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Gel dependence of electrophoretic mobilities of double‐stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis
Author(s) -
Gast FrankUlrich,
Sänger Heinz Ludwig
Publication year - 1994
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501501213
Subject(s) - viroid , electrophoresis , rna , dna , gel electrophoresis of nucleic acids , gel electrophoresis , agarose , agarose gel electrophoresis , base pair , rna silencing , nucleic acid , chemistry , microbiology and biotechnology , chromatography , biophysics , biology , biochemistry , gene , rna interference
Abstract Double‐stranded (ds) RNA normally exhibits a lower electrophoretic mobility than dsDNA having the same number of base pairs. This has been attributed to its net charge density that is lower than that of B‐form DNA. But we show here that dsRNA runs faster than corresponding DNA in gels containing either ≥ 2.5% agarose or ≥ 8% acrylamide with high crosslinking (19:1 acryl‐amide: N,N ′‐methylenebisacrylamide). However, the relative mobility of dsRNA as compared with DNA, extrapolated to 0% gel (0%T), remains constant (0.90 ± 0.03) in all systems, in support of the charge density hypothesis. In comparison to dsRNA standards, the potato spindle tuber viroid, a small ≈ 70% base‐paired rod‐like pathogenic RNA, is strongly retarded, presumably because of greater flexibility and/or stable curvature. Depending on the gel system, nonlinear extrapolation to 0% T leads to an apparent contour length of 140–230 bp, whereas 130 ± 20 bp can be determined from electron micrographs and 123–126 bp from secondary structure modeling. We attribute the variation of the electrophoretic behavior of both dsRNA and viroid RNA to interactions with the gel matrix. Nevertheless, extrapolation of the apparent contour length (in bp dsRNA) determined from low‐crosslinked polyacryl‐amide gels (2.6%C) is comparable to the determination by alternative methods.