Unit

UNIT 26.2 Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras

  1. Michelle M. Meyer1,
  2. Kaori Hiraga1,2,
  3. Frances H. Arnold1

Published Online: 1 JUN 2006

DOI: 10.1002/0471140864.ps2602s44

Current Protocols in Protein Science

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.

Author Information

  1. 1

    California Institute of Technology, Pasadena, California

  2. 2

    New York State Department of Health, Albany, New York

Publication History

  1. Published Online: 1 JUN 2006
  2. Published Print: MAY 2006

This is not the most recent version of the article. View current version (1 AUG 2010)

Abstract

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.

Keywords:

  • chimera;
  • recombination;
  • directed evolution;
  • protein design;
  • library creation;
  • combinatorial library