Single-strand conformation polymorphism (SSCP) analysis as a tool for viroid characterisation.
The potential of routine single-strand conformation polymorphism (SSCP) analysis for viroid characterization has been evaluated. Electrophoresis of 311 cloned full length viroid DNA inserts recovered from a field isolate of citrus exocortis viroid (CEVd) showed shifts in the migration of the cDNA or/and hDNA strands in non-denaturing 14% polyacrylamide gels. Using a single set of electrophoresis conditions seven different groups of variants (containing one to six changes), which did not represent the overall variability among clones, were identified. The relationship between the different SSCP profiles observed among clones and the variation in their nucleotide sequences was confirmed by sequence analysis. Variations in the dimensions of the gel allowed higher resolution and therefore the detection of additional single nucleotide variations among clones initially clustered into the same group. The viroid region affected by specific changes could be established by SSCP analysis of partial viroid length DNA. The potential use of SSCP analysis as a tool to screen existing viroid populations in infected hosts prior to sequencing is discussed.
Palacio
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Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain.
Duran-Vila
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D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005638:Fruit / Q000821:virology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010942:Plant Viruses / Q000235:genetics* / Q000302:isolation & purification*; D018807:Polymorphism, Single-Stranded Conformational; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014772:Viroids / Q000235:genetics* / Q000302:isolation & purification
D004279:DNA, Viral