Figure 1.

The TBC method of PCR-based gene synthesis of PKB2. (A) Generation of four sequential overlapping double-stranded DNA fragments from the TBC primer set. The 5′-blue- and 3′-red-colored regions of each primer correspond to the blue- and red-colored regions of primer overlap indicated in Table 1S. The size of each overlapping DNA fragment is indicated, and the blue-colored regions indicate the complementary nucleotides between adjacent fragments. PCR generates the fully elongated and amplified overlapping DNA fragments in two steps, gene assembly and gene amplification. (B) PCR assembly of sequential overlapping gene fragments. The four gene fragments are gel-purified and combined for further PCR assembly and amplification.