Analysis of DNA sequences using a single chemical cleavage procedure

• Published in: Biochemistry (Easton) Vol. 24; no. 22; pp. 6194 - 6200
• Main Authors: Ambrose, Barbara J. B, Pless, Reynaldo C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.10.1985
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Chemical Phenomena; Chemistry; Densitometry; DNA - genetics; Dna, deoxyribonucleoproteins; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Hydrolysis; Indicators and Reagents; Nucleic acids; Plasmids; Primary structure; Method; Nucleotide sequence; DNA; Electrophoretic mobility; Nucleotide fragment
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00343a025

A novel approach to sequence analysis of end-labeled, defined DNA fragments, using a single chemical cleavage procedure and electrophoretic separation in a single lane, has been developed. Prolonged treatment with hot aqueous piperidine results in partial cleavage of the DNA at all positions; the relative propensity for this cleavage is different for the various bases in the DNA. The hydrolysate is resolved on a DNA sequencing gel, and the distribution of radioactivity in the electrophoretic lane is analyzed (a) in terms of differential peak heights of the radioactive bands and (b) in terms of the spacings between successive bands. Simultaneous application of these two base-characteristic criteria allows the deduction of the nucleotide sequence with an accuracy approaching that of the established four-lane methods of DNA sequencing.