Chimera construction using multiple-template-based sequential PCRs

Chimera construction between different proteins is a useful method for investigating protein structure and function relationships. However, this technique, by its traditional application, has been daunting because of its complex procedure and low success rate. Here we describe a protocol for constructing chimeras between proteins that does not require the existence of restriction sites, or the purification of intermediate PCR products, which are essential in the traditional protocols. By introducing the “multiple-template” concept, this protocol only requires the use of two or three simple PCRs followed by general subcloning steps. Most importantly, the success rate is nearly 100%.