Extra-long PCR, an identifier of DNA adducts in single nematodes (Caenorhabditis elegans).
DNA adducts are frequently caused by chemical induced changes in DNA. If mis-repaired, they can lead to nucleotide substitutions, deletions or chromosomal rearrangements. Depending on adduct stereochemistry and properties of the DNA target, adducts can inhibit transcriptional mechanisms. Here we demonstrate how this phenomenon can be exploited to detect DNA adducts in individual nematodes (Caenorhabditis elegans). An extra-long (XL)-PCR (16,144 bp) target amplicon, the 11 exon spanning ced-1, could be amplified reliably from genomic lysate extracted from single nematodes. Amplification efficiency was assessed by means of a second, fully quantitative PCR. Following the normalization with an invariant control gene, adduct formation could be evaluated by the identification of XL-PCR amplifications that were, relative to the control gene, reduced or inhibited by >95%. No DNA adducts could be detected in C. elegans maintained under optimal growth conditions (no exposure controls) or nematodes exposed to 20 microg/g copper sulfate (exposure negative control). However, exposure to 5 mug/g benzo[a]pyrene induced a stark response, with 40% of nematodes displaying measurable DNA adducts. Similarly, adducts were identified in 10% of nematodes subjected to 3 microg/g fluoranthene or a mixture containing 0.5 microg/g benzo[a]pyrene and 1 microg/g fluoranthene.
Neher
Deborah A
DA
Department of Plant and Soil Science, University of Vermont, Burlington, VT 05405, USA.
Stürzenbaum
Stephen R
SR
D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity; D015415:Biomarkers / Q000032:analysis; D017173:Caenorhabditis elegans / Q000235:genetics*; D002875:Chromosomes / Q000235:genetics / Q000648:ultrastructure; D019327:Copper Sulfate / Q000633:toxicity; D018736:DNA Adducts / Q000032:analysis* / Q000235:genetics*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000633:toxicity; D005091:Exons / Q000235:genetics; D005449:Fluorenes / Q000633:toxicity; D016296:Mutagenesis / Q000187:drug effects; D009153:Mutagens / Q000633:toxicity; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D015415:Biomarkers; D018736:DNA Adducts; D017931:DNA Primers; D004791:Enzyme Inhibitors; D005449:Fluorenes; D009153:Mutagens; D001564:Benzo(a)pyrene; C007738:fluoranthene; D019327:Copper Sulfate