UBC Theses and Dissertations
Analysis of single telomeres using fluorescence microscopy Alhusari, Deema
Eukaryotic chromosome ends are protected by special DNA structures known as telomeres. In mammals the DNA of telomeres consist of TTAGGG repeats which, in cooperation with specialized proteins, “cap” the ends of chromosomes to protect the chromosomes from end-to-end fusion and erosion. Thus, telomeres are important to maintain chromosome stability and play a vital role in preserving the information in our genome. A key factor of telomeric function is the length of the telomeres. Short and dysfunctional telomeres with less than a few dozen repeats are associated with genomic instability and tumorigenesis. Furthermore, loss of telomere function is implicated in numerous diseases like bone marrow failure, hematological malignancies and other cancers. There is a substantial body of evidence indicating that the average length of telomeres can provide prognostic information in human diseases. However, limitations in the currently available technologies for detecting and measuring the length of telomeres has hampered progress in translating telomere length assays into clinical practice. Additionally, many questions about the relation between telomere length and telomere function remain to be answered. Consequently, novel approaches to study single telomeres are of significant interest. In this study, I investigated two cutting-edge technologies to assess properties of telomeres. I used quantitative fluorescence in situ hybridization (Q-FISH) to identify the length of telomeres based on fluorescence values. Using Q-FISH, I was able to generate DNA measurements using plasmids with different size telomeric inserts that served as a reference for length quantifications taken on different platforms. Also, I initiated explorations of a novel high-throughput method to study physical properties of single telomeres and, potentially, measure the length of telomere repeats. Convex Lens-induced Confinement technique (CLiC) is a technique developed to image individual biological molecules and study their dynamics. Using the CLiC platform, I sought to define the length of plasmid DNA based on diffusion coefficient values. My work has set the stage for others to explore the CLiC platform to study properties of telomeres including biological properties as well as their length. The latter can possibly be used as a prognostic tool in bone marrow failure, hematological malignancies and other disorders.
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