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. 2015 Jan 1:853:676-681.
doi: 10.1016/j.aca.2014.11.003. Epub 2014 Nov 6.

Simultaneous detection of ultraviolet B-induced DNA damage using capillary electrophoresis with laser-induced fluorescence

Jeffrey W Guthrie  1 , Robert T Limmer  2 , Eric A Brooks  2 , Chelsea C Wisnewski  2 , Nnekia D Loggins-Davis  2 , Abderraouf Bouzid  2
Affiliations

Simultaneous detection of ultraviolet B-induced DNA damage using capillary electrophoresis with laser-induced fluorescence

Jeffrey W Guthrie et al. Anal Chim Acta. .

Abstract

An immunoassay based on CE-LIF was developed for the simultaneous detection of cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) in genomic DNA irradiated with UVB or natural sunlight. Human cells were first exposed to varying amounts of UVB or natural sunlight to induce DNA damage. Genomic DNA was extracted and incubated with anti-CPD and anti-6-4PP primary antibodies attached to secondary antibodies with a fluorescent quantum dot (QD) reporter that emitted either red or yellow fluorescence. CE was used to separate the unbound antibodies from those bound to the photoproducts, and LIF with appropriate optical filters was used to separate the fluorescence signals from each QD to individual photomultiplier tubes for simultaneous photoproduct detection. Using this strategy, photoproducts were detected from ∼6 ng (200 ng μL(-1)) of DNA under a low UVB fluence of 65 J m(-2) for CPDs or 195 J m(-2) for 6-4PPs. This assay was also the first to demonstrate the detection of CPDs in human cells after only 15 min of irradiation under natural sunlight.

Keywords: 6-4 Photoproducts; Capillary electrophoresis; Cyclobutane pyrimidine dimers; Immunoassay; Laser-induced fluorescence; Quantum dots.

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