Location: Molecular Plant Pathology LaboratoryTitle: Cloning and sequencing of viroids
Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 7/9/2020
Publication Date: 11/30/2021
Citation: Hammond, R. 2021. Cloning and sequencing of viroids. Methods in Molecular Biology. 2316:237-242. https://doi.org/10.1007/978-1-0716-1464-8_20.
Interpretive Summary: Vioids are the smallest known infectious agents of plant diseases and are pathogens of food, industrial, and ornamental plants worldwide. Yield losses caused by viroids (small noncoding circular infectious RNAs) can reach 17-64% depending on the viroid strain and plant crop species. Sequence characterization of known and newly discovered viroids is critical to disease management and global trade, and has been facilitated by the ability to convert viroid RNAs into complementary DNAs. This chapter describes two easily performed methods to clone viroid RNAs and is intended as a reference for beginners in the field. This work will be of value to an international audience of researchers in industry, academia, and government organizations with an interest in plant pathology and virology and the detection and control of plant diseases.
Technical Abstract: Determining the sequence identity of viroid RNAs present in symptomatic or asymptomatic plant tissues is critical to obtaining knowledge of their distribution and 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.