Seamless cloning and domain swapping of synthetic and complex DNA

Abstract

The use of synthetic DNA can avoid problems that are sometimes encountered with conventional molecular biology techniques using DNA with high GC content, strong secondary structures, or repeat sequences. However, very complex DNA may still resist PCR and synthesis of DNA from oligonucleotides. In the method described here, separately synthesized DNA segments were seamlessly joined independently of the presence of restriction sites in the target DNA. This method allowed the reconstruction of complex DNA by concatenation of easily synthesized segments and permitted repeated swapping of segments, from a few nucleotides to large fragments of complex DNA for phenotypic analysis.