TY - JOUR
T1 - A sample preparation protocol for quantification of radiolabeled nucleoside incorporation into DNA by accelerator mass spectrometry
AU - Hah, Sang Soo
AU - Mundt, Janna M.
AU - Ubick, Esther A.
AU - Turteltaub, Ken W
AU - Gregg, Jeffrey
AU - Henderson, Paul
PY - 2007/6
Y1 - 2007/6
N2 - A general protocol is described for measuring the incorporation of radiocarbon-labeled 2′-deoxynucleosides into DNA using accelerator mass spectrometry (AMS). This technology provides attomole (10-18 mol) sensitivity, with detection limits for DNA analysis in the range of one 14C atom per 1011-1012 total carbons. In practice this corresponds to approximately 1 labeled nucleoside per 1011 normal bases. A key aspect of the method is the use of precautions aimed at prevention of artifactual DNA oxidation during the sample preparation by the use of antioxidants and chaotropic salts during the DNA isolation. In principle, any type of appropriately labeled nucleoside derivative can be studied using the described protocol, provided that there is incorporation of the deoxynucleoside into DNA. We demonstrated this protocol using MCF-7 human breast cancer cells and a mouse model for mammary carcinoma, which we dosed with 14C-labeled 2′-deoxyguanosine (dG) and 14C-labeled 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). The nucleoside 8-oxodG is a ubiquitous compound that forms in cells by the reaction of dG with reactive oxygen species which has been associated with numerous disease, carcinogenesis and aging. DNA from cells treated with 14C-labeled nucleosides was isolated and prepared for analysis by AMS in order to measure the DNA-bound radioactivity. The method allows the generation of reliable and sufficient yields of pure DNA from human cells and animal tissues for analysis of radiocarbon levels. Ultimately, this protocol will be applied to understanding the role of modified nucleoside incorporation into DNA in cancer initiation and progression, but could also be used to study any DNA metabolism process where 14C-labeled nucleosides are used.
AB - A general protocol is described for measuring the incorporation of radiocarbon-labeled 2′-deoxynucleosides into DNA using accelerator mass spectrometry (AMS). This technology provides attomole (10-18 mol) sensitivity, with detection limits for DNA analysis in the range of one 14C atom per 1011-1012 total carbons. In practice this corresponds to approximately 1 labeled nucleoside per 1011 normal bases. A key aspect of the method is the use of precautions aimed at prevention of artifactual DNA oxidation during the sample preparation by the use of antioxidants and chaotropic salts during the DNA isolation. In principle, any type of appropriately labeled nucleoside derivative can be studied using the described protocol, provided that there is incorporation of the deoxynucleoside into DNA. We demonstrated this protocol using MCF-7 human breast cancer cells and a mouse model for mammary carcinoma, which we dosed with 14C-labeled 2′-deoxyguanosine (dG) and 14C-labeled 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). The nucleoside 8-oxodG is a ubiquitous compound that forms in cells by the reaction of dG with reactive oxygen species which has been associated with numerous disease, carcinogenesis and aging. DNA from cells treated with 14C-labeled nucleosides was isolated and prepared for analysis by AMS in order to measure the DNA-bound radioactivity. The method allows the generation of reliable and sufficient yields of pure DNA from human cells and animal tissues for analysis of radiocarbon levels. Ultimately, this protocol will be applied to understanding the role of modified nucleoside incorporation into DNA in cancer initiation and progression, but could also be used to study any DNA metabolism process where 14C-labeled nucleosides are used.
KW - Accelerator mass spectrometry (AMS)
KW - Carbon-14
KW - DNA damage
KW - DNA digestion
KW - DNA isolation
KW - Nucleoside incorporation
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U2 - 10.1016/j.nimb.2007.01.214
DO - 10.1016/j.nimb.2007.01.214
M3 - Article
AN - SCOPUS:34248208233
VL - 259
SP - 763
EP - 766
JO - Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms
JF - Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms
SN - 0168-583X
IS - 1
ER -