Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution
Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution
复制标题
DOI：
10.1002/cbic.200400417
复制
发表时间：
2005-06-01
期刊：
影响因子：
3.2
通讯作者：
Jaeger, KE
中科院分区：
文献类型：
--
作者：
Eggert, T;Funke, SA;Jaeger, KE
提出了一种新的、简便的单点突变体外重组方法。这种方法有效地减少了新的点突变，这通常发生在重组过程中。多重聚合酶链反应（multiplex-PCR）产生含有预先形成的点突变的基因片段。这些片段随后通过重组PCR步骤组装成全长基因。基于多重PCR的重组（MUPREC）过程不需要DNA酶I消化进行基因片段化，因此即使使用少量靶DNA也很容易进行。该方案产生高频率的重组，而不产生野生型背景。此外，低错误率导致真重组体的高质量变体文库，从而最小化筛选工作并节省时间和金钱。MUPREC方法用于枯草芽孢杆菌脂肪酶的定向进化，该脂肪酶可以催化模型内消旋化合物的对映选择性水解。因此，该方法被证明是有用的，在生产一个可靠的第二代库的真正的重组体，更好地表现的变体，从使用高通量电喷雾电离质谱（ESI-MS）筛选系统进行鉴定。
A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments ore subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.