CaltechAUTHORS
  A Caltech Library Service

Functional and nonfunctional mutations distinguished by random recombination of homologous genes

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

[img]
Preview
PDF
See Usage Policy.

233Kb

Use this Persistent URL to link to this item: http://resolver.caltech.edu/CaltechAUTHORS:ZHApnas97

Abstract

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.


Item Type:Article
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
Record Number:CaltechAUTHORS:ZHApnas97
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:ZHApnas97
Alternative URL:http://www.pnas.org/cgi/content/abstract/94/15/7997
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:644
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:09 Sep 2005
Last Modified:26 Dec 2012 08:40

Repository Staff Only: item control page