Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA
Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA
复制标题
DOI：
10.1099/vir.0.80039-0
复制
发表时间：
2004-11-01
影响因子：
3.8
通讯作者：
Boland, CR
中科院分区：
文献类型：
--
作者：
Laghi, L;Randolph, AE;Boland, CR
以前的尝试，以确定致癌多瘤病毒在人类癌症产生了矛盾的结果，即使与PCR技术的应用。在此，考虑了多瘤病毒基因组的拓扑特征是否干扰有效的PCR扩增。质粒和SV40 DNA被用作比较超螺旋、环状松弛和线性模板的扩增效率的模型系统。发现检测环状模板比检测相同但线性的模板需要10倍多的分子。超螺旋阻碍了SV40环的体外扩增的10倍，并且在亚皮格兰量的模板下发生超螺旋SV40的不稳定扩增。因此，DNA的拓扑异构酶I处理改善了超螺旋SV40的PCR检测，显著降低了假阴性样品的数量。以前描述的，但有争议的，多瘤病毒存在于人体组织中应重新考虑和拓扑异构酶I敏感的多瘤病毒扩增可能有助于检测多瘤病毒基因组在哺乳动物组织。
Previous attempts to identify oncogenic polyomaviruses in human cancers have yielded conflicting results, even with the application of PCR technology. Here, it was considered whether the topological features of the polyomavirus genome interfere with efficient PCR amplification. Plasmid and SV40 DNAs were used as a model system for comparing the amplification efficiency of supercoiled, circular relaxed and linear templates. It was found that detection of circular templates required 10 times more molecules than detection of identical but linear templates. Supercoiling hindered the in vitro amplification of SV40 circles by a factor of 10, and erratic amplification of supercoiled SV40 occurred with subpicogrann amounts of template. Accordingly, topoisomerase I treatment of DNA improved the PCR detection of supercoiled SV40, significantly decreasing the number of false-negative samples. Previously described, yet controversial, polyomavirus presence in human tissues should be reconsidered and topoisomerase I-sensitive polyomavirus amplification might help to detect polyomavirus genomes in mammalian tissues.