Preparation of SCRATCHY Hybrid Protein Libraries: Size- and In-Frame Selection of Nucleic Acid Sequences

SCRATCHY is a combination of the incremental truncation for the creation of hybrid enzymes (ITCHY) technology (1 ) and DNA shuffling (2 ). It generates combinatorial libraries of hybrid proteins consisting of multiple fragments from two or more parental DNA sequences with no restriction to DNA sequence identity between the original sequences (3 ). Such multi-crossover hybrids can be of interest to the studies of fundamental questions of protein evolution and folding, as well as to the tailoring of enzymes for therapeutic and industrial applications.