Solid phase methods for polynucleotide production

Abstract

Polynucleotides having in excess of 1,000 nucleotides can be prepared using a solid phase synthesis technique. A feature of the technique is the use of a reusable solid support that contains covalently bound oligonucleotide. This covalently bound oligonucleotide is annealed to a bridge oligonucleotide, where the bridge is also annealed to a first oligonucleotide that forms a portion of the target polynucleotide. After the target polynucleotide is synthesized, it can be removed from the solid support under denaturing conditions, and the solid support re-used to prepare additional target polynucleotides. The yield of the target polynucleotide increases when shearing force is applied to the solid support that is linked to the growing oligonucleotide. This shearing force is thought to extend the growing end of the oligonucleotide away from contact with other oligonucleotide bound to the solid support and make that end more accessible to annealing with solution oligonucleotide. The synthesis is conveniently accomplished on a porous frit, where reagents and washing solutions are pumped through the frit.

1 . A device for automated gene fragment or gene assembly, comprising:
 (a) a reaction block comprising a plurality of cavities adapted to hold a plurality of reaction vessels, wherein each reaction vessel, when present within a cavity, comprises a first orifice, a second orifice, and a solid support positioned within the interior of the reaction vessel between the first and second orifice; and   (b) a reagent delivery and mixing unit in fluid communication with the plurality of reaction vessels when present.   
   
   
       2 . The device of  claim 1  wherein the reaction block further comprises one or more means to control or monitor the temperature of the reaction block. 
   
   
       3 . The device of  claim 1  wherein the reaction block further comprises one or more means to monitor the fluid level within a reaction vessel when present. 
   
   
       4 . The device of  claim 1  further comprising one or more means to immobilize the plurality of reaction vessels within the plurality of the cavities of the reaction block. 
   
   
       5 . The device of  claim 1  wherein the reagent delivery and mixing unit contains one or more pumps in fluid communication with one or more valves. 
   
   
       6 . The device of  claim 5  wherein a valve of the reagent delivery and mixing unit is positioned between the first orifice of a reaction vessel when present and one of the pumps of the reagent delivery and mixing unit, where the valve is in fluid communication with the pump, the reaction vessel and one or more liquid storage containers. 
   
   
       7 . The device of  claim 6  where a liquid in one of the liquid storage containers is transported from the liquid storage container through the valve into the reaction vessel by the action of the pump. 
   
   
       8 . The device of  claim 1  further comprising a microplate holding apparatus. 
   
   
       9 . The device of  claim 8  wherein the microplate holding apparatus further comprises a temperature monitoring and control means. 
   
   
       10 . The device of  claim 8  further comprising a multi-microplate storage system. 
   
   
       11 . The device of  claim 10  wherein the multi-microplate storage system further comprises a temperature monitoring and control means. 
   
   
       12 . The device of  claim 11  further comprising a means to transport a microplate from the multi-microplate storage system to the microplate holding apparatus. 
   
   
       13 . The device of  claim 8  further comprising a microplate-well seal-piercing means. 
   
   
       14 . The device of  claim 1  further comprising a barcode reader. 
   
   
       15 . The device of  claim 1  further comprising one or more means to analyze the products of the processes performed by the device. 
   
   
       16 . The device of  claim 15  where the means is a spectrophotometer. 
   
   
       17 . The device of  claim 15  where the means is a fluorescence plate reader. 
   
   
       18 . The device of  claim 1  further comprising a computer control unit, where the computer control unit executes pre-programmed commands to operate the device. 
   
   
       19 . The device of  claim 18  wherein the computer control unit executes pre-programmed commands to operate the action of reagent delivery and mixing unit. 
   
   
       20 . The device of  claims 2 ,  9  and  11  wherein the computer control unit executes pre-programmed commands to operate the temperature monitor and control means. 
   
   
       21 . The device of  claim 3  wherein the computer control unit executes pre-programmed commands to operate the fluid monitor means of the reaction block. 
   
   
       22 . The device of  claim 12  wherein the computer control unit executes pre-programmed commands to operate the means to transport a microplate from the multi-microplate storage system to the microplate holding apparatus. 
   
   
       23 . The device of  claim 13  wherein the computer control unit executes pre-programmed commands to operate the microplate-well seal-piercing means. 
   
   
       24 . A device for automated gene fragment or gene assembly, comprising:
 (a) a reaction block comprising a plurality of cavities adapted to hold a plurality of reaction vessels, wherein each reaction vessel, when present within a cavity, comprises a first orifice, a second orifice, and a solid support positioned within the interior of the reaction vessel between the first and second orifice;   (b) a reagent delivery and mixing unit in fluid communication with the plurality of reaction vessels when present;   (c) a microplate holding apparatus;   (d) one or more means to immobilize the plurality of reaction vessels when present within the plurality of cavities of the reaction block;   (e) one or more means to monitor or control the temperature of the reaction block, the multi-microplate storage system or the microplate holding apparatus;   (f) a computer control unit, where the computer control unit executes pre-programmed commands to operate the device;   (g) optionally, a multi-microplate storage system;   (h) optionally, a means to transport a microplate from a multi-microplate storage system to a microplate holding area; and   (i) optionally, a microplate well seal-piercing means.