One-lane sequence analysis of oligodeoxyribonucleotides.
Treatment of 5'-end 32P-labeled oligodeoxyribonucleotides with 0.4 M aqueous piperidinium formate, pH 2, at 37 degrees C for 6 h, followed by treatment with 1 M aqueous piperidine at 90 degrees C for 6 h, produces, after electrophoresis through 27% polyacrylamide sequencing gels, one-dimensional distributions of radioactivity from which the base sequences can be deduced. The order of intensities for the bands signaling the various bases is G greater than A greater than C greater than T. The spacing from a given band to the next higher band in the ladder was base characteristic, the order of band spacings being G greater than T greater than or equal to A greater than C. In contrast to the one-cleavage one-lane DNA sequencing method reported earlier (B. J. B. Ambrose and R. C. Pless, 1985, Biochemistry 24, 6194-6200), which was based on treatment of end-labeled DNA with hot aqueous piperidine in the presence of sodium chloride, the present method produces a salt-free hydrolysate, thus minimizing electrophoretic irregularities in the fastest moving bands.
D001345:Autoradiography; D001483:Base Sequence; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D009838:Oligodeoxyribonucleotides; D010880:Piperidines; D011089:Polydeoxyribonucleotides
D009838:Oligodeoxyribonucleotides; D010880:Piperidines; D011089:Polydeoxyribonucleotides; C032727:piperidine; D004247:DNA