Stemmer W P
Affymax Research Institute, Palo Alto, California 94304.
Nature. 1994 Aug 4;370(6488):389-91. doi: 10.1038/370389a0.
DNA shuffling is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly. Computer simulations called genetic algorithms have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis and error-prone PCR are not combinatorial and thus are limited in searching sequence space. We have tested mutagenic DNA shuffling for molecular evolution in a beta-lactamase model system. Three cycles of shuffling and two cycles of backcrossing with wild-type DNA, to eliminate non-essential mutations, were each followed by selection on increasing concentrations of the antibiotic cefotaxime. We report here that selected mutants had a minimum inhibitory concentration of 640 micrograms ml-1, a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme. Cassette mutagenesis and error-prone PCR resulted in only a 16-fold increase.
DNA改组是一种通过随机片段化和聚合酶链反应（PCR）重新组装对选定突变基因库进行体外同源重组的方法。称为遗传算法的计算机模拟已经证明了迭代同源重组对序列进化的重要性。寡核苷酸盒式诱变和易错PCR不是组合性的，因此在搜索序列空间方面受到限制。我们已经在β-内酰胺酶模型系统中测试了诱变DNA改组用于分子进化的情况。进行三轮改组和两轮与野生型DNA回交以消除非必需突变，每次之后都在浓度不断增加的抗生素头孢噻肟上进行选择。我们在此报告，选定的突变体的最低抑菌浓度为640微克/毫升，增加了32000倍，比任何已发表的源自TEM-1的酶高64倍。盒式诱变和易错PCR仅导致16倍的增加。
