Degenerate oligonucleotide gene shuffling

Peter L. Bergquist*, Moreland D. Gibbs

*Corresponding author for this work

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

5 Citations (Scopus)

Abstract

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
Pages191-204
Number of pages14
Volume352
ISBN (Electronic)9781597451871
ISBN (Print)9781588290724
DOIs
Publication statusPublished - 2007

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume52

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