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Permanent link (DOI): https://doi.org/10.7939/R3D611

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Development and application of a capillary electrophoresis immunoassay for DNA lesions induced by ultraviolet light Open Access

Descriptions

Other title
Subject/Keyword
ultraviolet light
capillary electrophoresis
DNA damage
cyclobutane pyrimidine dimer
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Goulko, Alevtina
Supervisor and department
Le, X. Chris (Department of Chemistry)
Examining committee member and department
Le, X. Chris (Department of Chemistry)
Weinfeld, Michael (Department of Oncology)
Loppnow, Glen (Department of Chemistry)
Chen, David (Department of Chemistry, UBC, external)
Lucy, Charles (Department of Chemistry)
Campbell, Robert (Department of Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2012-05-30T15:49:09Z
Graduation date
2011-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Ultraviolet (UV) light is one of the most abundant DNA damaging agents. The major DNA lesions, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine pyrimidone (64PPs) photoproducts, are carcinogenic and mutagenic. Studies of the formation, repair and mutagenicity of these DNA lesions induced by environmentally relevant doses of UV irradiation require sensitive and specific techniques for their detection. This thesis focuses on the development and application of an immunoassay combining capillary electrophoresis (CE) separation with laser induced fluorescence (LIF) detection. Primary monoclonal antibodies binding to either CPDs or 64PPs were used as the affinity recognition probes. A fluorescently labeled secondary antibody fragment (Fab) was used to bind to the primary antibody. The immunocomplexes of the DNA lesions with the antibodies were separated from the excess amounts of antibodies by using CE. The fluorescent intensities of the immunocomplexes containing the DNA lesions were measured for quantitative determination of CPDs and 64PPs. Synthetic standard oligonucleotides (16 bases in length) containing a single CPD or 64PP were used for quantitative calibration. Successful determination of CPDs in UV-irradiated 80-nucleotide (nt) DNA library, calf thymus DNA, human placenta DNA and human fibroblast cells demonstrated the applicability of this CE-LIF immunoassay. Irradiation of the random oligonucleotide sequences, naked DNA and cells with varying doses of UVB and/or UVC provided useful information on the relative yield of the formation of the UV-induced DNA lesions. The yield of CPD formation in the cellular DNA of CRL-2522 fibroblasts (3.6 ± 0.8 lesions per 103 nt per J/cm2) following irradiation with low doses of UVB (< 0.3 J/cm2) was approximately 7 times lower than that in the naked calf thymus DNA (26.8 ± 1.4 lesions per 103 nt per J/cm2). A fluorescently labeled oligonucleotide (90nt in length) containing a single CPD was designed, synthesized and partially characterized. It could potentially be used as a probe to study interactions between CPD-containing DNA and nucleotide excision repair proteins, or as a probe to screen for binding proteins or antibodies. The CE-LIF immunoassay takes advantage of antibody affinity, CE separation and highly sensitive LIF detection. Further potential applications include studies of DNA repair, monitoring of DNA damage and environmental biomarker development.
Language
English
DOI
doi:10.7939/R3D611
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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