Assembly and error reduction of synthetic genes from oligonucleotides

A process for reducing base pair error rate during the synthesis of a gene of interest or a fragment thereof, the process comprising the steps of:

• a) obtaining a pool of assembled gene segments that contain mismatched errors, wherein the assembled gene segments form the gene of interest or the fragment thereof,
• b) selecting error-less gene segments from the pool of assembled gene segments, comprising:
• i) denaturing the pool of assembled gene segments into single stranded nucleic acid sequences and forming the paired gene segments by random pairing complementary strands of the single stranded nucleic acid sequences, wherein some of the paired gene segments comprise mismatched base pairs;
• ii) generating a digest mixture containing error-less gene segments by mismatch digesting the paired gene segments with a T7 endonuclease I at 42° C.;
• iii) obtaining error-less gene segments by purifying the error-less gene segments from the digest mixture according to sizes of the error-less gene segments;
• and
• c) producing amplified error-less gene segments by amplifying the error-less gene segments using a high fidelity polymerase with proofreading activity, wherein the high fidelity polymerase with proofreading activity comprises a Q5 DNA polymerase, a Herculase DNA polymerase or a Phusion DNA polymerase,
• thereby reducing base pair error rate during the synthesis of the gene of interest or the fragment thereof, wherein the error rate of each of the amplified error-less gene segments is less than about 1 error per 4,000 base pairs.