Detection of DNA adducts at the DNA sequence level by ligation-mediated PCR.
Many carcinogens and mutagens interact with DNA to form specific adducts. Base-specificity and sequence-specificity of adduct formation has been analyzed previously with cloned, end-labelled DNA fragments. However, the distribution of adducts along a mammalian chromosome may be modulated by chromatin structure and could be different from that in naked plasmid DNA. Recently, a method has been developed that utilizes the sensitivity of the polymerase chain reaction (PCR) to detect DNA adducts at the DNA sequence level in mammalian cells. The sequence position of adducts can be mapped whenever it is possible to convert the adduct, either chemically or enzymatically, into a DNA strand break with a 5'-phosphate group. Fragments containing these ligatable breaks are amplified in a single-sided, ligation-mediated PCR reaction. We have used ligation-mediated PCR for detection of alkylguanine adducts and UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. We discuss the sensitivity of the method, its limitations, and its potential for mapping other DNA adducts at the DNA sequence level in mammalian cells.
Pfeifer
G P
GP
Beckman Research Institute of the City of Hope, Department of Biology, Duarte, CA 91010.
Drouin
R
R
Holmquist
G P
GP
D000818:Animals; D004247:DNA / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D008024:Ligands; D008322:Mammals; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D014466:Ultraviolet Rays
D008024:Ligands; D004247:DNA