Figure 1

Recombination techniques used in this work. a) DNA Shuffling: Parental genes are randomly fragmented using DNaseI. The resulting fragments are recombined using a primer-free PCR using denaturation at high temperature, followed by annealing to other fragments, and extension by DNA polymerase. Some of these annealing events result in skew extension without recombination of fragments from two homologous parents, leading to parental background. After 35 cycles of assembly, PCR amplification with primers is used to selectively amplify full-length sequences. b) RD-PCR with one skew primer per parent: The templates are extended by parent-specific sequences resulting in asymmetric products by attaching distinct "head" and "tail". These sequences are used in the recombination PCR as primers to ensure crossover events. After the template denaturation, a high number of short annealing and extension steps results in template switching. Based on the asymmetric primers a complete product formation can only be amplified if an odd number of crossovers occurs. The resulting product will always contain different parents at the exposed ends. c) RD-PCR with two skew primers per parent: Parental templates are amplified with two unique skew primers in the first step (solid and dashed). The protocol then proceeds as in b) above, but the presence of the unique sequence prevents skew extension without recombination from happening.