多重PCR在质粒拷贝数检测中的应用  

A New Method to Identify the Copy Number of Plasmid by Multiplex PCR

作　　者：李媛[1,2] 任长虹[1] 吴永红[1] 叶巧[1] 石锦平[1] 屈武斌[1] 郑晓飞[1] 刘虎岐[2] 张成岗[1,2] 

机构地区：[1]军事医学科学院放射与辐射医学研究所蛋白质组学国家重点实验室,北京100850 [2]西北农林科技大学生命科学学院,杨凌712100

出　　处：《中国生物工程杂志》2011年第6期93-98,共6页China Biotechnology

基　　金：国家“973”计划(2006CB504100);国家科技重大专项课题(2009ZX09503-002,2009ZX09301-002,2009ZX09103-616);国家自然科学基金(30900862,30800196)资助项目

摘　　要：聚合酶链式反应(PCR)作为常规分子克隆技术已在分子生物学的各个领域得到广泛应用。然而,多重PCR技术应用于质粒拷贝数检测的研究尚未见报道。为深入探索多重PCR在质粒拷贝数测定中的应用,首先利用构建的多重PCR引物设计及评估体系分别针对细菌基因组DNA和质粒载体DNA序列设计多重PCR引物;然后以转化有不同质粒载体的大肠杆菌菌液为模板进行多重PCR反应;最后利用凝胶成像仪采集图像后进行积分光密度分析以获得质粒的相对拷贝数。结果显示通过多重PCR得到的质粒相对拷贝数与Real-time PCR和Southern blot的检测结果基本一致,说明多重PCR可用于质粒拷贝数的定性检测。利用多重PCR技术建立了一种便捷的质粒拷贝数检测方法,为在宿主细胞中快速确定克隆载体或表达载体的相对含量提供了参考。The PCR technique has been widely used in various fields of molecular biological studies. However, multiplex PCR for determination of plasmid copy number has not been reported. To further explore the research in this field, based on the multiple PCR primer design and evaluation computer programs, Two-plex PCR primers were disigned to amplify the bacterial genome and plasmid DNA; then using bacterial liquid of E. coli with different plasmid vectors as the template for multiple PCR reactions. Finally, the integrated optical density of the gel image was analyzed to determine relative copy number of the plasmids. The data showed that the result of relative copy number of the plasmids determined by multiplex PCR was consistent with which determined by Real-time PCR and Southern blot, indicating that multiplex PCR was an alternative and valuable method compared with traditional techniques in routine experiments. So a rapid and efficient method in identification of the plasmid copy number was established, which provides a suitable way to host cell selection by easily and rapidly determining the relative copy number of clone vector and expression vector.

关 键 词：质粒拷贝数 多重PCR 菌液 实时定量PCR SOUTHERN BLOT 

分 类 号：Q52[生物学—生物化学]