Mutagenic libraries

Khalil, A.M., Julius, J.A. and Bachant, J. (2007) One step construction of PCR mutagenized libraries for genetic analysis by recombination cloning. Nucleic Acids Res. 35, el04. 

Similar sequence and binding data from mutagenic libraries for each of the four domains... 

Measure amount of retained Bt-Ag and scFv expression of the various control and mutagenic library reactions with fluorescence activated cell sorter instrument (e.g., FACSAria from Becton Dickinson). Load samples from Subheading 3.5.1 onto instrument and collect 50,000-100,000 events per reaction using analysis template developed above... 

Dissociation rate improvements of two to tenfold are typical for selections from initial mutagenic library. Two options are available if greater improvements are desired. The entire pool of mutant clones isolated from the original selection can be combined and used as template for another round of mutagenesis followed by selections. The mutations from individual clones can be combined in a site-specific manner to determine whether additive or synergistic effects are obtained. 

Siloto, R.M.R, Truksa, M., Brownfield, D., Good, A.G. and Weselake, R.J. 2009a. Directed evolution of acyl-CoA diacylglycerol acyltransferase Development and characterization of Brassica napus DGATl mutagenized libraries. Plant Physiol. Biochem. Al 456-461. 

TurboFP was also used as a basis for far-red fluorescent proteins (fRFPs). Residues surrounding the chromophore were mutagenized to create a library, which was subsequently subjected to random mutagenesis. A bright far-red variant with excitation and emission maxima at 588 and 635 nm, respectively was isolated and named Katushka [79]. This fast-maturing protein has an... 

Mutagenic PGR. More recendy, methods have been developed to use the PCR reaction to randomly mutagenize a defined sequence (25). The Taq polymerase used in PCR misincorporates nucleotides in a random fashion if manganese dichloride [7773-01-5], MnC, is included in the reaction buffer during PCR. The library of mutagenized PCR products can be screened for the desired phenotype. 

Llorens, O., Perez, J.J., and Villar, H.O., Toward the design of chemical libraries for mass screening biased against mutagenic compounds, J. Med. Chem., 44, 2793-2804, 2001. 

Sequential protocols in which one starts with a range of multivalent banks, and works towards higher affinities with monovalent libraries, mutational libraries or extension libraries, derived from the enriched clones, have been shown to be highly effective (e g. Refs. 33, 85 and 94). This and the evolutionary approach in highly mutagenic E. coli hosts over many selection rounds (e.g. Ref. 41) will surely influence further experimental design. 

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