Solid-phase methods for sequencing nucleic acids. III. Simultaneous sequencing of different long RNA fragments using DE 81 anion-exchange paper.
A solid-phase method for simultaneous sequencing of different long RNA fragments has been developed using Whatman DE 81 anion-exchange paper as the support. The approach involves 8 operations including: immobilization of heat-denatured 3'-end labeled RNA fragment on DE 81 paper; washing; modification reactions; washing; aniline reaction; washing; RNA desorption by salt and ethanol precipitation. For modifying the RNA, the following reactions were selected for the routine: G with dimethylsulfate at 90 degrees C/sodium borohydride at 0 degrees C, A + G with diethylpyrocarbonate at 90 degrees C, U + C with hydrazine at 0 degrees C and C with hydrazine/5M NaCl at 0 degrees C. The losses of RNA material during the reactions with large excess of reactants were 50% during the reduction with NaBH4 and 30% during C-reaction. Almost no losses were observed during aniline reaction. The RNA could be recovered by desorption with 2M NaClO4 in 50-70% yield. The whole solid-phase procedure up to the sequencing gel takes about 2 hours and is much faster and more convenient than chemical RNA sequencing in solution according to Peattie, especially if many fragments are to be processed.
D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D002854:Chromatography, Paper / Q000379:methods; D008099:Liver / Q000032:analysis; D009843:Oligoribonucleotides; D012335:RNA, Ribosomal; D051381:Rats; D012270:Ribosomes / Q000032:analysis
D009843:Oligoribonucleotides; D012335:RNA, Ribosomal