DOI
https://doi.org/10.25772/DSG6-XT54
Defense Date
2018
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Nanoscience and Nanotechnology
First Advisor
Jason C. Reed
Abstract
Genetic mutations in the regulatory genes responsible for cell proliferation, differentiation, and death play a significant role in cancer. Molecular diagnostic and sequencing techniques used to detect these abnormalities have limitations that need to be overcome. These issues can be addressed with the use of a novel High-Speed Atomic Force Microscope (HS-AFM) taking physical measurement of molecules labeled with a programmable nanoparticle (Cas9). This technique was used to demonstrate proof-of-concept using genetic mutations found in breast cancer and follicular lymphoma.
Current quantification methods for copy number variation (gene duplication) and gene expression analysis are based on amplification and fluorescence-based techniques (e.g., qPCR and FISH). These methods cannot easily and reliably detect multiple targets in a single reaction and produce amplification errors in the detection of low abundance targets. These issues were overcome using a multiplex amplification with low cycles of polymerase chain reaction (PCR), followed by single-molecule detection with Atomic Force Microscopy (AFM). The gene expression proles of two different total human RNA samples from our single reaction multiplex PCR/AFM aligned with the data from individual singleplex quantitative PCR reactions. The conventional AFM was replaced by a HS-AFM, invented by collaborators from the University of Bristol, to demonstrate the throughput capabilities necessary for use in clinical applications. The sensitivity of this instrument was able to detect contamination in three out of four popular commercial DNA purification kits, proving our technique useful for optimizing assay conditions.
To further increase features of this technique and broaden its application, a `nanomapping' labeling technique was established for physical mapping of DNA using HS-AFM. This approach increases multiplexing capabilities and is a complementary method for sequencing and other physical mapping techniques. Commercially available DNA was used to determine the efficiency and precision of CRISPR Cas9 labeling and applied to clinical biopsies for the detection and precise mapping of BCL2-IGH translocations present in follicular lymphoma tumors. To reduce the size and cost of the HS-AFM, the detection component was replaced by DVD optics and produced images of similar size, resolution, and quality. The successful demonstration and evolution of this technique proves promising for clinical implementation.
Rights
© Anita J. Olsen
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-10-2018