Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences

Open Access
Publisher Website
Google Scholar
Abstract
We have established a sensitive, monoclonal antibody (Mab)-based procedure permitting the selective enrichment of sequences containing the miscoding alkylation product O 6 -ethylguanine (O 6 -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 ≥25 O 6 -EtGua residues per diploid genome (corresponding to O 6 -EtGua/guanine molar ratios of ≥10 −8 ). For analysis, enzymatically restricted DNA from these cells is incubated with an antibody specific for O 6 -ethyl-2′deoxyguanosine, the resulting Mab-DNA complexes are separated from (O 6 -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 O 6 -EtGua is constant over a range of O 6 -EtGua/guanine molar ratios between 10 −5 and 10 −8 . (O 6 -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 hybrldisation. 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.