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

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Shuffling techniques can b e 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. O ne difficulty with current shuffling procedures is the predominance of unsh uffled ('parental') molecules in the pool of mutants. We describe a procedu re for gene shuffling using degenerate primers that allows control of the r elative levels of recombination between the genes that are shuffled and red uces 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 techniq ue with a diverse family of P-xylanase genes that possess widely different G + C contents. (C) 2001 Published by Elsevier Science B.V. All rights rese rved.