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.