Solid-phase methods for sequencing nucleic acids. VII. Chemical degradation of synthetic DNA-RNA hybrid fragments using CCS and DE 81 anion-exchange papers.
Synthetic oligo(ribo-deoxyribo)nucleotides were analyzed and characterized by different solid-phase chemical degradation procedures, 5'- and 3'-end labelled mixed fragments were degraded by a slightly modified DNA cleavage procedure using 1 and 10% piperidine for the chain scission reaction and CCS anion-exchange paper. Besides the normal degradation products obtained by the usual modification and strand cleavage reactions of both deoxy- and ribonucleotide residues, additional bands were identified in the sequence patterns resulting from the hydrolysis of the RNA moiety induced by piperidine. Since both degradation reactions cleave the backbone of the mixed DNA-RNA fragments differently and produce nucleotide components with different charges, the degradation products do not interfere and can be resolved by gel electrophoresis on polyacrylamide. In addition, 3'-end labelled DNA-RNA oligomers were degraded by a RNA cleavage procedure using DE 81 anion-exchange paper as solid support. The combination of all three degradation methods allows to confirm the nucleotide sequence.
D001483:Base Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D002854:Chromatography, Paper / Q000379:methods; D004247:DNA / Q000138:chemical synthesis*; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D012313:RNA / Q000138:chemical synthesis*
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D012313:RNA; D004247:DNA