Digestion-after-shuffling: a method for combinatorial library construction.

We have developed a convenient method for family shuffling of amino acid sequences, termed digestion-after-shuffling. After DNA shuffling of homologous genes, plasmid mixture is extracted from a library and used for several double digestions with restriction enzymes. For each double digestion, two restriction enzymes are selected, corresponding to the single restriction sites of different parental genes. After digestions, fragments with expected sizes are obtained by gel purification and religated to construct recombinant plasmids. Thus, the obtained genes should be chimeras and have at least two restriction sites originating from different parental sequences.