Figure 4

Comparative gene synthesis of polA coding region by methods of TBIO and SeqTBIO. The oligonucleotide pairs f₁-r₁, f₂-r₂, f₃-r₃ and f₄-r₄ were used to attempt the synthesis of a 351 bp central fragment of the polA coding region. The initial TBIO gene synthesis included the mixture of 8 primers and the reaction was subjected to 30 cycles of PCR amplification (lane 2). An early version of the SeqTBIO approach was performed in parallel (lanes 3–6) using the first 3 oligonucleotide pairs only. The central pair (f₁-r₁) was initially assembled (lane 3) followed by next pairs f₂-r₂ (lane 4, 5) and f3-r3 (lane 6). The TBIO reaction produced a smear and did not seem to generate a unique fragment but instead a range of product sizes ranging from 190 bp – 600 bp. In contrast, each SeqTBIO reaction generated a single-band product of the expected size. A PCR negative control is shown in lane 1. The reaction products were analyzed by electrophoresis on a 1.2% agarose gel stained with ethidium bromide and visualized by UV illumination.