Gene synthesis by circular assembly amplification.

Abstract

Here we report the development of a gene-synthesis technology, circular assembly amplification. In this approach, we first constructed exonuclease-resistant circular DNA via simultaneous ligation of oligonucleotides. Exonuclease- and subsequent mismatch cleaving endonuclease-mediated degradation of the resulting ligation mixture eliminated error-rich products, thereby substantially improving gene-synthesis quality. We used this method to construct genes encoding a small thermostable DNA polymerase, a highly repetitive DNA sequence and large (>4 kb) constructs.