Degenerate oligonucleotide gene shuffling

Peter L. Bergquist*, Moreland D. Gibbs

*Corresponding author for this work

    Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

    6 Citations (Scopus)


    Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products. One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation before shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G and C contents.

    Original languageEnglish
    Title of host publicationProtein engineering protocols
    PublisherHumana Press
    Number of pages14
    ISBN (Electronic)9781597451871
    ISBN (Print)9781588290724
    Publication statusPublished - 2007

    Publication series

    NameMethods in Molecular Biology
    PublisherHumana Press


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