United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Molecular Plant Pathology Laboratory, Beltsville, MD, USA.
Methods Mol Biol. 2022;2316:237-242. doi: 10.1007/978-1-0716-1464-8_20.
Determining the sequence identity of viroid RNAs present in symptomatic or asymptomatic plant tissues is critical to obtain knowledge of their distribution. It enables the development of tools for diagnostics and for studying the basic biology of viroids. With the advent of cDNA-based methods for cloning RNAs and cloning strategies that do not require prior knowledge of the viroid sequence, characterization of several newly discovered viroids has rapidly expanded our knowledge of these unusual pathogenic RNAs. This chapter describes two methods, using random primers or viroid-specific primers, to generate complementary DNA (cDNA) copies of viroid RNAs for subsequent cloning and sequence analysis.
确定在有症状或无症状植物组织中存在的类病毒 RNA 的序列同一性对于获得其分布知识至关重要。这使得能够开发用于诊断和研究类病毒基本生物学的工具。随着基于 cDNA 的 RNA 克隆方法和不需要预先了解类病毒序列的克隆策略的出现，对几种新发现的类病毒的特征描述迅速扩展了我们对这些异常致病 RNA 的认识。本章描述了两种方法，使用随机引物或类病毒特异性引物，生成类病毒 RNA 的互补 DNA(cDNA)拷贝，以便随后进行克隆和序列分析。
