Lambden P R, Cooke S J, Caul E O, Clarke I N
Department of Microbiology, University of Southampton Medical School, Southampton General Hospital, United Kingdom.
J Virol. 1992 Mar;66(3):1817-22. doi: 10.1128/JVI.66.3.1817-1822.1992.
A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.
已开发出一种新型的、不依赖序列的策略，用于扩增双链RNA（dsRNA）病毒基因的全长cDNA拷贝。以人（布里斯托尔）C组轮状病毒为例，使用T4 RNA连接酶将单个氨基连接的修饰寡核苷酸（引物1）连接到每个dsRNA基因组片段的两端。在进行逆转录、cDNA链退火和修复后，使用单个互补寡核苷酸（引物2）通过聚合酶链反应完成病毒dsRNA基因组的扩增。cDNA与病毒dsRNA的Northern（RNA）杂交表明，从非常小的临床样本中有可能生成代表完整基因组的cDNA。该技术用于确定基因10的完整核苷酸序列（728 bp）和编码分配，结果显示有一个212个氨基酸的开放阅读框，与人A组轮状病毒的NS26有有限的同源性。与以前的加尾方法不同，添加一个确定的引物可明确鉴定末端核苷酸，并且一般应适用于具有分段dsRNA基因组的病毒，特别是用于分析临床样本，因为临床样本中可获得的生物材料数量非常有限。
