A microscale procedure for isolating and sequencing the viroid RNA present in one gram of infected leaf tissue.
A microscale procedure for the isolation and purification of viroid RNA from one gram of viroid-infected leaf tissue and for its subsequent sequencing at the cDNA level is described using potato spindle tuber viroid (PSTV) as model system. Total nucleic acids are phenol-extracted and salt-fractionated with 2 M LiCl. The viroid-containing fraction is then subjected to bidirectional polyacrylamide gel electrophoresis. This removes all co-fractionated cellular RNAs from the circular viroid RNA which is finally recovered from the gel in pure form by isotachophoresis. Thus, from one gram of PSTV-infected tomato leaf tissue, about 100-250 ng of circular PSTV RNA can be obtained and used as template for several DNA primer-directed reverse transcription reactions. From the primer-extended overlapping cDNAs the entire sequence of the viroid progeny synthesized in an individual plant or plant leaf can thus be established by Maxam-Gilbert sequencing. This renders the procedure especially suited for the routine analysis of the in vivo fate of viroid mutants constructed in vitro.
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Max-Planck-Institut für Biochemie, Planegg-Martinsried, F.R.G.
Günther
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Sänger
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HL
D001345:Autoradiography; D001483:Base Sequence; D015152:Blotting, Northern; D005591:Chemical Fractionation; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000382:microbiology*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014772:Viroids / Q000235:genetics*
D004279:DNA, Viral; D012367:RNA, Viral