Optimized approaches for the sequence determination of double-stranded RNA templates
Optimized approaches for the sequence determination of double-stranded RNA templates
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DOI：
10.1016/j.jviromet.2010.08.013
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发表时间：
2010-11-01
影响因子：
3.1
通讯作者：
Adam, Guenter
中科院分区：
文献类型：
--
作者：
Darissa, Omar;Willingmann, Peter;Adam, Guenter
在许多情况下，双链RNA（dsRNA）是RNA病毒的分子和诊断研究的唯一可用模板。一种具有五个dsRNA片段基因组的新型真菌病毒用作使用几种基于PCR的方法扩增和克隆dsRNA片段的模型系统。通过经典方法获得的序列：随机PCR（rPCR），其中单个引物组装成5个片段中的4个重叠群。此外，使用改进的单引物扩增技术（SPAT）导致在一个RT-PCR中扩增全部或部分dsRNA片段。将这样的修饰引入FLAC方法（cDNA的全长扩增）导致与SPAT方法的扩增子相当的扩增子。代表五个基因组区段的全长PCR产物被克隆和测序。每种方法的最佳条件进行了介绍和讨论。在另一种方法中，将纯化的dsRNA片段直接克隆到平端pJET 1.2或pGEM（R）-T克隆载体中，但效率较低。这导致了几个长度高达2.2 kb的序列，它们可以构成其他方法的起始材料，如引物步移或作为诊断探针。(C)2010爱思唯尔有限公司版权所有。
Double-stranded RNA (dsRNA) is in many cases the only available template for molecular and diagnostic studies of RNA viruses. A novel mycovirus with a five dsRNAs segmented-genome served as a model system for the amplification and cloning of dsRNA segments using several PCR-based methods. Sequences obtained by the classical method: random PCR (rPCR) with a single primer assembled into 4 contigs out of the 5 segments. Moreover, using a modified single primer amplification technique (SPAT) resulted in the amplification of all or part of the dsRNA segments in one RT-PCR. Introducing such modifications into the FLAC method (full-length amplification of cDNA) resulted in amplicons comparable to those of the SPAT method. Full-length PCR products representing the five genomic segments were cloned and sequenced. The optimized conditions for each method are presented and discussed. In another approach, purified dsRNA segments were cloned directly into the blunt end pJET1.2 or the pGEM (R)-T cloning vectors with low efficiency though. This led to several sequences up to 2.2 kb in length, which could constitute a starting material for other methods like primer walking or as probes for diagnosis. (C) 2010 Elsevier B.V. All rights reserved.