ITCHY: Incremental Truncation for the Creation of Hybrid enzYmes

Methods Mol Biol. 2014:1179:225-44. doi: 10.1007/978-1-4939-1053-3_16.


Incremental Truncation for the Creation of Hybrid enzYmes (ITCHY) is a directed evolution technique for randomly recombining two genes. The chief advantage of ITCHY is that there is no requirement for the two genes to share any sequence similarity. This distinguishes ITCHY from directed evolution methods that are based on homologous recombination, such as DNA shuffling. In ITCHY, Escherichia coli exonuclease III is used to incrementally truncate one of the parental genes from its 3' end and the other from its 5' end. Ligation of the randomly truncated gene fragments yields a combinatorial library of chimeras. In this chapter, we provide detailed protocols for constructing libraries using both the user-friendly thio-ITCHY method and also time-dependent incremental truncation. We illustrate the protocols with the data that we obtained when we recombined two alcohol dehydrogenase genes that only share 47 % sequence identity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / genetics
  • Cloning, Molecular
  • DNA Shuffling
  • Directed Molecular Evolution / methods*
  • Gene Library
  • Peptide Library


  • Peptide Library
  • Alcohol Dehydrogenase