Compositions and methods for synthesis of high fidelity oligonucleotides

Abstract

Aspects of the invention relate to methods, compositions for synthesizing high fidelity oligonucleotides.

1 - 17 . (canceled) 
     
     
         18 . A method of synthesizing high fidelity oligonucleotides; the method comprising:
 a. providing a plurality of nucleic acids, each nucleic acid comprising a template sequence, a 5′ end flanking region and a 3′ end flanking region, wherein the 5′ end flanking region comprises a first primer binding sequence and the 3′ end flanking region comprises a second primer binding sequence and a restriction enzyme recognition site;   b. providing a first primer capable of annealing the first binding sequence and a second primer capable of annealing to the second primer binding sequence, the first primer comprising biotin at the 5′ end and uracil at the 3′ end;   c. amplifying the nucleic acids to form a plurality of double-stranded nucleic acids;   d. removing the second primer binding site by enzymatic cleavage using a suitable restriction enzyme;   e. isolating the plurality of digested nucleic acids using a solid support comprising streptavidin;   f. denaturing the plurality of double-stranded nucleic acids to form a plurality of single stranded nucleic acids;   g. removing the uracil at the 5′ end of the plurality of single-stranded nucleic acids; and   h. releasing the single-stranded nucleic acids from the solid support.   
     
     
         19 . The method of  claim 18  wherein the plurality of nucleic acids is provided on an array. 
     
     
         20 . The method according to  claim 18  or  19  wherein the primers are universal or semi-universal primers. 
     
     
         21 . The method according to  claim 18  or  19  further comprising subjecting the double-stranded nucleic acids to error removal. 
     
     
         22 . The method of  claim 21  comprising subjecting the double-stranded nucleic acids to MutS or MutS homologs. 
     
     
         23 . The method according to  claim 18  or  19  wherein in the step of denaturing, the double-stranded nucleic acids are subjected to denaturing conditions. 
     
     
         24 . The method according to  claim 18  or  19  wherein the uracil are removed using a DNA-glycosylase and endonuclease. 
     
     
         25 . The method according to  claim 18  or  19  wherein the step of releasing the single-stranded nucleic acid is by heating to break abasic bonds. 
     
     
         26 . The method of  claim 19  wherein the plurality of nucleic acids is synthesized on the array.