长片段融合PCR在构建烟曲霉rho1基因回补株中的应用

目的融合PCR是一种常用的构建重组片段或重组质粒的手段,但长片段融合PCR的难度较大。文中将探讨长片段融合PCR过程中引物设计及扩增条件对产物的影响。方法以构建烟曲霉rho 1基因回补株为例,采用融合PCR的方法扩增重组片段(长达6.5 kb),在引物设计时引入不同大小的同源区,并设置不同的扩增体系。结果当设计引物的同源区为35 bp,选用具有高扩增效率、高保真性的DNA聚合酶,以及各片段在融合PCR反应体系中的浓度为15 ng/μL时,实现了长达6.5 kb的片段扩增并完成了烟曲霉rho 1回补株的构建。结论在合适的PCR引物设计、片段浓度配比及聚合酶条件下,长片段融合PCR在丝状真菌的基因敲除及回补株的构建中是一种非常有效的工具。

The application of long-fragment fusion PCR in the construction of rhol-compelement strain of Aspergillus fumigatus

Objective To evaluate the effect of primer design and the amplification condition on long-fragment fusion PCR. Methods With the example of 6.5 kb-long fragment amplification by fusion-PCR during construction of rho 1 gene complement in Aspergillus fumigatus , primer design with diverse homologous regions and different condition for amplification were evaluated. Results The 6.5 kb-long fragment was successfully amplified by fusion-PCR under following conditions ： the primers with homologous region of 35 bp ; DNA polymerase with high amplification efficiency and high fidelity ; the concentration of template fragments higher than 15 ng/μL in whole reaction system. Conclusion With the appropriate primer, DNA polymerase and concentration of templates, the fusion PCR for the long-fragment might be a highly-efficient tool in gene-manufacturing of filamentous fungi.