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This protocol describes a directed evolution method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) is essentially a modified PCR that uses highly abbreviated annealing and extension steps to generate staggered DNA fragments and promote crossover events along the full length of the template sequence(s). The resulting library of chimeric polynucleotide sequence(s) is subjected to subsequent high-throughput functional analysis. The recombination efficiency of the StEP method is comparable to that of the most widely used in vitro DNA recombination method, DNA shuffling. However, the StEP method does not require DNA fragmentation and can be carried out in a single tube. This protocol can be completed in 4-6 h.