A new technique for determining the distribution of N7-methylguanine using an automated DNA sequencer.
We have developed a method to determine rapidly the sequence specificity of DNA alkylation resulting from chemical treatment. The utility of this approach is demonstrated here in a study of the sequence specificity of alkylation by dimethylsulphate (DMS). The method is independent of the sequence chosen and makes use of the polymerase chain reaction (PCR) to generate a fluorescently labelled DNA target. In this study, a 302 bp segment of the Escherichia coli lacI gene was amplified and the product purified by liquid chromatography on a Mono Q column. This DNA was alkylated with DMS and treated with hot piperidine to produce single-strand breaks at sites of N7 alkylation. The distribution of the break points, and hence the position and extent of alkylation, were determined on an Applied Biosystems 370A automated DNA sequencer.
Shoukry
S
S
Biology Department, York University, North York, Ontario, Canada.
Anderson
M W
MW
Glickman
B W
BW
D000478:Alkylation; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004926:Escherichia coli; D005784:Gene Amplification; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000096:biosynthesis; D010880:Piperidines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D013463:Sulfuric Acid Esters / Q000494:pharmacology
D009841:Oligonucleotides; D010880:Piperidines; D013463:Sulfuric Acid Esters; D006147:Guanine; C008450:7-methylguanine; C032727:piperidine; D004247:DNA; C007482:dimethyl sulfate