多重PCR在质粒拷贝数检测中的应用

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...

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

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.