Stevens Hans, Rector Annabel, Van Ranst Marc
Laboratory of Clinical and Epidemiological Virology, Rega Institute, University of Leuven, B-3000 Leuven, Belgium.
Cold Spring Harb Protoc. 2010 Apr;2010(4):pdb.prot5415. doi: 10.1101/pdb.prot5415.
The use of whole genome amplification and analysis of viruses is of increasing importance, as data generated using these methods are currently used for clinical diagnostics, epidemiological studies, phylogenetic analyses, and studies of genome organization and evolution. The best known amplification method for DNA is the polymerase chain reaction (PCR). This technique, however, has drawbacks: PCR produces relatively small amplicons and also requires prior knowledge of sequence data for the construction of the required consensus or degenerate primers. For circular DNA templates, it is possible to overcome these drawbacks by using the multiply primed rolling-circle amplification (RCA) technique, which mimics the rolling-circle mechanism that occurs in nature for replication of circular DNA molecules, e.g., plasmids. The RCA protocol described here is optimized for the amplification of circular DNA virus genomes.
全基因组扩增及病毒分析的应用愈发重要，因为目前利用这些方法生成的数据被用于临床诊断、流行病学研究、系统发育分析以及基因组组织与进化研究。最广为人知的DNA扩增方法是聚合酶链反应（PCR）。然而，该技术存在缺陷：PCR产生的扩增子相对较小，并且构建所需的共有引物或简并引物还需要序列数据的先验知识。对于环状DNA模板，可以通过使用多重引物滚环扩增（RCA）技术来克服这些缺陷，该技术模拟了自然界中环状DNA分子（如质粒）复制时发生的滚环机制。此处描述的RCA方案针对环状DNA病毒基因组的扩增进行了优化。
