ABSTRACT
This chapter reviews the mechanisms of viroid strain separation by return-polyacrylamide gel electrophoresis (R-PAGE) and temperature-gradient gel electrophoresis (TGGE) in view of viroid and RNA secondary structure. R-PAGE is a bidirectional electrophoretic procedure used to isolate viroids by exploiting their unique circular nature. Five functional domains have been proposed in native viroids. These include the conserved central region, the flanking pathogenicity and variable domains, and left and right terminal loops, which correspond in part to physical domains of the native viroid molecule. Nucleic acid samples are applied in a broad slot at one end of the gel and parallel to the temperature gradient. Experiments with gel-purified viroid preparations showed that strains exhibited differential mobilities in unidirectional electrophoresis under nondenaturing conditions, but that the was abolished under denaturing conditions. These experiments showed that viroid strain separation actually occurred during the initial nondenaturing run in R-PAGE.
