Cotton R G, Wright P J
Olive Miller Protein Laboratory, Murdoch Institute, Royal Children's Hospital, Australia.
J Virol Methods. 1989 Oct;26(1):67-76. doi: 10.1016/0166-0934(89)90075-x.
Osmium tetroxide and hydroxylamine (in combination with piperidine) have previously been shown to cleave mismatched T and C bases, respectively, in DNA.DNA heteroduplexes. In this work we report that mismatched T and C bases were similarly cleaved in DNA.RNA heteroduplexes of nucleic acids derived from different strains of dengue virus type 2. Further, some matched T or C bases one or two bases from mismatches were also chemically reactive and thus cleavable as detected by minor bands. Cleavages both at and near mismatches combined to generate a simply obtained pattern of difference between virus strains that could be used as a fingerprint of a given virus relative to another. The patterns obtained using viral RNA of one strain hybridized with the cDNA of another were similar for RNA prepared from purified virions and for total RNA extracted from infected cells. Use of probes of both senses should detect all differences. Two sequenced (NGC and PUO-218) and one unsequenced (D80-100) strains of virus were compared in these studies. The analyses allowed proof reading of the differences between NGC and PUO-218, ascertained from nucleotide sequencing, and demonstrated that D80-100 is more similar to PUO-218 than to NGC.
四氧化锇和羟胺（与哌啶联用）先前已被证明可分别切割DNA-DNA异源双链体中错配的T和C碱基。在本研究中，我们报告称，在源自2型登革病毒不同毒株的核酸的DNA-RNA异源双链体中，错配的T和C碱基也会被类似地切割。此外，一些与错配碱基相隔一两个碱基的匹配T或C碱基也具有化学反应活性，因此可通过小条带检测到被切割。错配处及其附近的切割共同产生了一种易于获得的病毒株间差异模式，可作为一种给定病毒相对于另一种病毒的指纹图谱。使用一种毒株的病毒RNA与另一种毒株的cDNA杂交获得的模式，对于从纯化病毒粒子制备的RNA和从感染细胞中提取的总RNA来说是相似的。使用两种方向的探针应能检测到所有差异。在这些研究中，对两种已测序（NGC和PUO-218）和一种未测序（D80-100）的病毒毒株进行了比较。这些分析对从核苷酸测序确定的NGC和PUO-218之间的差异进行了校正，并证明D80-100与PUO-218的相似性高于与NGC的相似性。
