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.