Published: August 27, 2019
Leona D. Scanlan, Pawel Jaruga, Sanem Hosbas Coskun, Christopher M. Sims, Shannon Hanna, Jamie L. Almeida, David N. Catoe, Bryant C. Nelson, M Miral Dizdar, Erdem Coskun
Caenorhabditis elegans (C. elegans) is used as a medical and systems toxicity model organism for environmental and developmental assays that include high-throughput methods and genetic studies. However, little is known about background levels of oxidatively induced DNA damage in the nematode or how culturing methods may affect DNA damage levels. Furthermore, C. elegans physiology makes it difficult to extract large amounts (> 50 µg) of high-quality genomic DNA. We therefore developed and optimized a DNA extraction method based on enzymatic digestion and high-salt phase separation that resulted in efficient extraction of > 50 µg DNA from 250000 to 500000 nematodes. Extracted DNA showed suitable RNA levels (< 10 %), functionality in polymerase chain reaction assays and reproducible fragmentation. The high-salt method and a phenol/chloroform method were used to extract DNA from nematodes grown in two liquid growth media and from irradiated nematodes. DNA yields from the high-salt and phenol/chloroform extraction methods were similar; however, high-salt extracts contained significantly more protein contamination. We utilized gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution to measure DNA base lesions in the extracted DNA. Lesion levels were similar in each liquid culture and were significantly decreased in high-salt extracts compared to previously published levels that utilized different extraction methods. The high-salt extracts also exhibited lower lesion levels than phenol/chloroform extracts; phenol caused a significant increase in 8-hydroxyguanine levels, a known effect, and, reported here for the first time, increases in 8-hydroxyadenine, R- and S-8,5'-cyclo-2'-deoxyguanosines, 2,6-diamino-4-hydroxy- 5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine. The high-salt DNA extraction method minimized artifactual DNA damage and is therefore ideal for molecular and toxicological studies where oxidative stress is under investigation.
Citation: Analytical Chemistry
Pub Type: Journals
Created August 27, 2019, Updated October 08, 2019