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Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

Kiianitsa K, Maizels N - Nucleic Acids Res. (2014)

Bottom Line: This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells.Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay.The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.

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Related in: MedlinePlus

Validation of mini-scale DNA and DPCC isolation for high-throughput screening applications. (A) DNA yield from untreated CCRF-CEM cells lysed in LS1 containing 5 M GTC and adjusted to the indicated LiCl and GTC concentrations by addition of 8 M LiCl, and precipitated following addition of 1 volume isopropanol at low RCF; rightmost samples included 8 mg silica gel per well. (B) Efficiency of DNA recovery. DNA yield was determined from HCT116 cells which had been untreated (NT) or treated for 30 min with 10 μM CPT, then lysed in LS1 containing 2.5 M GTC, 4 M LiCl and 8 mg silica gel per well, and DNA recovered by precipitation with 1 volume isopropanol. The x-axis indicates the number of cells seeded; assays were performed after overnight culture, so actual cell densities at treatment may be higher.
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Figure 2: Validation of mini-scale DNA and DPCC isolation for high-throughput screening applications. (A) DNA yield from untreated CCRF-CEM cells lysed in LS1 containing 5 M GTC and adjusted to the indicated LiCl and GTC concentrations by addition of 8 M LiCl, and precipitated following addition of 1 volume isopropanol at low RCF; rightmost samples included 8 mg silica gel per well. (B) Efficiency of DNA recovery. DNA yield was determined from HCT116 cells which had been untreated (NT) or treated for 30 min with 10 μM CPT, then lysed in LS1 containing 2.5 M GTC, 4 M LiCl and 8 mg silica gel per well, and DNA recovered by precipitation with 1 volume isopropanol. The x-axis indicates the number of cells seeded; assays were performed after overnight culture, so actual cell densities at treatment may be higher.

Mentions: Further tests of lysis buffers showed some sample-to-sample variability that was minimized if silica slurry (8 mg) was included in each well (e.g. Figure 2A). To determine cell numbers necessary for efficient DNA recovery, we performed an experiment modeling a high-throughput chemical library screen. Varying numbers of cells cultured in a 96-well plate were untreated or briefly treated with 10 μM CPT, lysed in LS1 containing 2.5 M GTC, 4 M LiCl and 8 mg silica gel per well, and DNA and DPCC recovered by precipitation with 1 volume of isopropanol and centrifugation of the microtiter plate at RCF 2740. Under these conditions, DNA was efficiently recovered from as few as 1–2 × 104 cultured human cells, with a yield of about 140 ng per 104 cells, and the DNA yield was not substantially affected by treatment with CPT (Figure 2B). The ability to culture cells and recover DNA on the same microplate greatly streamlines workflow.


Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

Kiianitsa K, Maizels N - Nucleic Acids Res. (2014)

Validation of mini-scale DNA and DPCC isolation for high-throughput screening applications. (A) DNA yield from untreated CCRF-CEM cells lysed in LS1 containing 5 M GTC and adjusted to the indicated LiCl and GTC concentrations by addition of 8 M LiCl, and precipitated following addition of 1 volume isopropanol at low RCF; rightmost samples included 8 mg silica gel per well. (B) Efficiency of DNA recovery. DNA yield was determined from HCT116 cells which had been untreated (NT) or treated for 30 min with 10 μM CPT, then lysed in LS1 containing 2.5 M GTC, 4 M LiCl and 8 mg silica gel per well, and DNA recovered by precipitation with 1 volume isopropanol. The x-axis indicates the number of cells seeded; assays were performed after overnight culture, so actual cell densities at treatment may be higher.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117749&req=5

Figure 2: Validation of mini-scale DNA and DPCC isolation for high-throughput screening applications. (A) DNA yield from untreated CCRF-CEM cells lysed in LS1 containing 5 M GTC and adjusted to the indicated LiCl and GTC concentrations by addition of 8 M LiCl, and precipitated following addition of 1 volume isopropanol at low RCF; rightmost samples included 8 mg silica gel per well. (B) Efficiency of DNA recovery. DNA yield was determined from HCT116 cells which had been untreated (NT) or treated for 30 min with 10 μM CPT, then lysed in LS1 containing 2.5 M GTC, 4 M LiCl and 8 mg silica gel per well, and DNA recovered by precipitation with 1 volume isopropanol. The x-axis indicates the number of cells seeded; assays were performed after overnight culture, so actual cell densities at treatment may be higher.
Mentions: Further tests of lysis buffers showed some sample-to-sample variability that was minimized if silica slurry (8 mg) was included in each well (e.g. Figure 2A). To determine cell numbers necessary for efficient DNA recovery, we performed an experiment modeling a high-throughput chemical library screen. Varying numbers of cells cultured in a 96-well plate were untreated or briefly treated with 10 μM CPT, lysed in LS1 containing 2.5 M GTC, 4 M LiCl and 8 mg silica gel per well, and DNA and DPCC recovered by precipitation with 1 volume of isopropanol and centrifugation of the microtiter plate at RCF 2740. Under these conditions, DNA was efficiently recovered from as few as 1–2 × 104 cultured human cells, with a yield of about 140 ng per 104 cells, and the DNA yield was not substantially affected by treatment with CPT (Figure 2B). The ability to culture cells and recover DNA on the same microplate greatly streamlines workflow.

Bottom Line: This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells.Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay.The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.

Show MeSH
Related in: MedlinePlus