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

• Author / Creator

  Goulko, Alevtina

• 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.

• Subjects / Keywords

  • Capillary electrophoresis
  • Cyclobutane pyrimidine dimer
  • Ultraviolet light
  • DNA damage

• Graduation date

  Spring 2011

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3D611

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.