Abstract    
     During evolution, organism speciation is accompanied by duplicated gene divergence (subfunctionalization or neofunctionalization), which can confer environmental adaptation advantages for new species. Diversity can subsequently also arise through the creation of single-crossover chimeras between the resulting paralogues. Recent studies in Drosophila have identified a significant number of such chimeric genes that appear to have originated from tandem duplication1. This study proposed that qualitative differences in expression, as well as structural differences between the chimeric and parental genes, could enhance rapid evolution. A number of in vivo methods2–5 have been developed to create single-crossover chimeric genes, while in vitro homologous recombination methods such as DNA shuffling6–8, staggered extension process (StEP)9,10, and other similar methods11–13 are best suited to creating multiple-crossover recombination libraries. The three-domain pore-forming Cry toxins of Bacillus thuringiensis show evidence of domain swapping through  ecombination14 that has resulted in novel specificities. Previous work has also indicated that the specificity of a Cry toxin can be altered through the creation of in vitro chimeras15–19 making this system ideal for developing an in vitro single-crossover method. An advantage of creating these chimeras in vitro is that the cell free systems that are increasingly being used in the discovery of therapeutic proteins20 can be employed to streamline the downstream screening processes. Here we present an in vitro template-change polymerase change reaction developed to enable the production of single-crossover recombination libraries that can mimic those produced in vivo.