Figure 1

SeqTBIO method of PCR-based gene synthesis for paz and polA. The nucleotide sequences paz and polA were assembled through a sequence of 4 and 22 reactions respectively and analyzed by agarose gel electrophoresis. Fragment sizes were compared against molecular markers ranging from 100 to 2000 bp. Two oligonucleotides were used for each amplification reaction starting with the central pair (f₁ and r₁), followed by the next pairs (f₂ and r₂, then f₃ and r₃, etc.) in the subsequent reactions, in an inside-out manner. The oligonucleotides overlapped by 15 to 23 nt. The entire paz sequence was synthesized in 4 reactions showing single products in each reaction with traces of unincorporated primers (A, lanes 1–4). The robustness of sequential step DNA assembly is shown for the synthesis of polA where single band fragments are clearly seen for each assembly step (B, lanes 1–22). Lane F shows the final synthesis product of polA. The assembled gene products for both proteins contained 30 nt of sequence at the 5'and 3' termini homologous to the terminal ends of a linearized plasmid vector of pET-3a for subsequent in vivo homologous recombination. These homologous regions (HR) were designed within the last outside primers sets used in the assembly process.