title: Combinatorial recombination of gene fragments to construct a library of chimeras.
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.
authors.affiliation: California Institute of Technology, Pasadena, California.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Meyer
authors.affiliation: California Institute of Technology, Pasadena, California.|New York State Department of Health, Albany, New York.
authors.forename: Kaori
authors.initials: K
authors.lastname: Hiraga
authors.affiliation: California Institute of Technology, Pasadena, California.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins