Wang Xiaohong, Zheng Zhi-Ming
Tumor Virus RNA Biology Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland.
Curr Protoc Microbiol. 2016 Feb 8;40:14B.6.1-14B.6.29. doi: 10.1002/9780471729259.mc14b06s40.
Papillomaviruses are a family of small, non-enveloped DNA tumor viruses. Knowing a complete transcription map of each papillomavirus genome can provide guidance for various papillomavirus studies. This unit provides detailed protocols to construct a transcription map of human papillomavirus type 18. The same approach can be easily adapted to other transcription map studies of any other papillomavirus genotype due to the high degree of conservation in genome structure, organization, and gene expression among papillomaviruses. The focused methods are 5'- and 3'-rapid amplification of cDNA ends (RACE), which are techniques commonly used in molecular biology to obtain full-length RNA transcript or to map a transcription start site (TSS) or an RNA polyadenylation (pA) cleavage site. Primer walking RT-PCR is a method for studying the splicing junction of RACE products. In addition, RNase protection assay and primer extension are also introduced as alternative methods in the mapping analysis.
乳头瘤病毒是一类小型无包膜的DNA肿瘤病毒。了解每种乳头瘤病毒基因组的完整转录图谱可为各种乳头瘤病毒研究提供指导。本单元提供了构建人乳头瘤病毒18型转录图谱的详细方案。由于乳头瘤病毒在基因组结构、组织和基因表达方面具有高度保守性，相同的方法可轻松适用于任何其他乳头瘤病毒基因型的其他转录图谱研究。重点方法是5'和3' cDNA末端快速扩增（RACE），这是分子生物学中常用的技术，用于获得全长RNA转录本或绘制转录起始位点（TSS）或RNA聚腺苷酸化（pA）切割位点。引物步移RT-PCR是一种研究RACE产物剪接连接的方法。此外，核糖核酸酶保护分析和引物延伸也作为定位分析中的替代方法被引入。
