DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.
A protocol is described for the isolation of DNA and subsequent preparation of samples for the measurement of adduct levels by accelerator mass spectrometry (AMS). AMS is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. However, special precautions must be taken to avoid cross-contamination of isotope between samples and to produce a sample that is compatible with AMS. The DNA isolation method described is based on digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen DNA isolation columns. DNA is then precipitated with isopropanol, washed repeatedly with 70% ethanol to remove salt, and then dissolved in water. This method has been used to generate reliably good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. For quantification of adduct levels from 14C-labeled compounds, DNA samples are then converted to graphite, and the 14C content is measured by AMS.
Dingley
Karen H
KH
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, CA, USA.
Ubick
Esther A
EA
Vogel
John S
JS
Haack
Kurt W
KW
D000818:Animals; D002250:Carbon Radioisotopes; D015197:Carcinogenicity Tests; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D018736:DNA Adducts / Q000032:analysis*; D004249:DNA Damage; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009152:Mutagenicity Tests
D002250:Carbon Radioisotopes; D018736:DNA Adducts; D004247:DNA