Date of Award

January 2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Agricultural and Biological Engineering

First Advisor

Joseph M.K. Irudayaraj

Committee Member 1

Chris J Gilpin

Committee Member 2

Wen Jiang

Committee Member 3

Abigail S Engelberth

Abstract

Structure determines function. Properly ordered chromatin organization is fundamental for accurate regulation of gene expression inside eukaryotes. A native chromatin extraction method is developed based on solid phase reversible immobilization (SPRI) using magnetic nanoparticles as carriers. This method provides a quick, simple, inexpensive and an environmental-friendly means of chromatin extraction, and preserves the native structure of chromatin. The purified chromatin consists of DNA, histones, chromatin associated RNAs (CARs) and other non-histone proteins. Using the purified chromatin as a source, we developed a biotin-streptavidin mediated enzyme-based immunosorbent assay for simultaneous detection of epigenetic modifications on DNA and histone proteins. In order to analyze the CARs, we also developed a magnetophoretic chromatography method to remove the free RNAs, and the CARs were further extracted from the collected chromatin. The extracted CARs were reverse transcribed as cDNA and further characterized by real-time qPCR. The total assay time taken for cell separation, chromatin purification and chromatin associated RNAs extraction can be accomplished in less than 2h. Finally, we further applied cryogenic electron microscopy to analyze higher order chromatin structure in near-native state. We found that the chromatin fibers hold a branching structure and this structure generally exist in eukaryotic cells.

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