultaneously chemically linking a plurality of monomers to a plurality of parent molecules. A method of making at least one type of polymers by chemically linking a plurality of monomers to a parent molecule attached in a synthesis column one at a time in an apparatus comprising an airtight cabinet with a lid, a block having a plurality of waste columns disposed on a horizontal plane having x and y axles, a plurality of synthesis columns connected to waste columns of said block by their bottom extremity, and reagents being delivered from a plurality of reagent containers through reagent dispense lines and wherein the reagents are dispensed with tip dispensers into synthesis columns, the waste columns being controlled by waste valves, the method comprises displacing the block along both y axis and x axis to dispense reagents in synthesis columns through tip dispensers with means to displace said block, draining the synthesis columns by the opening of waste valves, repeating the dispensing of reagents and the draining of synthesis columns as needed for a polymer. 
         [0023]    The chemical linkage may be, for example, a phosphodiester bond, a phosphorothioate bond, a phosphonate bond, a phosphoramidate bond, an amide bond, an imine bond, a carbamate bond, an azo bond, a sulfone bond, a sulfonide bond, a sulfonamide bond, a sulfide bond, a disulfide bond, an ether bond, an ester bond, a thiourea bond, a urea bond or a carbon-carbon bond. 
         [0024]    The polymer synthesized may be nucleic acids, DNA, RNA, peptide nucleic acids (PNA) or polypeptides. The monomer may be a modified nucleotide comprising a minor groove binder. 
         [0025]    The steps to create a molecule by chemical linkage include, in sequence:
       a. washing the support on which is attached to one or more parent molecules;   b. dispensing a liquid comprising a deblocking agent to remove the protecting group attached to the parent molecule;   c. draining the liquid comprising the deblocking agent;   d. washing the support;   e. dispensing a liquid comprising a coupling activator;   f. dispensing a liquid comprising a protected nucleotide;   g. draining the liquid comprising a protected nucleotide;   h. dispensing a liquid comprising a capping agent;   i. draining the liquid comprising the capping agent;   j. washing the support;   k. dispensing a liquid comprising an oxidizer;   l. draining the liquid comprising the oxidizer.       
 
         [0038]    It is to be noted that the previous sequence is for only one monomer added. Any of the operations of this sequence may be repeated when necessary depending of the resulting polymers to be synthesized. 
         [0039]    The features of the present invention, which are believed to be novel, are set forth with particularity in the appended claims. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0040]    The above and other objects, features and advantages of the invention will become more readily apparent from the following description, reference being made to the accompanying drawings in which: 
           [0041]      FIG. 1  is a cross-sectional schematic side view showing the polymer synthesizer and its principal components. 
           [0042]      FIG. 2  is a schematic top view showing the waste lines organization. 
           [0043]      FIG. 3  is a schematic diagram of the control system of the polymer synthesizer. 
           [0044]      FIG. 4  is a cross-sectional side view showing a first embodiment of the block. 
           [0045]      FIGS. 5   a  and  5   b  are perspective views showing the motion of the plate and of the block. 
           [0046]      FIGS. 6   a  and  6   b  are cross-sectional side views showing a second embodiment of the block,  FIG. 6   a  being an exploded view of the different parts and the  FIG. 6   b  a view as assembled. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0047]    A novel polymer synthesizer will be described hereinafter. Although the invention is described in terms of specific illustrative embodiment(s), it is to be understood that the embodiment(s) described herein are by way of example only and that the scope of the invention is not intended to be limited thereby. 
         [0048]    The  FIG. 1  shows the polymer synthesizer and its principal components. The process occurs in the airtight cabinet  170  which defines an internal chamber  172 . The chamber  172  is filled with an inert gas such as argon or nitrogen. There is a hermetic lid  174  on the top of the cabinet  170 . The window  160 , which is fixed in the lid  174  holds the tip dispensers  162 . These tip dispensers  162  are connected to the reagent dispense lines  164  which are connected to the pressurized reagent containers  166  through the reagent valves  168 . 
         [0049]    The synthesis occurs in the synthesis column  140  which are connected to the block  110 . The reagents are carried to the synthesis column through the dispenser tips  162 . The block  110  comprises the waste columns  112  and the waste channel  116  that are connected to a waste container (not shown). The block  110  is displaced by the plate  150 . A purge waste port  130  is connected to a waste container (not shown) with the outlet  132 . 
         [0050]    The wastes are drained with a plurality of lines forming the waste system shown in  FIG. 2 . In a first embodiment, the waste columns are regrouped in a two dimensional matrix for the waste evacuation. As seen in the  FIG. 2 , the waste channel  224  connects all the waste column of a row. The valve  220  controls the evacuation of the channel  224  to the waste container  240 . The valve  230  controls the inlet of the waste container  240 . All the channels are connected to the common waste line  234 . The purge waste port  210  is connected to the waste container  240  through the purge waste line  214  and is controlled by the valve  212 . 
         [0051]    The polymer synthesizer is controlled by a control system as shown in  FIG. 3 . The computer  310  controls the reagent container valves  320 , the waste valves  330  and the motion of the plate  340 . 
         [0052]    A first embodiment for the block  410  is shown in  FIG. 4 . The synthesis column  440  receives the reagent product from the tip dispensers (not shown) by the inlet  450 . The synthesis column  440  is adapted to hermetically fit with a waste column  412  and the waste are expelled into a waste container (not shown) through the waste channel  414 . 
         [0053]    The motion of the plate  520  is shown in  FIG. 5   a , the surface of the plate  520  is adapted to receive a block. The surface  510  represents the bottom of the airtight cabinet (not shown). A second embodiment is shown in  FIG. 5   b , where the block itself  530  is displaced without a plate. 
         [0054]      FIGS. 6   a  and  6   b  show a second embodiment for the block which is constituted by two part, the spacer block, or top part,  610  and the o-ring block, or bottom part,  660 . The synthesis columns  640  are hold by a support  670 . The bottom parts of the synthesis column  640  are forced into the o-rings  650  to make an airtight fit. The synthesis columns  640  are inserted in the top part  610  of the block through the openings  680 . The wastes are expelled thought the wastes columns  612  and drained into a waste container (not shown) by the waste channel  614 . It is to be noted that the block may be constituted by only one part also with the o-rings fixed in the waste columns. 
         [0055]    While illustrative and presently preferred embodiment(s) of the invention have been described in detail hereinabove, it is to be understood that the inventive concepts may be otherwise variously embodied and employed and that the appended claims are intended to be construed to include such variations except insofar as limited by the prior art.