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首页> 外文期刊>Biomolecular engineering >Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): Two complementary techniques for enzyme evolution
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Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): Two complementary techniques for enzyme evolution

机译：简并寡核苷酸基因改组（DOGS）和随机漂移诱变（RNDM）：酶进化的两种互补技术

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Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. 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 shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G + C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space. (C) 2005 Published by Elsevier B.V.
机译：酶掺入突变和DNA改组已经帮助改善了酶的生化特性。许多基因改组技术主要导致未改组（亲本）分子的再生。我们描述了使用简并引物进行基因改组的程序，该程序允许控制改组基因之间相对重组的相对水平，并减少未改组亲本基因的再生。该改组程序避免了在改组之前使用核酸内切酶进行基因片段化，并允许将嵌合基因的选定部分随机诱变包括在内。我们举例说明了改组技术与拥有广泛不同的G + C含量的β-木聚糖酶基因家族的使用。此外，我们介绍了一种新方法（RNDM），用于从没有对细胞进行自适应选择的文库中快速筛选突变体。它们仅通过保留酶活性来鉴定。 RNDM和DOGS的组合可以对蛋白质的功能序列空间进行全面探索。 （C）2005由Elsevier B.V.发布

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