Attoui H, Billoir F, Cantaloube J F, Biagini P, de Micco P, de Lamballerie X
Unité des Virus Emergents, Laboratoire de Virologie Moléculaire, EFS Alpes-Méditerranée, 27 Boulevard Jean Moulin, 13005 cedex 5, Marseille, France.
J Virol Methods. 2000 Sep;89(1-2):147-58. doi: 10.1016/s0166-0934(00)00212-3.
The genetic study of viruses having dsRNA genomes is hampered by the technical difficulty of complete sequence determination of dsRNA. Optimised methods are described here for sequencing dsRNAs, which meet three different situations: (1) genomes that can be obtained in fairly high amounts (>20 ng per separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that cannot be detected by electrophoretic gel separation and staining. These methods include improved Single Primer Amplification Technique protocols, an adaptation of the SMART methodology, and a new method permitting the selective enzymatic removal of dsRNA segments. Strategies permitting adaptation of these protocols to the full-length determination of dsRNA viral genomes are described. Each of the protocols is described for sequence determination of a chosen dsRNA virus.
双链RNA基因组病毒的基因研究因双链RNA完整序列测定的技术难题而受阻。本文描述了用于双链RNA测序的优化方法，这些方法适用于三种不同情况：（1）能够以相当高的量获得的基因组（每个分离片段>20 ng）；（2）通过电泳凝胶分离和染色可检测到的RNA量有限的基因组；（3）通过电泳凝胶分离和染色无法检测到的基因组。这些方法包括改进的单引物扩增技术方案、SMART方法的改编以及一种允许选择性酶促去除双链RNA片段的新方法。还描述了使这些方案适用于双链RNA病毒基因组全长测定的策略。针对选定的双链RNA病毒的序列测定描述了每个方案。
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