一种利用整合酶位点特异性重组构建随机突变库的方法

A Random Mutation Library Construction Method Combined with Site-Specific Recombination Based Tandem Assembly Mediated by Serine Integrase

易错PCR利用的低保真聚合酶不具有3'到5'的校对功能,能够引入较高几率的随机突变,经常被用来构建突变库。位点特异性串联重组(SSRTA)方法拥有丰富的整合位点配对组合,可以为大片段基因簇重组提供多种模块组合。该串联重组方法特异性高、灵活性强、重组发生效率高。在通过易错PCR构建突变库时,引入用整合酶的体外多片段串联重组拼装系统,能够突破PCR长度的限制,灵活的组装各个突变模块,从而获得大片段的基因簇突变库。本研究以番茄红素基因簇的突变为例,阐释了这两种方法结合的应用。

Error-prone PCR is usually utilized to build genetic mutation library in vitro, which based on the low-fidelity polymerase does not have 3＇ to 5＇ proofreading function, and make it possible to introduce a higher probability of random mutations. Site-Specific Recombination based Tandem Assembly （SSRTA） method with its high diversity of integration sites pairs, provides a variety of combinations in the recombination of large-scare multi-gene cluster, especial in microbial secondary metabolism cluster, which has been proven possessing highly specificity, flexibility, and efficiency in recombination. The introduction of integrase mediated SSRTA system while using error-prone PCR to construct mutant libraries in vitro, can break through the limitation of the length of the PCR, assembling each module more flexible, and achieving the construction of a large fragment of the mutant gene cluster library. In this research, we take lycopene gene cluster as an example to illustrate the application of these two methods combination.