Summary: UNIT 26.2Combinatorial Recombination of Gene
Fragments to Construct a Library of
and Frances H. Arnold1
Caltech, Pasadena, California
Recombination of distantly related and nonrelated genes is difŪcult 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 speciŪc 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 speciŪc gene fragments. Curr. Protoc. Protein Sci.
61:26.2.1-26.2.20. C 2010 by John Wiley & Sons, Inc.