Abstract
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.
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References
Owens RA (1978) In vitro synthesis and characterization of DNA complementary to potato spindle tuber viroid. Virology 89:380–387
Palukaitis P, Symons RH (1978) Synthesis and characterization of a complementary DNA probe for chrysanthemum stunt viroid. FEBS Lett 92:262–272
Owens RA, Diener TO (1981) Sensitive and rapid diagnosis of potato spindle tuber viroid by nucleic acid hybridization. Science 213:670–672
Owens RA, Cress DE (1980) Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences. Proc Natl Acad Sci U S A 77:5302–5306
Navarro B, Daros JA, Flores R (1996) A general strategy for cloning viroids and other small circular RNAs that uses minimal amounts of template and does not require prior knowledge of its sequence. J Virol Methods 56:59–66
Verhoeven JTJ, Jansen CCC, Willemen TM, Kox LFF, Owens RA, Roenhorst JW (2004) Natural infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. Eur J Plant Pathol 110:823–831
Yoon J-H, Cho I-S, Choi G-S, Choi S-K (2014) Construction of an infectious cDNA clone of a Chrysanthemum stunt viroid Korean isolate. Plant Pathol J 30:68–74
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 574.87324 1/1989
Verhoeven JTJ, Meekes ETM, Roenhorst JW, Flores R, Serra P (2013) Dahlia latent viroid: a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification. J Gen Virol 94:711–719
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Hammond, R.W. (2022). Cloning and Sequencing of Viroids. In: Rao, A.L.N., Lavagi-Craddock, I., Vidalakis, G. (eds) Viroids. Methods in Molecular Biology, vol 2316. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1464-8_20
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DOI: https://doi.org/10.1007/978-1-0716-1464-8_20
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