Microfluidic techniques for DNA melting analysis and digital polymerase chain reaction

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Publication Type dissertation
School or College College of Engineering
Department Bioengineering
Author Sundberg, Scott Owen
Title Microfluidic techniques for DNA melting analysis and digital polymerase chain reaction
Date 2010-08
Description Microfluidic methods were applied to nucleic acid mutation identification and quantification. DNA melting analysis interrogation volumes were reduced 4 orders of magnitude (down to 1 nL volumes) from commercial instrumentation, allowing less reagent consumption while yielding adequate signal for genotyping and scanning of polymerase chain reaction (PCR) products. A microfluidic instrument was developed for digital PCR applications, using a spinning plastic disk patterned by xurography. Theplatform offers faster thermocycling times (30 cycles in ~12 min), simplified fluid partitioning, and a less expensive disposable when compared to currently available digital PCR platforms. PCR within the disk was validated by quantifying plasmid DNA sample using "on/off" fluorescence interrogation across 1000 wells (30 nL/well) at varying template concentration. A 94% PCR efficiency and product amplification specificity were determined by aggregate real-time PCR and melting analysis. The technique of quasi-digital PCR was also applied within this platform, wherein a single mutation copy was preferentially amplified from a large background of wild-type DNA, to detect and quantify low levels of rare mutations. This method demonstrated a sensitivity of 0.01% (detecting a mutant to wild-type DNA ratio of 43:450000), by mixing known concentrations of an oncogene mutation with thousands of wild-type template copies. Statistic analysis tools were constructed in order to interpret digital PCR data, with results comparing well to DNA absorption measurements.
Type Text
Publisher University of Utah
Subject Circulating tumor cells; Digital PCR; DNA melting analysis; Microfluidics; PCR
Dissertation Institution University of Utah
Dissertation Name Doctor of Philosophy
Language eng
Rights Management Copyright © Scott Owen Sundberg 2010
Format Medium application/pdf
Format Extent 15,745,599 bytes
Source Original in Marriott Library Special Collections, QH9.7 2010 .S86
ARK ark:/87278/s60k2qb9
Setname ir_etd
Date Created 2012-04-24
Date Modified 2018-03-28
ID 194712
Reference URL https://collections.lib.utah.edu/ark:/87278/s60k2qb9