Titlebook: Directed Evolution Library Creation; Methods and Protocol Elizabeth M.J. Gillam,Janine N. Copp,David Ackerle Book 2014Latest edition Spring

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Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolutioninsertion. Each approach has a common initial step that utilizes an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion into target DNA during in vitro transposition. Removal of the transposon using a type IIS restricti

Galactogogue

Restriction Enzyme-Mediated DNA Family Shufflingat could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length v

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Assembly of Designed Oligonucleotides: A Useful Tool in Synthetic Biology for Creating High-Quality protein, metabolic, and genome engineering. In directed evolution of proteins, ADO benefits from using reduced amino acid alphabets for saturation mutagenesis and/or DNA shuffling, but all 20 canonical amino acids can be also used as building blocks. ADO is performed in a two-step reaction. The firs

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USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homolmbled into full-length genes, proven for up to ten fragments. USERec requires a minimal crossover sequence (a 5′-AN. T-3′ motif) of the fragments that can be implemented wherever structural or functional comparisons suggest a fragment boundary. The greatly reduced sequence constraints of this method

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Generating Targeted Libraries by the Combinatorial Incorporation of Synthetic Oligonucleotides Durinhat encode designed, site-specific mutations by assembly PCR. This protocol enables a researcher to “hedge the bets,” namely, to explore a large number of potentially beneficial mutations in a combinatorial manner such that individual library variants carry a limited number of mutations.