Chimera construction or insertion. Top left: PCR amplification of both ends of the parent cDNA along with the vector. For chimera construction, one fragment of the cDNA needs to be replaced. Thus, the forward primer is immediately downstream of the fragment to be substituted, and the reverse primer will be immediately upstream of the fragment to be substituted. For insertion, however, two primers will be next to each other without skipping a single base. Top right: Insert amplification of the equivalent region of a homologous gene (cDNA) for chimera construction. However, for insertion of a cDNA encoding a full length protein, such as green fluorescent protein, the insert amplification will cover the entire cDNA. The remaining procedure is the same as in Figure 1.