DNA-lesion mapping in mammalian cells.
Formation of DNA damage is a crucial event in carcinogenesis. Irreparable DNA lesions have the potential to cause mispairing during DNA replication, thereby giving rise to mutations. Critically important mutations in cancer-related genes, i.e., oncogenes and tumor suppressor genes, are key contributors to carcinogenesis. Theoretically, co-localization(s) of persistent DNA lesions and mutational hotspots in cancer-relevant genes can be used for causality inference. The inferred causality can be validated if a suspected carcinogen can similarly produce corresponding patterns of DNA damage and mutagenesis in vitro and/or in vivo. DNA-lesion footprinting (mapping) in conjunction with mutagenicity analysis is used for investigating cancer etiology. Ligation-mediated polymerase chain reaction (LM-PCR) is a versatile DNA-lesion footprinting technique, which enables sensitive and specific detection of DNA damage, at the level of nucleotide resolution, in genomic DNA. Here, we describe an updated protocol for LM-PCR analysis of the mammalian genome. This protocol can routinely be used for DNA-lesion footprinting of a variety of chemical and/or physical carcinogens in mammalian cells.
Besaratinia
Ahmad
A
Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, CA 91010, USA. ania@coh.org
Pfeifer
Gerd P
GP
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D002273:Carcinogens / Q000633:toxicity; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced / Q000235:genetics; D002477:Cells; D004247:DNA / Q000235:genetics; D018736:DNA Adducts / Q000032:analysis; D004249:DNA Damage; D018983:DNA Footprinting / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D016158:Genes, p53; D015894:Genome, Human; D006801:Humans; D008322:Mammals; D016296:Mutagenesis; D009154:Mutation / Q000187:drug effects; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011740:Pyrimidine Dimers / Q000528:radiation effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic; D014466:Ultraviolet Rays
D002273:Carcinogens; D018736:DNA Adducts; D017931:DNA Primers; D011740:Pyrimidine Dimers; D004247:DNA; D011088:DNA Ligases