Saleem M, Pelcher L E
Can J Biochem Cell Biol. 1985 May;63(5):382-6. doi: 10.1139/o85-055.
DNA oligomer directed ribonuclease H (RNase H) methodology is applied to specifically cleave tobacco mosaic virus (TMV) RNA. Using a synthetic DNA oligomer P(dT8)dCdC, complementary to a region from nucleotide 5545 to nucleotide 5554 at the 3' end of TMV RNA, we have cleaved the RNA at the site of polynucleotides complementary to the DNA oligomer. Factors such as secondary structure of the RNA, concentrations of DNA oligomer, RNase H and magnesium ions in the reaction mixture, and time of incubation were optimized for the RNase H cleavage of TMV RNA-DNA oligomer complex. Denaturation of TMV RNA with 50% dimethyl sulphoxide at 50 degrees C is essential for the site-specific cleavage.
DNA寡聚物导向的核糖核酸酶H（RNase H）方法被应用于特异性切割烟草花叶病毒（TMV）RNA。使用与TMV RNA 3'端核苷酸5545至核苷酸5554区域互补的合成DNA寡聚物P(dT8)dCdC，我们已在与DNA寡聚物互补的多核苷酸位点切割了RNA。对于TMV RNA-DNA寡聚物复合物的RNase H切割，优化了诸如RNA的二级结构、反应混合物中DNA寡聚物、RNase H和镁离子的浓度以及孵育时间等因素。在50℃用50%二甲基亚砜使TMV RNA变性对于位点特异性切割至关重要。
