Single Cell And Single Molecule Techniques For The Analysis Of The Epigenome
Epigenetic regulation is a critical biological process for the health and development of a cell. Epigenetic regulation is facilitated by covalent modifications to the underlying DNA and chromatin proteins. A fundamental understanding of these epigenetic modifications and their associated interactions at the molecular scale is necessary to explain phenomena including cellular identity, stem cell plasticity, and neoplastic transformation. It is widely known that abnormal epigenetic profiles have been linked to many diseases, most notably cancer. While the field of epigenetics has progressed rapidly with conventional techniques, significant advances remain to be made with respect to combinatoric analysis of epigenetic marks and single cell epigenetics. Therefore, in this dissertation, I will discuss our development of devices and methodologies to address these pertinent issues. First, we designed a preparatory polydimethylsiloxane (PDMS) microdevice for the extraction, purification, and stretching of human chromosomal DNA and chromatin from small cell populations down to a single cell. The valveless device captures cells by size exclusion within the micropillars, entraps the DNA or chromatin in the micropillars after cell lysis, purifies away the cellular debris, and fluorescently labels the DNA and/or chromatin all within a single reaction chamber. With the device, we achieve nearly 100% extraction efficiency of the DNA. The device is also used for in-channel immunostaining of chromatin followed by downstream single molecule chromatin analysis in nanochannels (SCAN). Second, using multi-color, time-correlated single molecule measurements in nanochannels, simultaneous coincidence detection of 2 epigenetic marks is demonstrated. Coincidence detection of 3 epigenetic marks is also established using a pulsed interleaved excitation scheme. With these two promising results, genome-wide quantification of epigenetic marks was pursued. Unfortunately, quantitative SCAN never materialized. Reasons for this, including poor signal to background, are explained in detail. Third, development of mobility-SCAN, an analytical technique for measuring and analyzing single molecules based on their fluorescent signature and their electrophoretic mobility in nanochannels is described. We use the technique to differentiate biomolecules from complex mixtures and derive parameters such as diffusion coefficients and effective charges. Finally, the device is used to detect binding interactions of various complexes similar to affinity capillary electrophoresis, but on a single molecule level. Fourth, we conclude by briefly discussing SCAN-sort, a technique to sort individual chromatin molecules based on their fluorescent emissions for further downstream analysis such as DNA sequencing. We demonstrate a 2-fold enrichment of chromatin from sorting and discuss possible system modifications for better performance in the future.
Ph.D. of Applied Physics
Doctor of Philosophy
dissertation or thesis