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Measurement of Oxidatively Induced DNA Damage in Caenorhabditis elegans with High-Salt DNA Extraction and Isotope-Dilution Mass Spectrometry.
Scanlan, Leona D; Coskun, Sanem Hosbas; Jaruga, Pawel; Hanna, Shannon K; Sims, Christopher M; Almeida, Jamie L; Catoe, David; Coskun, Erdem; Golan, Rachel; Dizdaroglu, Miral; Nelson, Bryant C.
Afiliación
  • Scanlan LD; Material Measurement Laboratory - Biomolecular Measurement Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Coskun SH; Material Measurement Laboratory - Biosystems and Biomaterials Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Jaruga P; Gazi University, Faculty of Pharmacy , Ankara , 06330 , Turkey.
  • Hanna SK; Material Measurement Laboratory - Biomolecular Measurement Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Sims CM; Material Measurement Laboratory - Biosystems and Biomaterials Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Almeida JL; Material Measurement Laboratory - Biosystems and Biomaterials Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Catoe D; Material Measurement Laboratory - Biosystems and Biomaterials Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Coskun E; Material Measurement Laboratory - Biosystems and Biomaterials Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Golan R; Material Measurement Laboratory - Biomolecular Measurement Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Dizdaroglu M; Material Measurement Laboratory - Biomolecular Measurement Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
  • Nelson BC; Material Measurement Laboratory - Biomolecular Measurement Division , National Institute of Standards and Technology , Gaithersburg , Maryland 20899 , United States.
Anal Chem ; 91(19): 12149-12155, 2019 10 01.
Article en En | MEDLINE | ID: mdl-31454479
Caenorhabditis elegans is used extensively as a medical and toxicological model organism. However, little is known about background levels of oxidatively induced DNA damage in the nematode or how culturing methods affect DNA damage levels. The tough C. elegans cuticle makes it challenging to extract genomic DNA without harsh procedures that can artifactually increase DNA damage. Therefore, a mild extraction protocol based on enzymatic digestion of the C. elegans cuticle with high-salt phase-separation of DNA has been developed and optimized. This method allows for efficient extraction of >50 µg DNA using a minimum of 250000 nematodes grown in liquid culture. The extracted DNA exhibited acceptable RNA levels (<10% contamination), functionality in polymerase chain reaction assays, and reproducible DNA fragmentation. Gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution measured lower lesion levels in high-salt extracts than in phenol extracts. Phenolic extraction produced a statistically significant increase in 8-hydroxyguanine, a known artifact, and additional artifactual increases in 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine, and 8-hydroxyadenine. The high-salt DNA extraction procedure utilizes green solvents and reagents and minimizes artifactual DNA damage, making it more suitable for molecular and toxicological studies in C. elegans. This is, to our knowledge, the first use of GC-MS/MS to measure multiple 8,5'-cyclopurine-2'-deoxynucleosides in a toxicologically important terrestrial organism.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Daño del ADN / Caenorhabditis elegans / ADN de Helmintos / Fraccionamiento Químico Tipo de estudio: Prognostic_studies Límite: Animals / Female / Humans Idioma: En Revista: Anal Chem Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Daño del ADN / Caenorhabditis elegans / ADN de Helmintos / Fraccionamiento Químico Tipo de estudio: Prognostic_studies Límite: Animals / Female / Humans Idioma: En Revista: Anal Chem Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos