Zhao, Huimin and Arnold, Frances H. (1997) Functional and nonfunctional mutations distinguished by random recombination of homologous genes. Proceedings of the National Academy of Sciences of the United States of America, 94 (15). pp. 7997-8000. ISSN 0027-8424 http://resolver.caltech.edu/CaltechAUTHORS:ZHApnas97
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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.
|Additional Information:||© 1997 by The National Academy of Sciences Communicated by Peter B. Dervan, California Institute of Technology, Pasadena, CA, May 16, 1997 (received for review March 3, 1997) We thank Prof. Steven Benner (University of Florida) for helpful discussions. This work was supported by the U. S. Department of Energy’s program in Biological and Chemical Technologies Research within the Office of Industrial Technologies, Energy Efficiency and Renewables. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||adaptive evolution, neutral mutations, DNA shuffling, in vitro evolution|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Archive Administrator|
|Deposited On:||09 Sep 2005|
|Last Modified:||26 Dec 2012 08:40|
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