Abstract
Accelerator mass spectrometry (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. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 147-157 |
| Number of pages | 11 |
| Journal | Methods in molecular biology (Clifton, N.J.) |
| Volume | 1105 |
| DOIs | |
| State | Published - 2014 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Medicine(all)
Cite this
DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry. / Dingley, Karen H.; Ubick, Esther A.; Vogel, John S.; Ognibene, Ted J.; Malfatti, Michael A.; Kulp, Kristen; Haack, Kurt W.
In: Methods in molecular biology (Clifton, N.J.), Vol. 1105, 2014, p. 147-157.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.
AU - Dingley, Karen H.
AU - Ubick, Esther A.
AU - Vogel, John S.
AU - Ognibene, Ted J.
AU - Malfatti, Michael A.
AU - Kulp, Kristen
AU - Haack, Kurt W.
PY - 2014
Y1 - 2014
N2 - Accelerator mass spectrometry (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. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.
AB - Accelerator mass spectrometry (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. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.
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U2 - 10.1007/978-1-62703-739-6_12
DO - 10.1007/978-1-62703-739-6_12
M3 - Article
VL - 1105
SP - 147
EP - 157
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
SN - 1064-3745
ER -