Degenerate oligonucleotide gene shuffling (DOGS): A method for enhancing the frequency of recombination with family shuffling

Moreland D. Gibbs, K. M Helena Nevalainen, Peter L. Bergquist*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

76 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 prior to 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 β-xylanase genes that possess widely different G + C contents.

Original languageEnglish
Pages (from-to)13-20
Number of pages8
JournalGene
Volume271
Issue number1
DOIs
Publication statusPublished - 13 Jun 2001

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