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DNA Extraction and DNA Damage Measurement in the Nematode Caenorhabditis elegans

Published

Author(s)

Leona D. Scanlan, Pawel Jaruga, Sanem Hosbas Coskun, Jamie L. Almeida, David N. Catoe, Jennifer McDaniel, Miral M. Dizdar

Abstract

Little is known about endogenous DNA damage in the nematode. In this work, we standardized the growth of the nematode in two different growth media (axenic CeHR supplemented with 20% milk and S-basal with E. coli), and developed a novel high-salt, phenol-free DNA extraction protocol. We extracted DNA from aliquots of worms grown in both media with both the high-salt method and with a phenol-based extraction. We then used gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope dilution to measure the levels of four oxidatively-induced DNA lesions [5-hydroxy-5-methylhydantoin (5-OH-5-MeHyd), 4,6-diamino-5-formamidopyrimidine (FapyAde), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), and 8-hydroxyguanine (8-OH-Gua)] and R- and S-diastereomers of two 8,5'-cyclopurine 2'-deoxynucleoside lesions: (5'R)- and (5'S)-8,5'-cyclo-2'-deoxyadenosine (R-cdA and S-cdA) and (5'R)- and (5'S)-8,5′-cyclo-2′-deoxyguanosine (R-cdG and S-cdG). We also exposed aliquots of nematodes to 10 Gy ionizing radiation, and measured the DNA lesions in those samples. The nematode grows faster in S-basal (3.5 days from egg to reproducing adult in S-basal as compared to 5 days in CeHR) but is able to reach a higher density in CeHR (80 nematodes per µL in CeHR versus 35 per µL in S-basal). DNA extraction showed no significant difference between the amount of DNA (ng DNA per 1000 nematodes) extracted using the high-salt method as compared to the phenol method (p-value = 0.243). Significantly more DNA (p-value <0.0001) was extracted from mixed-stage nematodes grown in CeHR medium compared to S-basal: extracts from nematodes grown in CeHR averaged 547 ng/1000 nematodes, while S-basal averaged 90 ng/ 1000 nematodes. We are currently performing digital PCR on DNA extracts to determine the purity and verify identity of the DNA (bacteria, bovine or nematode) and running pulse-field gels to analyze the size of fragmented DNA in the DNA extracts. Initial lesion analysis shows the presence of
Proceedings Title
Society of Environmental Toxicologists and Chemists North American chapter
Conference Dates
November 1-5, 2015
Conference Location
Salt LAke City, UT, US
Conference Title
SETAC North America 36th nnual Meeting

Keywords

C. elegans, DNA extraction, DNA damage, mass spectrometry

Citation

Scanlan, L. , Jaruga, P. , Hosbas Coskun, S. , Almeida, J. , Catoe, D. , McDaniel, J. and Dizdar, M. (2015), DNA Extraction and DNA Damage Measurement in the Nematode Caenorhabditis elegans, Society of Environmental Toxicologists and Chemists North American chapter, Salt LAke City, UT, US (Accessed May 23, 2022)
Created October 31, 2015, Updated October 12, 2021