Editing Protein engineering (section)

====Incremental truncation for the creation of hybrid enzymes (ITCHY)====
Fragments of parental genes are created using controlled digestion by exonuclease III. These fragments are blunted using endonuclease, and are ligated to produce hybrid genes. THIOITCHY is a modified ITCHY technique which utilized nucleotide triphosphate analogs such as α-phosphothioate dNTPs. Incorporation of these nucleotides blocks digestion by  exonuclease III. This inhibition of digestion by exonuclease III is called spiking. Spiking can be accomplished by first truncating genes with exonuclease to create fragments with short single stranded overhangs. These fragments then serve as templates for amplification by DNA polymerase in the presence of small amounts of phosphothioate dNTPs. These resulting fragments are then ligated together to form full length genes. Alternatively the intact parental genes can be amplified by PCR in the presence of normal dNTPs and phosphothioate dNTPs. These full length amplification products are then subjected to digestion by an exonuclease. Digestion will continue until the exonuclease encounters an α-pdNTP, resulting in fragments of different length. These fragments are then ligated together to generate chimeric genes.<ref name=PoluriBook/>{{page needed|date=May 2017}}