Rapid full-length cloning of nonpolyadenylated RNA virus genomes.
Rapid full-length cloning of nonpolyadenylated RNA virus genomes.
复制标题
非聚腺苷酸化 RNA 病毒基因组的快速全长克隆。
DOI：
10.1002/9780471729259.mc16f03s4
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发表时间：
2007
影响因子：
--
通讯作者：
Miller,WAllen
中科院分区：
文献类型：
--
作者：
Beckett,Randy;Miller,WAllen
对于旨在了解病毒复制和基因表达机制的研究来说，获得病毒基因组的全长感染性克隆实际上是必需的。虽然构建全长克隆可能很简单，但获得具有传染性的克隆绝非例行公事。在这里，作者描述了最大限度地提高获得全长感染性克隆的可能性的方法。这些协议包括 (1) 对非聚腺苷酸化 RNA 基因组末端进行测序，(2) 在一次反应中直接从病毒 RNA 获得全长 PCR 产物，以及 (3) 将 PCR 产物有效克隆到载体中，该载体允许包含完美或接近完美末端的病毒 RNA 的体外转录。鉴于传统上获得 RNA 基因组感染性克隆（尤其是非多腺苷酸化 RNA 病毒）的困难，该单元对于所有研究动植物非多腺苷酸化和多腺苷酸化病毒的病毒学家来说应该很有价值。
Access to a full‐length infectious clone of a viral genome is a virtual necessity for research aimed at understanding virus replication and gene expression mechanisms. While construction of a full‐length clone may be straightforward, obtaining one that is infectious is by no means routine. Here the authors describe methods to maximize the likelihood of obtaining a full‐length infectious clone. These include protocols to (1) sequence the ends of nonpolyadenylated RNA genomes, (2) obtain a full‐length PCR product in a single reaction directly from viral RNA, and (3) efficiently clone the PCR product into a vector that allows in vitro transcription of viral RNA containing perfect or near‐perfect termini. Given the traditional difficulty of obtaining infectious clones of RNA genomes (especially of nonpolyadenylated RNA viruses), this unit should be valuable to all virologists working with nonpolyadenylated as well as polyadenylated viruses of plants and animals.