Methods and Devices for Nucleic Acid Synthesis

Abstract

Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.

1 - 41 . (canceled) 
     
     
         42 . A nucleic acid array system comprising:
 a. one or more solid support;   b. a plurality of discrete features associated with the one or more solid support wherein each feature comprises a plurality of support-bound oligonucleotides having a predefined sequence wherein a first support-bound oligonucleotide comprises a terminal region that is complementary to a terminal region of a second support-bound oligonucleotide, wherein a Nth support-bound oligonucleotide comprises a terminal sequence complementary to a terminal sequence region of a (N-1)th support-bound oligonucleotide, wherein N is no less than 3; and   c. at least a first plurality of anchor oligonucleotides comprising a terminal sequence that is identical to a sequence region of the first support-bound oligonucleotide.   
     
     
         43 . The nucleic acid array system of  claim 42  further comprising a second plurality of support-bound anchor oligonucleotide having a sequence region that is identical to the Nth support-bound oligonucleotide. 
     
     
         44 - 65 . (canceled) 
     
     
         66 . The nucleic acid array system of  claim 42  wherein the first to the Nth support-bound oligonucleotides together comprise a target nucleic acid having a predetermined sequence. 
     
     
         67 . The nucleic acid array system of  claim 66  wherein each solid support comprises a plurality of features for assembling two or more target nucleic acids in parallel. 
     
     
         68 . The nucleic acid array system of  claim 42  further comprising an anchor support wherein the anchor oligonucleotides are immobilized on the anchor support. 
     
     
         69 . The nucleic acid array system of  claim 68  further comprising a microvolume of reagents localized on each feature for synthesizing construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template. 
     
     
         70 . The nucleic acid array system of  claim 69  further comprising a fluid medium for transferring the construction oligonucleotides to the anchor support. 
     
     
         71 . A system for producing at least one target nucleic acid, comprising:
 a. one or more solid supports having at least a first and a second features thereon, the first feature having a first plurality of support-bound oligonucleotides, the second feature having a second plurality of support-bound oligonucleotides, wherein each of the first plurality of support-bound oligonucleotides comprises a terminal sequence region complementary to that of the second plurality of support-bound oligonucleotides;   b. an anchor support comprising a plurality of anchor oligonucleotides, each having a terminal region identical to that of the first plurality of support-bound oligonucleotides; and   c. a solution of reagents for synthesizing construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template.   
     
     
         72 . The system of  claim 71  further comprising a Nth plurality of support-bound oligonucleotides comprising a terminal sequence complementary to that of a (N-1)th plurality of support-bound oligonucleotides, wherein N>=3. 
     
     
         73 . The system of  claim 72  wherein the first to the Nth pluralities of support-bound oligonucleotides together comprise a target nucleic acid having a predetermined sequence. 
     
     
         74 . The system of  claim 73  wherein each solid support comprises a plurality of features for assembling two or more target nucleic acids in parallel. 
     
     
         75 . The system of  claim 71  wherein the solution comprises a plurality of microvolumes each localized on a feature. 
     
     
         76 . The system of  claim 71  further comprising a fluid medium for transferring the construction oligonucleotides to the anchor support. 
     
     
         77 . The system of  claim 71  wherein the support-bound oligonucleotides are synthesized or spotted on the one or more solid supports. 
     
     
         78 . A method for producing at least one target nucleic acid, comprising:
 a. providing the nucleic acid array system of  claim 42 ;   b. synthesizing a first to Nth construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template;   c. transferring the first to Nth construction oligonucleotides to the first plurality of anchor oligonucleotides; and   d. hybridizing the first to Nth construction oligonucleotides with the first plurality of anchor oligonucleotides, thereby producing at least one target nucleic acid.   
     
     
         79 . A method for producing at least one target nucleic acid, comprising:
 a. providing the nucleic acid array system of  claim 71 ;   b. synthesizing a first and second construction oligonucleotides using the support-bound oligonucleotides as template;   c. transferring the first and second construction oligonucleotides to the anchor support; and   d. hybridizing the first and second construction oligonucleotides with the anchor oligonucleotides, thereby producing at least one target nucleic acid.   
     
     
         80 . A method for producing at least one target nucleic acid, comprising:
 a. providing the nucleic acid array system of  claim 73 ;   b. synthesizing a first to a Nth construction oligonucleotides using the support-bound oligonucleotides as template;   c. transferring the first to the Nth construction oligonucleotides to the anchor support; and   d. hybridizing the first to the Nth construction oligonucleotides with the anchor oligonucleotides, thereby producing at least one target nucleic acid.