| Autor: |
Trevor J. Carnelley, Woei G. Tan, Sharon Barker, Jane Lee, Michael Weinfeld, Paula Murphy, X. Chris Le, Hailin Wang |
| Rok vydání: |
2001 |
| Předmět: |
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| Zdroj: |
Journal of Chromatography A. 924:377-386 |
| ISSN: |
0021-9673 |
| DOI: |
10.1016/s0021-9673(01)00987-6 |
| Popis: |
Detection of benzo[a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6 x 10(5) and 1 x 10(6) plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-10) M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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