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Detection of BPDE-DNA Adducts on Specific Genes

  • Author / Creator
    Wang, Chuan
  • Benzo[a]pyrene diol epoxide (BPDE), a known carcinogen, is a reactive metabolite of benzo[a]pyrene (BP). BP is usually formed during combustion of organic substances, such as cigarette smoke, automobile emission exhaust, and industrial waste burning. BPDE can covalently bind to the nucleobases of DNA. The investigation of the formation of BPDE-DNA adducts in the p53 gene revealed a correlation between the profiles of adduct hotspots and p53 mutation hotspots in cancers, suggesting a probable cancer-related mutation caused by BPDE-DNA adducts. Detection of BPDE-DNA adducts on specific genes is important for monitoring human exposure to this carcinogen and for studying environmental carcinogenesis. In this thesis, two binding-induced DNA assembly (BINDA) assays for BPDE-DNA were developed to quantify the BPDE-DNA adducts in the specific p53-exon7. The focus on this gene was because of its relevance to cancer. In a homogeneous BINDA assay, two probes were constructed by attaching one DNA motif to an antibody against BPDE and another DNA motif to a hybridization sequence of p53-exon7. Cooperative binding of the two probes with the p53-exon7 containing BPDE adducts triggered the assembly of the two DNA motifs. Real-time qPCR quantification of the assembled DNA motifs enabled the detection of BPDE adducts in the specific gene. The second BINDA assay involved the immobilization of one DNA motif and the BPDE antibody on magnetic beads. Taking advantage of the pre-concentration and washing procedures of magnetic beads, this assay was able to achieve higher sensitivity. Both the homogeneous BINDA and the magnetic bead-mediated BINDA assays were coupled with solid-phase extraction to analyze the BPDE-DNA adducts in the presence of complex genomic DNA. The magnetic bead-mediated BINDA assay was also applied to the detection of the BPDE-DNA adducts in the p53-exon7 of cells treated with BPDE. The results demonstrated the potential of the new assays for detection of DNA adducts on specific genes. The ability to quantify the DNA adducts formed on a specific sequence, rather than the overall adducts on the whole genome, opens up opportunities for further investigation into the specific DNA damage and its involvement in environmental carcinogenesis.

  • Subjects / Keywords
  • Graduation date
    2014-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3PD7V
  • 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.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Medical Sciences- Laboratory Medicine and Pathology
  • Supervisor / co-supervisor and their department(s)
    • Li, Xing-Fang (Laboratory Medicine and Pathology)
  • Examining committee members and their departments
    • Li, Liang (Chemistry)
    • Tyrrell, Gregory (Laboratory Medicine and Pathology)
    • Keelan, Monika (Laboratory Medicine and Pathology)
    • Le, X. Chris (Laboratory Medicine and Pathology)
    • Bowser, Michael (Chemistry, University of Minnesota)
    • Li, Xing-Fang (Laboratory Medicine and Pathology)