A Versatile Platform for Highly Sensitive Analysis of DNA Size, Conformation, and Binding Interactions in Free Solution
Embargo until
2019-05-01
Date
2018-03-15
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Publisher
Johns Hopkins University
Abstract
Nucleic acid analysis has enhanced our understanding of biological processes and disease progression, elucidated the association of genetic variants and disease, and led to the design and implementation of new treatment strategies. These diverse applications require analysis of a variety of characteristics of nucleic acid molecules including size or length, detection or quantification of specific sequences, analysis of conformations or conformational changes, and observation of interactions between nucleic acids and other biomolecules. In addition to this variability in measurement modality, samples themselves can contain multiple species, further convoluting the complexity of analysis. Strategies that can detect rare or transient species, characterize population distributions, offer high sensitivity and quantification capabilities, and analyze small sample volumes enable the collection of rich multiparametric data from a single biosample. Platforms that integrate micro- and nano- fluidic operations with high sensitivity single molecule detection facilitate manipulation and detection of individual nucleic acid molecules and are well poised to fulfill this need.
In this thesis, we present a single molecule free solution hydrodynamic separation (SML-FSHS) platform for highly sensitive, quantitative, and versatile analysis of DNA molecules. Coupling a microfluidic size separation strategy with single molecule detection enables the unique ability to sensitively and quantitatively analyze multiple nucleic acid properties in free solution with extremely high detection sensitivity and low reagent consumption. The separation strategy achieves separation and sizing over a wide dynamic range, while single molecule burst analysis enables layered insight into DNA conformation and packing. Moreover, size-based separation of intermolecular interactions enables detection and quantification of binding properties in a free solution environment. The simplicity of operation, free solution conditions and potential for automation make the platform attractive for a variety of lab-based analysis. Such a system can be used to garner diverse information about DNA conformation, structure, and interactions.
We start by outlining the various DNA properties of interest and discuss some of the existing alternative microfluidic single molecule analysis methods (Chapter 1). Then, we introduce our SML-FSHS platform and demonstrate its utility for DNA length separation and sizing (Chapter 2). Next, we explore layered conformational analysis through a combination of hydrodynamic mobility and single molecule burst analysis (Chapter 3). Then, we further expand the platform’s application to analyze intermolecular interactions including DNA hybridization and DNA-protein binding analysis (Chapter 4). Finally, we develop an integrated in-line preconcentration technique to further increase the concentration sensitivity beyond the capabilities of traditional methods (Chapter 5).
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Keywords
DNA, microfluidics, single molecule detection, separation, molecular rheotaxis, preconcentration