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" Integrated Microfluidic Systems for Genetic Analysis "
Cronier, Samantha Anne
Mathies, Richard A
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Latin Dissertation
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English
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| Record Number
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897852
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TL8db0c12q
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| Main Entry
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Gerber, Matthew J
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Integrated Microfluidic Systems for Genetic Analysis\ Cronier, Samantha AnneMathies, Richard A
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2011
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2011
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| Abstract
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Recent work in the areas of microfluidic technology and innovative new chemistries have made possible tremendous improvements in our ability to determine the nucleotide sequence of DNA. These advances are already changing the way doctors diagnose and treat human disease and enabling scientists to undertake genetic studies never before possible. In this dissertation I provide a brief history of DNA sequencing methods as well as a general description of microfluidic technologies, in particular those used for genetic analysis. Next, I describe my dissertation research on the development of a highly efficient fully integrated microfluidic platform for Sanger DNA sequencing, including automated thermal cycling, purification, concentration and in-line injection of the extension fragments for microchip capillary electrophoresis separation. The two-layer glass device that I developed features two independently operated valve-free systems, comprised of a 200 nL thermal cycling reactor with resistive temperature detector, a 1.2 nL in situ photopolymerized oligonucleotide affinity capture gel for post reaction clean-up and inline injection, and an 18-cm long capillary electrophoresis channel for separation. Integration of the efficient photopolymerized affinity gel capture allows sequencing from only 350 - 500 attomoles of starting DNA template. Using this device, I was able to sequence 507 ± 31 bases at 99% accuracy. In addition, I show that this method is compatible with single cell genetic analysis techniques (SCGA) by sequencing from the small amounts (~100 attomoles) of amplified DNA bound to agarose microbeads that can be produced from single cells. This dissertation concludes with a discussion of the future of DNA sequencing and the feasibility of performing single cell DNA sequencing using the Microbead Integrated DNA Sequencing (MINDS) method.
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Cronier, Samantha Anne
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UC Berkeley
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