Functional and nonfunctional mutations distinguished by random recombination of homologous genes.
We describe a convenient method for distinguishing functional from nonfunctional or deleterious mutations in homologous genes. High fidelity in vitro gene recombination ("DNA shuffling") coupled with sequence analysis of a small sampling of the shuffled library exhibiting the evolved behavior allows identification of those mutations responsible for the behavior in a background of neutral and deleterious mutations. Functional mutations are expected to occur in 100% of the sequenced screened sample; neutral mutations are found in 50% on average, and deleterious mutations do not appear at all. When used to analyze 10 mutations in a laboratory-evolved gene encoding a thermostable subtilisin E, this method rapidly identified the two responsible for the observed protease thermostability; the remaining eight were neutral with respect to thermostability, within the precision of the screening assay. A similar approach, coupled with selection for growth and survival of the host organism, could be used to distinguish adaptive from neutral mutations.
Zhao
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Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Arnold
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FH
D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011995:Recombination, Genetic; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
D013381:Subtilisins