abstract: There are many biological questions that require single-cell analysis of gene sequences, including analysis of clonally distributed dimeric immunoreceptors on lymphocytes (T cells and B cells) and/or the accumulation of driver/accessory mutations in polyclonal tumors. Lysis of bulk cell populations results in mixing of gene sequences, making it impossible to know which pairs of gene sequences originated from any particular cell and obfuscating analysis of rare sequences within large populations. Although current single-cell sorting technologies can be used to address some of these questions, such approaches are expensive, require specialized equipment, and lack the necessary high-throughput capacity for comprehensive analysis. Water-in-oil emulsion approaches for single cell sorting have been developed but droplet-based single-cell lysis and analysis have proven inefficient and yield high rates of false pairings. Ideally, molecular approaches for linking gene sequences from individual cells could be coupled with next-generation high-throughput sequencing to overcome these obstacles, but conventional approaches for linking gene sequences, such as by transfection with bridging oligonucleotides, result in activation of cellular nucleases that destroy the template, precluding this strategy. Recent advances in the synthesis and fabrication of modular deoxyribonucleic acid (DNA) origami nanostructures have resulted in new possibilities for addressing many current and long-standing scientific and technical challenges in biology and medicine. One exciting application of DNA nanotechnology is the intracellular capture, barcode linkage, and subsequent sequence analysis of multiple messenger RNA (mRNA) targets from individual cells within heterogeneous cell populations. DNA nanostructures can be transfected into individual cells to capture and protect mRNA for specific expressed genes, and incorporation of origami-specific bowtie-barcodes into the origami nanostructure facilitates pairing and analysis of mRNA from individual cells by high-throughput next-generation sequencing. This approach is highly modular and can be adapted to virtually any two (and possibly more) gene target sequences, and therefore has a wide range of potential applications for analysis of diverse cell populations such as understanding the relationship between different immune cell populations, development of novel immunotherapeutic antibodies, or improving the diagnosis or treatment for a wide variety of cancers. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2017
| Identifer | oai:union.ndltd.org:asu.edu/item:46219 |
| Date | January 2017 |
| Contributors | Schoettle, Louis Noble (Author), Blattman, Joseph N (Advisor), Yan, Hao (Committee member), Chang, Yung (Committee member), Lindsay, Stuart (Committee member), Arizona State University (Publisher) |
| Source Sets | Arizona State University |
| Language | English |
| Detected Language | English |
| Type | Doctoral Dissertation |
| Format | 239 pages |
| Rights | http://rightsstatements.org/vocab/InC/1.0/, All Rights Reserved |
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