UNIT 26.2 Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras
Published Online: 1 JUN 2006
Copyright © 2006 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Meyer, M. M., Hiraga, K. and Arnold, F. H. 2006. Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras. Current Protocols in Protein Science. 44:26.2:26.2.1–26.2.17.
- Published Online: 1 JUN 2006
- Published Print: MAY 2006
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Recombination of distantly related and nonrelated genes is difficult using traditional PCR-based techniques, and truncation-based methods result in a large proportion of nonviable sequences due to frame shifts, deletions, and insertions. This unit describes a method for creating libraries of chimeras through combinatorial assembly of gene fragments. It allows the experimenter to recombine genes of any identity and to select the sites where recombination takes place. Combinatorial recombination is achieved by generating gene fragments with specific overhangs, or sticky ends. The overhangs permit the fragments to be ligated in the correct order while allowing independent assortment of blocks with identical overhangs. Genes of any identity can be recombined so long as they share 3 to 5 base pairs of identity at the desired recombination sites. Simple adaptations of the method allow incorporation of specific gene fragments.
- directed evolution;
- protein design;
- library creation;
- combinatorial library