Dissertation
High-throughput single-molecule telomere length characterization
Doctor of Philosophy (Ph.D.), Drexel University
28 Jun 2018
DOI:
https://doi.org/10.17918/00000165
Abstract
A method was developed to measure telomere lengths at the single-molecule level. This was accomplished through modification of the type II CRISPR/Cas9 system for RNA-directed Cas9 nickase (Cas9n) to nick-label the telomere repeat while simultaneously barcoding the rest of the genome with a separate nickase enzyme that recognizes a 7bp motif. High-throughput imaging of the labeled single-molecules in nanochannel arrays allows for genome mapping using the subtelomere repeat elements (SREs) to unique chromosomal DNA and measuring the telomere length at the ends of the DNA molecules. This results in single subtelomere specific telomere lengths for approximately 30-35 chromosome arms. The method was performed on fibroblasts to mimic the aging process and two telomerase positive cells lines. The expected telomere attrition was observed in the aging model and telomere-specific biases for critically short telomeres were noted. Telomere lengths were also measured and analyzed for the Alternative Lengthening of Telomeres (ALT) positive cell line U2OS. Telomere length, the heterogeneity of telomere length, the frequency of signal-free ends, and the frequency and size of internal telomere-like sequences (ITSs) within recombinant molecules varied depending on the subtelomere. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were observed along with punctate labeling of the telomere on long telomeres indicating the inclusion of stretches of non-canonical telomere repeats. Differences in structural changes in repose to telomere damage and repair were observed between telomerase positive and ALT positive cancer cell lines. In addition, the method can be used to identify previously uncharacterized subtelomeres and make improvements to the human reference genome.
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Details
- Title
- High-throughput single-molecule telomere length characterization
- Creators
- Jennifer McCaffrey
- Contributors
- Ming Xiao (Advisor) - Drexel University, School of Biomedical Engineering, Science, and Health Systems
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xvi, 98 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Biomedical Engineering, Science, and Health Systems (1997-2026); Drexel University
- Other Identifier
- 991014743748604721