title: Directed evolution through DNA shuffling for the improvement and understanding of genes and promoters.
abstract: Unlike rational protein engineering, directed evolution provides an a priori approach toward the engineering of improved proteins and novel promoters. This minimally recursive technique builds upon small improvements by selecting and combining the best changes. Protein-protein/DNA interactions, catalytic efficiency, or resilience to inhibitors can be improved by thousands of times. By working within a subspace of homologous sequences, DNA shuffling recombines that subspace. Individuals are screened for a particular trait or two and selected for when they meet a set threshold. Here we explain basic principles to follow and provide procedures for the preparation, fragmentation, efficient size fractionation, and purification of parental material, as well as for the reassembly and rescue polymerase chain reactions (PCRs).
authors: Department of Plant and Soil Sciences, Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, KY, USA. jrwerkman@uky.edu
authors: Joshua R
authors: JR
authors: Werkman
authors: Sitakanta
authors: S
authors: Pattanaik
authors: Ling
authors: L
authors: Yuan
mesh_terms: D032621:DNA Shuffling / Q000379:methods*; D018744:DNA, Plant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D019143:Evolution, Molecular; D015723:Gene Library; D014644:Genetic Variation; D016296:Mutagenesis; D031344:Perilla frutescens / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011995:Recombination, Genetic
chemical_list: D018744:DNA, Plant; D004268:DNA-Binding Proteins