Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence. Analysis of A549 cells.
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.
Tan
W G
WG
Department of Public Health Sciences, Faculty of Medicine, University of Alberta, Edmonton, Canada.
Carnelley
T J
TJ
Murphy
P
P
Wang
H
H
Lee
J
J
Barker
S
S
Weinfeld
M
M
Le
X C
XC
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis*; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry; D018736:DNA Adducts / Q000032:analysis*; D017931:DNA Primers; D006801:Humans; D007122:Immunoelectrophoresis / Q000379:methods*; D007834:Lasers; D009691:Nucleic Acid Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014407:Tumor Cells, Cultured
D018736:DNA Adducts; D017931:DNA Primers; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA