摘要
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.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2017年第6期2440-2446,共7页
Genomics and Applied Biology
基金
中国国家基础研究发展973计划(2012CB721102)
自然科学基金(31300034)共同资助