Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences.
We have established a sensitive, monoclonal antibody (Mab)-based procedure permitting the selective enrichment of sequences containing the miscoding alkylation product O6-ethylguanine (O6-EtGua) from mammalian DNA. H5 rat hepatoma cells were reacted with the N-nitroso carcinogen N-ethyl-N-nitrosourea in vitro, to give overall levels of greater than or equal to 25 O6-EtGua residues per diploid genome (corresponding to O6-EtGua/guanine molar ratios of greater than or equal to 10(-8). For analysis, enzymatically restricted DNA from these cells is incubated with an antibody specific for O6-ethyl-2'-deoxyguanosine, the resulting Mab-DNA complexes are separated from (O6-EtGua)-free fragments by filtration through a nitrocellulose (NC) membrane, and the DNA is recovered from the filter-bound complexes quantitatively. The efficiency of Mab binding to DNA fragments containing O6-EtGua is constant over a range of O6-EtGua/guanine molar ratios between 10(-5) and 10(-8). (O6-EtGua)-containing restriction fragments encompassing known gene sequences (e.g., the immunoglobulin E heavy chain gene of H5 rat hepatoma cells used as a model in this study) are subsequently amplified by PCR and quantified by slot-blot hybridisation. The content and distribution of a specific carcinogen-DNA adduct in defined sequences of genomic DNA can thus be analyzed as well as the kinetics of intragenomic (toposelective) repair of any DNA lesion for which a suitable Mab is available.
Hochleitner
K
K
Institute of Cell Biology (Cancer Research), University of Essen Medical School, FRG.
Thomale
J
J
Nikitin AYu
Rajewsky
M F
MF
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification*; D004249:DNA Damage / Q000235:genetics; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D005038:Ethylnitrosourea / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; C018534:O(6)-ethyl-2'-deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine; D005038:Ethylnitrosourea