Weng Z, Xiong Z
Department of Plant Pathology, University of Arizona, Tucson 85721, USA.
Genome Res. 1995 Sep;5(2):202-7. doi: 10.1101/gr.5.2.202.
A combination of ligation-anchored PCR and anchored cDNA cloning techniques were used to clone the termini of the saguaro cactus virus (SCV) RNA genome. The terminal sequences of the viral genome were subsequently determined from the clones. The 5' terminus was cloned by ligation-anchored PCR, whereas the 3' terminus was obtained by a technique we term anchored cDNA cloning. In anchored cDNA cloning, an anchor oligonucleotide was prepared by phosphorylation at the 5' end, followed by addition of a dideoxynucleotide at the 3' end to block the free hydroxyl group. The 5' end of the anchor was subsequently ligated to the 3' end of SCV RNA. The anchor-ligated, chimerical viral RNA was then reverse-transcribed into cDNA using a primer complementary to the anchor. The cDNA containing the complete 3'-terminal sequence was converted into ds-cDNA, cloned, and sequenced. Two restriction sites, one within the viral sequence and one within the primer sequence, were used to facilitate cloning. The combination of these techniques proved to be an easy and accurate way to determine the terminal sequences of SCV RNA genome and should be applicable to any other RNA molecules with unknown terminal sequences.
采用连接锚定PCR和锚定cDNA克隆技术相结合的方法，克隆了巨人柱仙人掌病毒（SCV）RNA基因组的末端。随后从克隆中确定了病毒基因组的末端序列。5′末端通过连接锚定PCR克隆，而3′末端则通过一种我们称为锚定cDNA克隆的技术获得。在锚定cDNA克隆中，通过在5′末端磷酸化制备一个锚定寡核苷酸，随后在3′末端添加一个双脱氧核苷酸以封闭游离羟基。锚定的5′末端随后连接到SCV RNA的3′末端。然后使用与锚定互补的引物将连接了锚定的嵌合病毒RNA逆转录为cDNA。将包含完整3′末端序列的cDNA转化为双链cDNA，进行克隆和测序。使用病毒序列内的一个限制性酶切位点和引物序列内的一个限制性酶切位点来促进克隆。这些技术的结合被证明是一种确定SCV RNA基因组末端序列的简便而准确的方法，并且应该适用于任何其他末端序列未知的RNA分子。
