The Creation of ITCHY Hybrid Protein Libraries

Incremental truncation is a method for creating a library of every one base truncation of dsDNA. Incremental truncation libraries can be created by time dependent Exo III digestions (1 ) or by the incorporation of α-phosphorothioate dNTPs (αS-dNTPs) (2 ). The fusion of two incremental truncation libraries is called an ITCHY library (3 ). An ITCHY library created from a single gene consists of genes with internal deletions and duplications. An ITCHY library created between two different genes consists of gene fusions created in a DNA-homology independent fashion. ITCHY libraries, as well as an incremental truncation-like method called SHIPREC (4 ), have the potential to create proteins with improved or novel properties as well as to generate artificial families for in vitro recombination in a method called SCRATCHY (5 ) (see Chapter 17 ).