Fracture-Based Fabrication of a Size-Controllable Micro/Nanofluidic Platform for Mapping of DNA/Chromatin.

摘要

In the years since the launch of The Human Genome Project (HGP), which significantly increased our understanding of biological inheritance by revealing the structure and function of genetic material, tangential research efforts have revealed mechanisms of inheritance that extend beyond the sequence of nucleic acids within an individual’s genome. The study of these mechanisms, referred to as epigenetics, now lies at the frontier of biomedical research. While much is known regarding genetic inheritance, the complexity of chromosome structure and lack of appropriate methodologies have long hindered mechanistic dissection of epigenetic inheritance. The work in this dissertation seeks three fundamental objectives: (1) the development of appropriate tools for chromatin mapping, (2) the identification of a well-defined model system, and (3) the use of ‘super-resolution imaging’. First, a unique micro/nanofluidics platform was developed utilizing fracture-based fabrication techniques. The use of such techniques, combined with the careful selection of appropriate materials, enabled the formation of channels with dimensions that could be modified by simply modifying the magnitude of the uniaxial strain applied. By integrating stress focusing notch micro-features into the soft elastomer, polydimethylsiloxane (PDMS), nano-scale fractures were generated at desired positions, producing an array of nano-channels.These adjustable channels were then utilized to achieve the efficient pre-concentration, capturing, and linearization of DNA and chromatin via nano-confinement and a squeezing flow. In the tuneable channel device, DNA molecules were pre-concentrated up to 10,000 fold at the defined position using electrophoresis, and were successfully trapped and linearized up to its contour length for epigenetic marker profiling.Finally, Tetrahymena was selected as an optimal biological system, and was used to elucidate the spatial distribution of histones along replicated DNA, as well as to characterize specific histone-DNA interactions occurring during replication by the super-resolution microscopy. This multi-disciplinary dissertation project provides insight into both the unknown epigenetic changes occurring during DNA replication, and the biological machinery underlying fundamental DNA-histone interactions. The application of this adjustable fluidics platform to other biological model species may provide a means to establish other epigenetic marker maps including patterns of post-translational modifications of histone and DNA methylation to study yet unknown epigenetic mechanisms.