High-throughput Single-Molecule Telomere Length Characterization
Wednesday, June 13, 2018
11:00 AM-1:00 PM
BIOMED PhD Thesis Defense
Title:
High-throughput Single-Molecule Telomere Length Characterization
Speaker:
Jennifer McCaffrey, PhD Candidate, School of Biomedical Engineering, Science and Health Systems
Advisor:
Ming Xiao, PhD, Associate Professor, School of Biomedical Engineering, Science and Health Systems
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.
Contact Information
Ken Barbee
215-895-1335
barbee@drexel.edu