Methods and devices for nucleic acid synthesis

Abstract

Disclosed are devices and methods to synthesize polynucleotides and libraries of polynucleotides such as libraries of oligonucleotides. In exemplary embodiments, the device includes a support having a plurality of features. Each feature contains a plurality of oligonucleotides. Within each feature, each of the plurality of oligonucleotides includes an identical predetermined subunit sequence of X nucleosides and a degenerate sequence of Y nucleosides. A predetermined combination of a subset of the features can be used to produce a polynucleotide having a predetermined sequence of Z nucleosides.

1 . A device for polynucleotide synthesis, comprising:
 a solid support;   a plurality of features on the solid support, and   a plurality of oligonucleotides situated at each feature, each oligonucleotide having a primer binding site and an M-mer payload, wherein all payloads from the plurality of features provide up to 4 M  different sequences, wherein M is an integer between 2 and 50.   
     
     
         2 . The device of  claim 1 , wherein the solid support is an oligomer array. 
     
     
         3 . The device of  claim 1 , wherein the solid support comprises a plurality of beads. 
     
     
         4 . The device of  claim 1 , wherein the solid support has up to 4 M  features. 
     
     
         5 . The device of  claim 1 , wherein the oligonucleotides are immobilized on each feature. 
     
     
         6 . The device of  claim 1 , wherein the oligonucleotides are synthesized in situ on each feature. 
     
     
         7 . The device of  claim 1 , wherein the oligonucleotides each comprise a further primer binding site such that the two primer binding sites flank the M-mer payload. 
     
     
         8 . The device of  claim 1 , wherein the primer binding site is universal for all oligonucleotides. 
     
     
         9 . The device of  claim 1 , wherein the primer binding site comprises a degenerate sequence. 
     
     
         10 . The device of  claim 1 , wherein the primer binding site comprises a restriction enzyme recognition site, such that upon restriction enzyme digestion, the primer binding site can be removed. 
     
     
         11 . The device of  claim 1 , wherein M is an integer between 4 and 20. 
     
     
         12 . A system for polynucleotide synthesis, comprising:
 the device of  claim 1 , comprising a plurality of solid supports, wherein all payloads from the plurality of solid supports provide 4 M  different sequences.   
     
     
         13 . A method of for polynucleotide synthesis, comprising:
 providing the system of  claim 12 ;   selecting, based on the sequence of a predetermined target polynucleotide to be synthesized, one or more oligonucleotides on the plurality of solid supports;   synthesizing construction oligonucleotides complementary to the selected one or more oligonucleotides, using a primer that binds to the primer binding site in a chain extension reaction;   removing the primer from the construction oligonucleotides; and   assembling the construction oligonucleotides to form the target polynucleotide.   
     
     
         14 . The method of  claim 13 , wherein in the selecting step, the one or more oligonucleotides are selected such that the payloads therein together comprise the target polynucleotide. 
     
     
         15 . The method of  claim 14 , wherein the one or more oligonucleotides are selected such that the payloads therein have overlapping sequences between two adjacent payloads. 
     
     
         16 . The method of  claim 15 , wherein the overlapping sequences between two adjacent payloads are identical. 
     
     
         17 . The method of  claim 15 , wherein the overlapping sequences between two adjacent payloads are complementary. 
     
     
         18 . The method of  claim 13 , wherein the plurality of oligonucleotides each comprise a further primer binding site such that the two primer binding sites flank the M-mer payload. 
     
     
         19 . The method of  claim 13 , wherein in the synthesizing step, the primer is universal for the selected one or more oligonucleotides. 
     
     
         20 . The method of  claim 13 , wherein in the synthesizing step, the primer comprises a degenerate sequence. 
     
     
         21 . The method of  claim 13 , wherein the removing step comprises digesting with a restriction enzyme that recognizes a restriction enzyme recognition site within the primer. 
     
     
         22 . The method of  claim 13 , wherein the removing step comprises digesting the primer with uracil deglycosylase that recognizes one or more cleavable uracil groups within the primer. 
     
     
         23 . The method of  claim 13 , wherein the assembling step comprises annealing the construction oligonucleotides and ligating ligatable ends. 
     
     
         24 . A method of for polynucleotide synthesis, comprising:
 providing the system of  claim 12 ;   synthesizing construction oligonucleotides complementary to the plurality of oligonucleotides, using a primer that binds to the primer binding site in a chain extension reaction;   selectively releasing, based on the sequence of a predetermined target polynucleotide to be synthesized, one or more construction oligonucleotides from the solid support;   removing the primer from the released construction oligonucleotides; and   assembling the released construction oligonucleotides to form the target polynucleotide.   
     
     
         25 . The method of  claim 24 , wherein the selectively releasing step comprises selectively heating features containing the one or more construction oligonucleotides.