Construction of full-length cDNA copies of viral double-stranded RNA.
Construction of full-length cDNA copies of viral double-stranded RNA.
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DOI：
10.1007/bf00572703
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发表时间：
1988-06-01
期刊：
影响因子：
1.6
通讯作者：
Bruenn, J A
中科院分区：
文献类型：
--
作者：
Nemeroff, M E;Pietras, D F;Bruenn, J A
描述了一种用于构建dsRNA的全长cDNA克隆的方法。所有dsRNA病毒都有一个与衣壳蛋白相关的转录酶，负责合成正链，然后从病毒颗粒中挤出。我们已经在体外转录合成的分段酿酒酵母病毒（ScV）作为模板的第一链cDNA合成。通过33个碱基的合成寡核苷酸引发合成。它含有与来自一个ScV病毒dsRNA片段（S14）的正链的3'端互补的27个核苷酸，以及在5'端编码XbaI限制性位点的6个额外核苷酸。使用类似的XbaI接头合成寡核苷酸合成第二条cDNA链，并通过标准连接技术克隆ds cDNA。通过序列分析表征的所有四种cDNA质粒分离物均含有S14的完整5'末端序列。其中两个在3'端是完整的，一个在这里缺少一个基地。在这四个克隆中，有一个还保留了两端的XbaI位点。通过使用合成寡核苷酸制备具有独特限制性位点接头的全长cDNA克隆允许更容易地筛选完整cDNA克隆，如果载体和cDNA都不具有所选择的限制性位点的话。它还为以后的研究提供了更容易的序列分析和基因组操作，例如克隆到表达载体中。该方法比任何先前描述的用于生产全尺寸cDNA克隆的方法更有效。
A method is described for the construction of full-length cDNA clones of dsRNAs. All dsRNA viruses have a capsid-associated transcriptase that is responsible for synthesis of the plus strand that is then extruded from viral particles. We have used in vitro transcripts synthesized by the segmented Saccharomyces cerevisiae virus (ScV) as templates for first-strand cDNA synthesis. Synthesis was primed by a 33-base synthetic oligonucleotide. This contained 27 nucleotides complementary to the 3' end of the plus strand from one ScV viral dsRNA segment (S14), and 6 additional nucleotides encoding an XbaI restriction site at the 5' end. The second cDNA strand was synthesized using a similar XbaI linker-synthetic oligonucleotide and the ds cDNA was cloned by standard ligation techniques. All four cDNA plasmid isolates characterized by sequence analysis contained the complete 5' end sequence of S14. Two of these were complete at the 3' end, and one lacked a single base here. Of these four clones, one also retained the XbaI sites at either end. Preparing full-length cDNA clones with unique restriction-site linkers by the use of synthetic oligonucleotides allows for easier screening for complete cDNA clones if neither the vector nor the cDNA has the chosen restriction site. It also provides for easier sequence analysis and manipulation of the genome for later studies, such as cloning into expression vectors. This method is more efficient than any previously described for production of full-sized cDNA clones.