Chimera assembly by overlapping PCR products

Primers used for this technique required two important features: target site amplification and overlap sequences. Each part has a minimum length of 20 base pairs (bp). Oligonucleotide design considered the 3′-A overhang ends produced by Platinum Taq High Fidelity DNA polymerase (Table ​(Table2).2). In order to fuse two DNA segments, we proceeded as follows: (1) amplify each fragment independently by PCR, (2) purify each band from agarose gel (Purification kit, Roche), (3) mix the purified fragments into the assembly PCR, where the overlapping region will produce the 3′-OH DNA end required for polymerization, (4) make an enrichment reaction using external oligonucleotides, and (5) purify the assembled DNA from an agarose gel. After purification, the DNA was digested with KpnI-XbaI restriction enzymes, cloned into pRK415 and transformed into DH5α. Transformants were selected on LB/Tet plates at 37°C. Chimeras 05 through 14 were assembled by this method, yielding the constructs pRK415chim05 to chim14 (pRKch05-ch14).